Abstracts of the10th International ISSX meeting, September 29–October 3, 2013, Toronto, Ontario, Canada.

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ABSTRACTS FROM THE INTERNATIONAL ISSX MEETING

September 29–October 3, 2013 Toronto, Ontario, Canada

Volume 45, Supplement 1, 2013

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10th International ISSX Meeting September 29 – October 3, 2013 Toronto, Ontario, Canada Contents ISSX Overview Upcoming ISSX Meetings Short Courses (Sunday, September 29, 2013)


Short Course 1: Metabolomic Profiling as a Tool for Identifying Novel Drug-Target Interactions Abstracts: SC1.1 – SC1.4 Short Course 2: Model Systems and Methods for Assessing Uptake and Efflux of Small Molecules Abstracts: SC2.1 – SC2.5 Short Course 3: Strategies and Techniques for Predicting Human Drug Metabolism and Drug-Drug Interactions Abstracts: SC3.1 – SC3.4 Short Course 4: Application of Novel Mass Spectrometric Methods to Drug Metabolism and Pharmacokinetics Studies Abstracts: SC4.1 – SC4.4 Keynote Presentation (Sunday, September 29, 2013) Keynote Lecture 1: Systems Pharmacology – Systems Biology Meets Drug Discovery & Development Abstract: S1 Plenary Sessions and Parallel Sessions Presentations (Monday, September 30, 2013) Plenary Session 1: Drug-Metabolizing Enzymes as Potential Therapeutic Targets Abstracts: S2 – S4 Parallel Session 1: Nuclear Receptors as Regulators of Drug Metabolism and as Therapeutic Targets Abstracts: S5 – S8 Parallel Session 2: Pharmacogenomics and Personalized Medicine: Progress in Clinical Implementation Abstracts: S9 – S12 Parallel Session 3: Regulation of Drug Transporter Expression and Function Abstracts: S13 – S16 Parallel Session 4: Application of Structural Biology to the Prediction of Drug Response, Metabolism and Toxicity Abstracts: S17 – S20 Plenary Sessions and Parallel Sessions Presentations (Tuesday, October 1, 2013) Plenary Session 2: Utility of Genetic and Epigenetic Technologies in Drug Development and Safety Assessment Abstracts: S21 – S23 Parallel Session 5: Metabolism, Pharmacokinetics and Interactions of Traditional Medicines and Natural Products Abstracts: S24 – S27 Parallel Session 6: Cell Type Specific Mechanisms of Adverse Drug Reactions Abstracts: S28 – S30, P29 and P442

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Plenary Sessions and Parallel Sessions Presentations (Wednesday, October 2, 2013) Plenary Session 3: Identification and Use of Novel Biomarkers for Xenobiotic-Induced Toxicity Abstracts: S31 – S33 Parallel Session 7: Targeted Drug Delivery for Enhanced Therapy Abstracts: S34 – S37 Parallel Session 8: Use of PB-PK Modeling in Pharmaceutical Safety Assessment Abstracts: S38 – S40, P264 and P458 Parallel Session 9: Regulatory Perspectives in Drug-Drug Interactions and Metabolites in Safety Testing Abstracts: S41 – S44


Parallel Session 10: Toxicogenomic and Metabolomic Profiling: Current Status and Future Utility in Assessing Drug Efficacy and Safety Abstracts: S45 – S47, P24 and P60 Plenary Sessions and Parallel Sessions Presentations (Thursday, October 3, 2012) Plenary Session 4: Genetically Modified Animal Models for Predicting Human Drug Disposition and Response Abstracts: S48 – S50 Parallel Session 11: Tissue Imaging and Distribution Studies in Drug Development Abstracts: S51 – S54 Parallel Session 12: Development and Uses of Hepatocyte-Derived and Hepatocyte-Like Cells for Predicting Human Drug Metabolism Abstracts: S55 – S58 Finalists for the Predoctoral (Graduate) Poster Awards Competition (Monday, September 30 – Thursday, October 3, 2013) Abstracts: A2 – A6 Finalists for the Postdoctoral Poster Awards Competition (Monday, September 30 – Thursday, October 3, 2013) Abstracts: A1, A7 – A12 Poster Presentations (Monday, September 30 – Wednesday, October 2, 2013) Abstracts: P1 – P470 Author Index Terms Index

About ISSX The International Society for the Study of Xenobiotics (ISSX) is the premier scientific organization for researchers interested in the metabolism and disposition of xenobiotics. ISSX was formed in 1981 when a small group of scientists, brought together during the 1970’s under the aegis of the Gordon Research Conferences on Drug Metabolism, took the bold step of creating an international society to promote the interaction of scientists dedicated to the study of xenobiotics in living systems. The study of xenobiotics (medicinal drugs, agricultural chemicals, industrial chemicals, environmental contaminants and other exogenous substances) encompasses the introduction into, distribution and transport throughout, interactions with and elimination of these compounds from biological systems. The studies on xenobiotics include the rates and extents of the processes and the biological consequences.

Society Mission


ISSX is an international association that promotes the understanding of the interactions of medicines and chemicals with living systems.

Goals of the Society

To facilitate and encourage the assembly, acquaintanceship and association of scientists engaged in research in xenobiotic metabolism and in other related disciplines.

To disseminate, discuss and publish results of research and related matters of interest in metabolism.

To promote public awareness of chemical metabolism and its social and environmental implications.

To promote education and training in the field.

UPCOMING ISSX MEETINGS

2014 5th Asia Pacific ISSX Meeting Nankai University Tianjin, China May 9–12, 2014


19th North American ISSX Meeting/29th JSSX Meeting Hilton San Francisco San Francisco, California, USA October 19–23, 2014 2015 13th European Meeting The University of Strathclyde Glasgow, Scotland, UK June 21–25, 2015 20th North American Meeting Hilton Orlando Bonnet Creek Orlando, Florida, USA October 18–22, 2015 2016 11th International ISSX Meeting Busan Exhibition and Convention Center Busan, Korea June 12–16, 2016

http://informahealthcare.com/dmr ISSN: 0360-2532 (print), 1097-9883 (electronic) Drug Metab Rev, 2013; 45(S1): 1–25 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/03602532.2013.868114

ABSTRACTS

Speaker Abstracts


S1. SYSTEMS PHARMACOLOGY – SYSTEMS BIOLOGY MEETS DRUG DISCOVERY & DEVELOPMENT Douglas A. Lauffenburger Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA Definitions of systems biology are as broad ranging as the field itself. An early offering was from the Institute for Systems Biology: Systems biology does not investigate individual genes or proteins one at a time, as has been the highly successful mode of biology for the past 30 years. Rather, it investigates the behavior and relationships of all the elements in a particular biological system while it is functioning. The National Institute of General Medical Sciences offered a different perspective: Systems biology seeks to predict the quantitative behavior of an in vivo biological process under realistic perturbation, where the quantitative treatment derives its power from explicit inclusion of the process components, their interactions, and local states. A central unifying concept calls for understanding how complex biological entities function by integrating quantitative information about multiple molecular- and cellular-level components and properties via computational modeling in order to generate hypotheses, predictions, and insights. The notion of integration as central to bioscience and bioengineering is set forth in the 2009 National Research Council report A New Biology for the 21st Century. But there are multiple dimensions of integration along which the analysis of biological systems must be pursued. One axis represents horizontal integration, moving from the study of individual components (e.g., a single molecule) to that of multiple components (in principle, up to the full genome-wide complement). This axis is manifested in -omic biology. A second axis represents vertical integration, featuring studies of cell-level behavior dependence on molecular properties, tissue- or organ-level behavior dependence on cell properties, organism-level behavior dependence on tissue/organ properties, and so forth. A third axis represents dynamic integration, characterized by the depth or intensity of information addressed in measurement and modeling efforts to understand complex molecular ‘‘machines’’ or ‘‘circuits’’ at the deepest scale. This axis moves from sequence and structure at the most basic degrees, through thermodynamic information, to kinetic and transport information, to comprehensive dynamical systems operation. The necessity of emphasizing these integrative approaches in drug discovery and development has been manifest all along, of course, so ‘systems pharmacology’ is more a matter of ‘‘how to do it’’ than ‘‘whether to do it’’. This talk will describe some examples of work pursued in the laboratory of the presenter along with colleagues at MIT and elsewhere.

S2. HUMAN CYTOCHROME P450S AS THERAPEUTIC TARGETS FOR MECHANISM-BASED INACTIVATORS Paul F. Hollenberg Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA Cytochrome P450s catalyze the metabolism of a wide variety of drugs and other xenobiotics. In this process, they form a variety of reactive intermediates which covalently bind to cellular macromolecules, ultimately leading to toxicity. These reactive intermediates may also react with the P450s that catalyze their formation to form covalent adducts and inactivate the P450, a process referred to as mechanism-based inactivation. A variety of different types of drugs undergo metabolism by P450s to form highly reactive intermediates that can inactivate the proteins by binding to the heme or to the apoprotein. Mechanism-based inactivators can be used to target specific P450s to alter the metabolism of drugs as well as endogenous substrates including steroid hormones and they can be used to probe the active sites of P450s in their catalytically functional conformations. This presentation will focus on studies on the mechanism-based inactivators of P450s, their use to target and affect metabolism of endogenous and exogenous substrates including drugs and steroid hormones, the trapping and identification of the reactive intermediates responsible for inactivation, the determination of the sites of modification on the protein

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responsible for the loss of activity. P450s to be discussed will include 2B6, 2E1, 3A4, 17A1 and 19A1. The identification of the site of modification can provide valuable information regarding the three-dimensional structure of the active site. Recent advances in trapping procedures for the identification of reactive intermediates have greatly facilitated these types of studies. We will also address the mechanisms of inactivation and the use of combined structural and computational analytical methods to obtain insights into the three-dimensional structures of the active sites and how the modified P50s may show changes in their product profiles while retaining partial metabolic activity. (Supported in part by NIH Grant CA 16954).

S3. TARGETING THE ARYLAMINE N-ACETYLTRANSFERASES IN CANCER


Rodney F. Minchin and Neville J. Butcher School of biomedical sciences, Univerity of Queensland, Brisbane, Australia The arylamine N-acetyltransferases are a family of enzymes that catalyze the acetylation of many aromatic and heterocyclic amine compounds. In humans, there are two N-acetyltransferase isozymes, NAT1 and NAT1. NAT2 is a typical drug metabolizing enzyme that is located primarily in the liver and the gastrointestinal tract. By contrast, NAT1 is more widely distributed in the body. Interest in the potential role of NAT1 in cancer began with the observation that this gene is often differentially expressed in subtypes of breast and prostate cancer. Indeed, NAT1 has been reported as one of the most robust biomarkers for luminal breast cancer. Further studies have established that overexpression of NAT1 can confer growth advantage and chemo-resistance in some cancer cells. In our laboratory, we have found that inhibition of NAT1 with either small molecule inhibitors or siRNA can lead to a marked phenotypic change in many different cancer cell types. For example, in the human colon cancer line HT-29, inhibition of NAT1 leads to the up-regulation of E-cadherin, contact inhibition, and reduced growth at confluence. We have also observed using invasive breast cancer cell lines that inhibition of NAT1 is associated with a change in cell shape, and decreased proliferation. Moreover, these cells lose their ability to invade in an in vitro invasion assayed. Finally, inhibition of NAT1 prevented the formation of colonies in the lungs of nude mice injected intravenously with MDA-MB-231 cells, suggesting a decrease in metastatic potential. In addition to over changes in cell phenotype, growth and invasion, we have also observed a change in the metabolic capacity of cells where NAT1 has been knocked down with siRNA. In particular, cells lose the ability to utilize MTA in the methionine salvage pathway, suggesting they become methionine dependent. We propose that NAT1 plays an important role in major metabolic pathways that are particularly important in tumor cell growth. Consequently, this enzyme is a novel drug target in the treatment of cancer and other proliferative diseases.

S4. INHIBITION OF CYTOCHROME P450 17A1: TARGETING ANDROGEN PRODUCTION IN PROSTATE CANCER Emily E. Scott Medicinal Chemistry, The University of Kansas, Lawrence, KS, USA Human steroidogenesis is a complex biochemical pathway involving six different cytochrome P450 enzymes: 11A1, 17A1, 21A2, 11B1, 11B2, and 19A1. Modulation of each enzyme has distinct therapeutic potential with respect to the balance of mineralocorticoid, glucocorticoid, androgen, and estrogen synthesis, but inhibitor selectivity among these enzymes can be a significant concern. Abiraterone, an FDA-approved inhibitor of 17A1 for prostate cancer treatment, has two different types of selectivity issues. First, abiraterone also binds to other human steroidogenic P450 enzymes, including several required for glucocorticoid and mineralocorticoid synthesis. Secondly, abiraterone blocks not only the 17,20-lyase activity of 17A1 to prevent androgen biosynthesis as desired in prostate cancer, but this compound also halts the enzyme’s 17-hydroxylation activity, which occurs in the same active site and is necessary for glucocorticoid synthesis. As a result abiraterone has non-optimal, complex effects on steroidogenesis in prostate cancer patients. Abiraterone binds via coordination of its nitrogen heterocycle to the 17A1 heme iron. This binding mode appears to be shared among several steroidogenic P450 enzymes and is likely a factor in the lack of selectivity between steroidogenic P450 enzymes and between the two different 17A1 reactions. Other 17A1 inhibitors in development reported to be selective for the 17A1 lyase reaction over its 17-hydroxylase reaction also bind to 17A1 in this mode, raising concerns over their selectivity between the two 17A1 reactions and for 17A1 over other steroidogenic P450 enzymes. To facilitate an understanding of 17A1 inhibition, three complementary approaches have been applied: 1) mutation of key amino acids in the 17A1 active site that interact with inhibitors, 2) evaluation of steroidal and non-steroidal inhibitors, and 3) modifications to inhibitor scaffolds with the goal of optimizing interactions with 17A1. Functional and structural analysis of mutants, existing inhibitors, and new analogs are providing key information that may be helpful in the design of more selective 17A1 inhibitors for use in treating prostate cancer.

DOI: 10.3109/03602532.2013.868114

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S5. ADP-RIBOSYLATION BY ARTD14 DIFFERENTIALLY MODULATES NUCLEAR RECEPTOR FUNCTION


Jason Matthews Department of Pharmacol/Med Sci Bldg 4336, University of Toronto, Toronto, ON, Canada The activity of nuclear receptors and other transcription factors is regulated by numerous post-translational modifications (PTMs), including phosphorylation, ubiquitination and acetylation. Recently, ADP-ribosylation has emerged as another PTM that regulates nuclear receptor function. ADP-ribosylation is catalysed by members of the ADP-ribosyltransferase diphtheria-like (ARTD; also known as poly(ADP-ribose) polymerase [PARP]) family that transfer single or chains of ADP-ribose from NADþ onto target proteins or on to themselves. Although 22 genes in humans encode proteins that may exhibit ADP-ribosyltransferase activity, the cellular targets and functional characterization of the majority of these proteins especially family members that exhibit mono- rather than poly(ADP-ribose) activity remain to be described. Here we show that ARTD14 (also known as TiPARP/PARP7) is a mono-ADP-ribosyltransferase that acts as a mixed function coregulator exhibiting corepressor or coactivator activity in a receptor-dependent manner. Using RNAi, overexpression, transient transfection and mouse embryonic fibroblasts isolated from Artd14/ mice we provide evidence that ATRD14 is a ligand-regulated corepressor of aryl hydrocarbon receptor (AHR) activity. Deletion and mutagenesis studies showed that ARTD14-mediated inhibition of AHR required its catalytic domain. In contrast, ARTD14 acts as a coactivator of liver X receptor a (LXRa; NR1H3) and LXRb (NR1H2) transactivation. The coactivation function of ARTD14 was dependent on its catalytic domain. Deletion studies identified a putative coactivator domain of ARTD14 (a.a. 200–225), which was distinct from a putative repressor domain (a.a. 225–245). Mass spectrometry confirmed our previously reported mono-ADP-ribosyltransferase activity of ARTD14. In vitro ribosylation assays revealed that ARTD14 mono-ADP-ribosylated both LXRa and LXRb. Taken together, our findings reveal that ARTD14 is a mono-ADP-ribosyltransferase that modifies nuclear receptor action potentially opening new avenues for therapeutic intervention.

S6. THE NUCLEAR RECEPTOR CAR AS A NOVEL THERAPEUTIC TARGET IN HEMATOLOGICAL MALIGNANCIES Hongbing Wang Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA The constitutive androstane receptor (CAR, NR1i3) has been well established as a central component in the coordinate response to xenobiotic stimulations by governing the transcription of numerous hepatic genes associated with xenobiotic metabolism, detoxification and clearance. Although activation of CAR generally increases the metabolism of drugs and leads to therapeutic failures, in the case of prodrugs, such as cyclophosphamide (CPA), increasing cytochrome P450 (CYP)-mediated biotransformation may generate more therapeutic metabolites and benefit CPA-based chemotherapy. As an alkylating prodrug, CPA has been used extensively in the treatment of hematologic malignancies, in particular, as an important component in the front-line regimens for non-Hodgkin lymphoma and chronic lymphocytic leukemia. Unfortunately, despite aggressive chemotherapy, a significant number of patients remain uncured due to development of drug resistance and/or intolerable toxicities. The need for further optimization of the current regimens is evident. One potential means to improve the therapeutic efficacy of CPA-based chemotherapy is to enhance metabolic conversion of CPA to the pharmacologically active 4-hydroxylcyclophosphamide (4-OH-CPA) via CYP2B6, but not to the N-dechloroethyl-cyclophosphamide and the toxic chloroacetaldehyde by CYP3A4. Towards this end, we have shown that activation of the human CAR preferentially induced the expression of hepatic CYP2B6 over CYP3A4 and increased the formation of 4-OH-CPA. We have also developed a unique human primary hepatocyte-leukemia coculture model and demonstrated that co-administration of CPA with selective CAR activators lead to significantly enhanced apoptosis in leukemia cells without increasing hepatotoxicity. Together, these findings offer proof of concept that CAR as a novel molecular target can facilitate CPA-based chemotherapy and may ultimately benefit the development of new therapeutic strategies.

S7. NUCLEAR RECEPTORS PXR AND CAR IN THE CROSSROAD OF DRUG METABOLISM AND ENERGY METABOLISM Wen Xie Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA, USA This presentation will focus on the endobiotic function of xenobiotic receptors in energy metabolism and the use of xenobiotic receptors as potential therapeutic targets to manage metabolic disease. The functional crosstalk between energy metabolism and drug metabolism will also be discussed. PXR and CAR were isolated as ‘‘xenobiotic receptors’’ that regulate the expression of xenobiotic enzymes. We have recent reported that PXR and CAR have important yet opposite role in energy metabolism.

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We showed that PXR ablation inhibited high fat diet (HFD)-induced obesity, hepatic steatosis, and insulin resistance, which were accounted for by increased oxygen consumption, increased mitochondrial b-oxidation, inhibition of hepatic lipogenesis and inflammation, and sensitization of insulin signaling. In an independent model, introducing the PXR-/- allele into the ob/ob background also improved body composition and relieved the diabetic phenotype. The ob/ob mice deficient of PXR showed increased oxygen consumption and energy expenditure, as well as inhibition of gluconeogenesis and increased rate of glucose disposal during euglycemic clamp. Mechanistically, the metabolic benefits of PXR ablation were associated with the inhibition of c-Jun N-terminal kinase (JNK) activation and down-regulation of lipin-1, a novel PXR target gene. The metabolic benefit of PXR ablation was opposite to the reported pro-diabetic effect of CAR ablation. Our results may help to establish PXR as a novel therapeutic target, and PXR antagonists may be used for the prevention and treatment of obesity and type 2 diabetes.


S8. REGULATION OF DRUG METABOLISM AND ENDOGENOUS FUNCTIONS BY PPARA VERSUS CAR/PXR Ulrich M. Zanger1, Maria Thomas2, Benjamin A. Kandel1 and Kathrin Klein2 1 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany, 2Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany Peroxisome proliferator-activated receptor alpha (PPARa) is a key regulator of lipid metabolism and energy homeostasis. In a systematic pharmacogenomic pathway analysis we identified PPARa as a new candidate gene regulating expression of CYP3A4 (Klein et al., Clin Pharmacol Ther 91:1044-52, 2012). Using chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter-gene (LUC) assays in HepG2 cells in the absence or presence of the synthetic PPARa ligand WY14643 we identified three functional PPARa-response elements within 10 kb of the CYP3A4 promoter that conferred inducible and/or constitutive transcriptional activation. Lentiviral gene knock-down showed regulation of several other P450s (CYPs 1A2, 2B6, 2C8, 7A1) by PPARa. In addition, not only WY14643 but also the endogenous PPARa ligand, phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0/18:1 GPC, POPC) induced drug metabolizing enzymes and transporters in human hepatocytes (Thomas et al., Mol Pharmacol 83:709-18, 2013). We furthermore investigated a splice variant PPARa-tr that lacks exon 6, resulting in a truncated PPARa protein that may exert a dominant negative effect on target genes. To investigate this hypothesis, we used a cohort of surgical human liver samples (N = 150) and observed that PPARa-tr and wild-type mRNA transcripts have diametrical effects on various target genes, indicating that PPARa-tr contributes to finetuning of gene regulation by PPARa. To compare the transcriptional regulomes of PPARa versus the traditional xenoreceptors CAR/PXR, human hepatocytes from several donors were treated with prototypical agonists of CAR (CITCO), PXR (rifampicin) and PPARa (WY14643) and gene expression changes were quantified using genome-wide Affymetrix microarray profiling and Taqman real-time PCR technology (Fluidigm). These data show that in humans PPARa and CAR/PXR regulate many drug detoxifying P450s and other ADME genes in the same direction whereas their effects on lipid and carbohydrate metabolism are in part diametrically opposed. In conclusion our studies suggest that in humans, but apparently less so in mice in mice, PPARa belongs to a network of nuclear receptors that regulates xenobiotic metabolism in a coordinated fashion with lipid/energy homeostasis. Supported by the German Federal Ministry of Education and Research [Virtual Liver Network 0315755] and by the Robert Bosch Foundation, Stuttgart, Germany.

S9. CLINICAL PHARMACOGENOMICS Munir Pirmohamed Department of Molecular and Clinical Pharmacology, The University of Liverpool, Liverpool, United Kingdom, L69 3GE Pharmacogenomics/Pharmacogenetics, as a discipline, has been around for over 50 years. Although many drug-phenotype associations have been described, the area has been criticised for making little impact on clinical practice. Research in the discipline has gone through several transitions, with the latest involving genome-wide approaches (which are beginning to show some tractable associations). Many reasons have been cited for the failure to impact on clinical practice including poor phenotypic characterisation of patients, and small sample sizes, which lead to barely significant findings that cannot be replicated. Such issues are now being addressed through consensus phenotype guidelines (in some cases) and multi-centre international consortia. Additionally, the evidential base required to introduce genetic predictors into clinical practice seems to be higher than that used for non-genetic predictors. It is often suggested that a randomised controlled trial (RCT) is needed to demonstrate the clinical utility of a genetic biomarker – however, if a RCT is going to be needed for every SNP (let alone multiple SNPs leading to a phenotype), then it is neither going to be practical nor economically viable. We need to think of better ways of generating evidence and more intelligent ways of interpreting it. Many of these points will be illustrated in the presentation with key examples.

DOI: 10.3109/03602532.2013.868114

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S10. CLINICAL IMPLEMENTATION OF PHARMACOGENETICS IN CHILDREN: FROM GUIDELINES TO CLINICAL DECISION SUPPORT


James M. Hoffman Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, TN, USA, 38103 While barriers remain, progress is being made towards broader use of pharmacogenetics in clinical practice. In the past, there was a lack of freely available, peer-reviewed, updatable, and detailed gene/drug clinical practice guidelines, but now the Clinical Pharmacogenetics Implementation Consortium (CPIC), a shared project of PharmGKB and the Pharmacogenomics Research Network, provides guidelines that enable the translation of genetic laboratory tests results into specific prescribing decisions for patients. Instead of focusing on when genetic tests should be ordered, the nine CPIC guidelines published to date help clinicians understand how available genetic test results should be used to optimize drug therapy. Computational tools, such as clinical decision support (CDS) delivered through an electronic health record (EHR) is essential to facilitate gene-based drug prescribing. Active CDS that provides concise gene-based drug prescribing recommendations at the time an affected drug is ordered is particularly important because pharmacogenetic tests can be conducted preemptively and have lifetime implications. Through our PG4KDS protocol, St. Jude Children’s Research Hospital provides a model for migrating pharmacogenetic tests (array-based) into routine care to be available pre-emptively in the care of children with catastrophic diseases. Genotyping is performed in a CLIA-approved laboratory using the Affymetrix DMET Plus array with a CYP2D6 copy number assay, testing for variants in 225 genes. Once clear prescribing recommendations can be made for a gene-drug pair, such as from consensus guidelines through CPIC, an automated system is used to incorporate the genetic results and clinical interpretations into the EHR. High-risk phenotypes automatically populate the EHR Problem List, and the problem list is the unambiguous triggers that drive alert firing to clinicians when high-risk drugs are prescribed. By only interrupting clinicians when a high priority phenotype and high-risk drug are both present, our CDS approach is carefully crafted to limit alert fatigue, a common shortcoming of CDS. Over 50 CDS rules have been developed and implemented as part of PG4KDS.

S11. WARFARIN PHARMACOGENETICS – EVIDENCE AND CLINICAL IMPLEMENTATION Larisa H. Cavallari Pharmacy Practice, University of Illinois at Chicago, Chicago, IL, USA, 60612 Warfarin is a challenging drug to dose, and inappropriate dosing increases the risk for adverse events, namely bleeding and thrombosis. In fact, warfarin ranks as a leading cause of hospitalization admissions due to adverse drug events among older individuals. The difficulty in warfarin dosing stems from the large inter-patient variability in the dose necessary for optimal anticoagulation. The genes for vitamin K epoxide reductase complex 1 (VKORC1), the target of warfarin, and cytochrome P450 2C9 (CYP2C9), which metabolizes S-warfarin, are important contributors to the variability in warfarin dose. Guidelines and algorithms including the CYP2C9 and VKORC1 genotypes in addition to clinical factors are available to assist with the translation of genetic information into warfarin prescribing decisions. However, warfarin pharmacogenetic dosing algorithms were derived from predominately European populations and perform less well in those of African descent. Efforts by individual groups as well as collaborative efforts by the International Warfarin Pharmacogenetics Consortium (IWPC) have led to the discovery of novel genetic variants contributed to warfarin dose requirements in African Americans. In particular, a recent GWAS revealed the rs12777823 A variant on chromosome 10 that occurs in approximately 40% of African Americans and is associated with a warfarin dose reduction of 6.9 mg/week in heterozygotes and 9.3 mg/week in homozygotes. African Americans comprise the majority of patients at the University of Illinois Hospital, where genotype-guided warfarin dosing is now the standard-of-care. Specifically, we recently developed and implemented a coordinated, multidisciplinary, genotype-guided warfarin dosing and management approach. This initiative is a combined effort across the Colleges of Pharmacy and Medicine and involves routine genotyping for variants influencing warfarin dose requirements in conjunction with a clinical pharmacogenetics consult service for patients newly initiating warfarin. Clinical decision support tools were integrated into the electronic medical record to automatically order genotyping and pharmacogenetic consultation in response to an initial warfarin order for a warfarin-naı¨ve patient. Genotyping is done on site, with results expected to be available in time to inform the second warfarin dose. The pharmacogenetics consult team provides a genotype-guided dose recommendation and subsequent daily dose recommendations based on INR response. Through the provision of personalized warfarin dosing, our goal is to improve the quality of anticoagulation management and reduce the risk for adverse events as a result of inappropriate warfarin dosing. An additional goal is to optimize genotyping to account for variants of importance for those of African descent, including the newly discovered rs12777823 variant.

S12. IMPLEMENTING PERSONALIZED MEDICINE IN AN ELECTRONIC MEDICAL RECORD ENVIRONMENT: LESSONS BEING LEARNED Dan M. Roden Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA

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It is a sine qua non of contemporary pharmacotherapy that patients display substantial variability in the extent of benefit and in adverse drug reactions. While an increasing body of knowledge links common DNA polymorphisms to these variable drug outcomes, methods to translate that knowledge into clinical workflow, and to assess the impact on healthcare outcomes, have not been developed. The Vanderbilt PREDICT (Pharmacogenomic Resource for Enhanced Decisions in Care and Treatment) program addresses this issue by embedding preemptive genotype data for common pharmacogenomic variants into an electronic medical record that incorporates advanced clinical decision support capabilities. In the first three years of operation, over 13,000 subjects have been genotyped on a multiplexed platform that includes variants in CYP2C19 (that modulates response to the widely-used antiplatelet drug clopidogrel), SLCO1B1 (simvastatin myopathy), CYP2C9 and VKORC1 (warfarin steady state dose), TPMT (thiopurines), and CYP3A5 (tacrolimus): each of these drug-gene pairs are now deployed in the Vanderbilt electronic medical record. Barriers that were addressed and overcome included selecting patients for genotyping because they are at risk for receiving target drugs, engaging the practitioner community, identifying actionable drug-gene pairs, deploying genotyping in a CLIA environment, and developing and deploying clinical decision support. Other academic and non-academic medical centers are adopting similar programs and these programs will allow accrual of sufficiently large patient subsets to begin to assess impact on healthcare outcomes.

S13. EVALUATION OF DRUG TRANSPORTER REGULATION IN DRUG DISCOVERY AND DEVELOPMENT Joseph A. Ware Genentech Inc, South San Francisco, CA, USA Despite progress to elucidate the molecular and cellular mechanisms of drug transporter regulation, there remains a considerable gap between in vitro studies, preclinical models, and clinical prediction and outcome. However, it is quite common in drug discovery and development to encounter complex drug disposition and toxicity scenarios that are not solely explained by the regulation of drug metabolizing enzymes, thereby suggesting the involvement of drug transporters and/or their regulatory genes. The objective of this presentation will be to: 1) Discuss the impact of an anti-FGF19 antibody on bile acid synthesis, transporter expression and preclinical toxicity (Pai et al., Tox. Sciences, 126 (2)446–456, 2012. 2) Review the impact of inflammatory disease and PGx on sulfasalazine PK/PD and 3) Highlight the ITC2 working group’s recommendation regarding induction and regulation of transporters and changes in transporter expression/function in disease states.

S14. REGULATION OF DRUG TRANSPORTERS IN LIVER BY ACTIVATION OF NUCLEAR RECEPTORS Curtis Klaassen Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS, USA Drug transporters are critical membrane proteins that mediate the uptake and efflux of numerous pharmaceuticals and other xenobiotic compounds. Transcriptional regulation of drug transporters by nuclear receptors is a critical detoxification mechanism to enhance the hepatic uptake and elimination of toxic chemicals. Forty-five years ago, we demonstrated in rats that phenobarbital enhanced plasma disappearance of phenol-3,6-dibromphthalein disulfonate (DBSP), which reflects increased biliary transport in liver (Klaassen and Plaa, 1968). We also demonstrated that pregnenolone-16a-carbonitrile (PCN) increased the hepatic uptake and excretion of cardiac glycosides in rats (Klaassen, 1974), and increased the uptake of ouabain in isolated rat hepatocytes (Eaton and Klaassen, 1979). It was later discovered in the 1990s that phenobarbital and PCN act through critical xenobiotic-sensing nuclear receptors (mainly the constitutive androstane receptor [CAR/Nr1i3] and the pregnane X receptor [PXR/Nr1i2] respectively) to modulate the target gene transcription. In fact, many nuclear receptor activators, which were originally termed microsomal enzyme inducers due to their abilities to increase the transcription of phase-I and –II drug metabolizing enzymes, are also transcriptional activators of transporters. Using rat and mouse models, we have systematically characterized the mRNAs of many hepatic uptake transporters (such as the organic anion transporting polypeptides [Oatps/ Slcos]) and efflux transporters (such as the multidrug resistance-associated proteins [Mrps/Abccs]), and demonstrated that they are increased following treatment of the prototypical ligands of PXR, CAR, and peroxisome proliferator activated receptor alpha (PPARa/Nr1c1), as well as other xeno-sensors such as the aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2/Nfe2l2). Certain xeno-sensors (e.g. AhR and Nrf2) act in concert to regulate common gene transporter gene batteries (Yeager et al., 2009), and using a Nrf2 ‘‘gene-dose response’’ model, we have shown that the mRNAs of certain transporters increases dose-dependently following increased nuclear levels of Nrf2 proteins (Wu et al., 2012). More importantly, using the corresponding xeno-sensor null mice, we have demonstrated that the chemical-mediated mRNA increase of many drug transporters is dependent on the presence of the corresponding nuclear receptors (Aleksunes and Klaassen, 2012). To determine whether the nuclear receptor-mediated up-regulation of transporters is due to a direct effect, we have quantified the genomicDNA binding of PXR in mouse liver in control and PCN-treated mice using ChIP-Seq, and discovered novel PXR-DNA binding sites to transporter genes in vivo; and interestingly, we have unveiled a novel DR(5n þ 4) periodic-DNA binding pattern of PXR in mouse liver (Cui et al., 2010). We have also quantified the true mRNA abundance and novel isoforms of all liver transporters

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in liver by RNA-Seq during development (Cui et al., 2012), and following treatment of PCN and the CAR ligand TCPOBOP. In conclusion, we and others have demonstrated that activation of certain critical xenobiotic-sensing nuclear receptors increases the transcription of many uptake and efflux transporters in liver, and this leads to enhanced hepatic uptake and elimination of drugs. Using xeno-sensor null mice and second-generation sequencing, we have also shown that many of these chemicalmediated gene regulations are dependent on the presence of that corresponding nuclear receptor, and are due to direct DNA-binding.

S15. REGULATION OF ABC TRANSPORTERS AT THE BLOOD-BRAIN AND BLOOD-SPINAL CORD BARRIERS


David S. Miller Lab of Toxicology and Pharmacology, NIH/NIEHS, Research Triangle Park, NC, USA The blood-brain and blood-spinal cord barriers reside within the brain and spinal cord capillary endothelia. These tissues function as gatekeepers of the CNS. They provide ready access to needed nutrients, ions and proteins, are responsible for the brain to blood transport of metabolic wastes and limit entry of neurotoxicants. Low junctional and transcellular permeabilities along with high expression of multiple ATP Binding Casssette (ABC) transporters make these tissues formidable barriers to the delivery of small and large therapeutics to the CNS. Several ABC transporters that are ATP-driven efflux pumps, e.g., P-glycoprotein, BCRP, MRP2, are expressed on the luminal plasma membrane of the capillary endothelial cells, where they present a barrier to small molecule drugs. This presentation will review recent work showing that CNS barrier ABC transporter expression is a moving target. Available evidence indicates that similar mechanisms are at work in both the bloodbrain and blood-spinal cord barriers. Changes in transporter protein expression are signaled through multiple ligand-activated receptors and transcription factors that respond to peripheral and CNS disease, xenobiotics (drugs and toxicants) and inflammatory and oxidative stress. Identified receptors and transcription factors that upregulate expression of ABC transporters at CNS barriers include, estrogen receptor, glucocorticoid receptor, tumor necrosis factor-alpha receptor, pregnane-X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), NF-kB, AP-1, and Nrf2.

S16. REGULATION OF DRUG TRANSPORTERS IN DISEASE Micheline Piquette-Miller University of Toronto, Toronto, ON, Canada Underlying disease states are often key sources of inter-subject variability of drug response in patient populations. Inflammation, which is a component of many diseases such as infection, arthritis, atherosclerosis, diabetes and cancer, has been reported to impart changes in drug disposition. Transport proteins which are highly expressed in the membrane barriers of intestine, blood brain barrier, placenta, kidney and liver can profoundly impact the absorption, distribution and clearance of numerous drugs, toxins and metabolic products and thus may play an important role in these changes. Indeed, our studies in animal models of protozoal, bacterial and viral infection as well as chronic conditions such as diabetes and obesity have demonstrated significant changes in the expression of many of the ABC drug efflux transporters, the SLC family of organic anion uptake transporters as well as the CYP3A drug metabolizing enzymes. These changes are associated with altered distribution and clearance of their substrates. Cytokine and nuclear-receptor mediated pathways play a key role. Recent studies in human placenta also indicate disease or infection induced changes in transporter expression. As transporters are involved in the distribution and elimination of a large number of clinically important drugs and endogenous compounds, our studies suggest that disease-induced changes in transporter activity is an important source of inter-subject variability in the disposition and clearance of their substrates.

S17. STRUCTURAL DETERMINANTS OF P450 MEDIATED METABOLISM: A MOVING TARGET Eric F. Johnson1, Ying Fan1 and C. David Stout2 1 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA, 2Department of Integrative Structural & Computational Biology, The Scripps Research Institute, La Jolla, CA, USA Conformational dynamics are important for the binding of small molecules in P450 active sites as the catalytic center of the protein is buried in the protein. Characterization of P450 dynamics at the atomic level is largely based on x-ray crystal structures exhibiting distinct conformations of the proteins with different ligands bound in the active site. P450 2D6 has been crystallized in open and closed forms that exhibit differences for conformations of the N-terminal and C-terminal loops, b-sheet 1, the helix F-G loop and the helix B-C loop that form surfaces of the substrate binding cavity. Our current studies have used a single crystal form of 2D6 to develop detailed information about substrate and inhibitor binding to the enzyme. A key aspect of the underlying crystal lattice is that interactions between neighboring 2D6 molecules allow dynamic changes in the conformation of the protein

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when substrates and inhibitors bind, whereas the overall crystal lattice remains stable so that the quality of the x-ray diffraction is maintained. In this crystal form, P450 2D6 exhibits an open conformation with one molecule of thioridazine bound in the active site and with another thioridazine molecule bound in a substrate access channel. Addition of ligands directly to the mother liquor or soaking crystals in artificial mother liquors containing a different ligand allow the new ligand to bind in the active site of the crystallized P450 2D6 without a significant loss of diffraction quality. Results obtained by x-ray diffraction using these crystals identified ionic, hydrogen bonding and hydrophobic interactions between P450 2D6 and a variety of ligands. As result, a relatively comprehensive pattern of adaptive responses is evident from these results. (Supported by NIH Grant GM031001).

S18. ARYLAMINE N-ACETYLTRANSFERASES STRUCTURE IN DRUG METABOLISM AND DRUG DISCOVERY


Edith Sim1,2 1 Faculty of Science, Engineering and Computing, Kingston University, Kingston-upon-Thames, United Kingdom, 2Department of Pharmacology, University of Oxford, Oxford, United Kingdom Arylamine N-acetyltransferases (NAT) were first identified as xenobiotic conjugating enzymes responsible for pharmacogenetic variation. They transfer an acetyl group from acetyl CoA to arylamines, arylhydrazines and arylhydroxylamines. Classically human NAT2 polymorphism is the cause of isoniazid and hydralazine slow acetylation. Slow acetylation is also linked to drug and xenobiotic toxicity, including arylamine carcinogenesis. Two human NATs, NAT1 and NAT2 are over 80% identical and structural studies have helped elucidate specificity differences(Wu et al., 2007). Mammals and birds can have up to three NAT gene homologues encoded at adjacent polymorphic loci. Homologues in bacteria and now in fungi have been described and Salmonella typhimurium NAT, which increases the sensitivity of Ames tester strains for carcinogens, was the first NAT structure to be determined (Sinclair et al., 2000). The X-ray structure established the mechanism of the reaction with an active site catalytic triad of cys, his and asp which is conserved in all NAT structures to date (over 20). The activation of the cys residue is poised to accept the acetyl group from the donor acetylCoA. The three domain fold (alpha, beta, alpha/beta lid) is also conserved. The active site residues are in the first two domains whilst the loops between domains may vary and the third domain shows most variation in sequence and in length. The binding site of the co-factor acetylCoA has been identified in three structures and differs amongst homologues which may underlie functional differences. The third domain appears to be an integral part of the acetylCoA binding domain and binding can be strong enough to survive recombinant protein purification and crystallisation (Pluvinage et al., 2011). NATs can have different roles with human NAT1 having a role in endogenous metabolism and it has been identified as a target for breast cancer diagnostics (Laurieri et al., 2010) and therapy (Minchin & Butcher, 2012). NAT in Mycobacterium tuberculosis, the most recently solved structure (Abuhammad et al., 2013), is a target for anti-tubercular therapy. Structural studies can define enzyme/drug interactions for xenobiotic metabolism and influence drug design for specific inhibitors of different NAT isoenzymes from man and M. tuberculosis (Abuhammad et al., 2012).

References 1. Abuhammad A, et al. (2012) PLoS ONE 7: e52790. doi:10.1371/journal.pone.0052790 Piperidinols That Show AntiTubercular Activity as Inhibitors of Arylamine N-Acetyltransferase 2. Abuhammad, A. et al., (2013) Acta Crystallographica D (in press) Structure of arylamine N-acetyltransferase from M. tuberculosis 3. Laurieri, N et al., (2010) J. Am. Chem. Soc., 132, 3238–9. Small molecule colorimetric probes for specifici detection of Human arylamine N-acetyltransferase 1, a potential breast cancer marker. 4. Minchin, R. & Butcher, N. (2012) Pharmacological Reviews. 64, 147–65. Arylamine N-acetyltransferase 1: a novel drug target in cancer development. 5. Pluvinage et al., (2011) FEBS Letters, 585, 3947–52. The Bacillus anthracis arylamine N-acetyltransferase 6. Sinclair et al., (2000) Nature Structural Biology Nat Struct Biol 7, 560–4 Structure of arylamine N-acetyltransferase reveals a catalytic triad. 7. Wu, H. et al. (2007) J. Biol. Chem. 282, 30189–97. Strcutural basis of substrate binding specificity of human arylamine N-acetyltransferases.

S19. USING CRYSTAL STRUCTURES OF DRUG METABOLIZING ENZYMES IN MECHANISM-BASED MODELING FOR DRUG DESIGN Hao Sun Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Reseach and Development, Groton, CT, USA

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Significant progress has been made in structure-based drug design at different stages of drug discovery thanks to the advancement of X-ray crystallography. P450-catalyzed drug metabolism is a major consideration for metabolic clearance-related dose adjustment and a primary source for drug metabolite-induced toxicity. During the last decade, major human cytochrome P450 structures including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 have been characterized, one of the biggest science breakthroughs in drug metabolism study and making it possible to understand P450 catalysis at a protein three-dimensional level. At Pfizer, we have successfully applied these findings in structure-based drug metabolism modeling for various aspects of drug design across our drug discovery projects. For example, they have been used to elucidate clearance mechanisms for clearancedirected drug design, to reduce reactive metabolite formation without removing the potential ‘‘warhead’’, and also to avoid drugdrug interactions. Considering the flexible nature of P450s in general, many challenges are present in the application of a standardized protein template for each P450 enzyme. In practice, our modeling approaches were validated using co-crystal structures, analog-based template selection, and also strong support from those high-quality experimental data in biotransformation and enzymology. In addition, other non-P450 drug-metabolizing enzymes such as aldehyde oxidase and glutathione transferase have been modeled most recently to help us explore novel clearance mechanisms for the observed disconnect of in vitro-in vivo extrapolation and related structure-based drug design. Thus, the status, usefulness, challenges and strategies of these mechanism-based modeling of drug metabolizing enzymes will be discussed.

S20. CAN WE UNDERSTAND AND PREDICT LIGAND BINDING TO A HIGHLY FLEXIBLE P450? James R. Halpert Skaggs School of Pharmacy and Pharmaceutical Sciences, UC San Diego, La Jolla, CA, USA Currently, methods for predicting cytochrome P450-mediated metabolism of new compounds are in great demand. Progress in this area is dependent on sophisticated understanding of the structural determinants and mechanisms of cytochrome P450 function. Our research has focused on the structure and function of cytochromes P450 2B. In particular we have utilized X-ray crystallography in conjunction with a variety of solution biophysical approaches to probe the role of conformational changes in determining substrate specificity. Structures of rabbit P450 2B4 and human P450 2B6 in complex with a variety of drugs and model compounds have revealed how rearrangement of secondary structure elements and of active site side chains reshapes the substrate binding pocket. In addition the structures have revealed the likely pathways for drug access to the active site. Most recently, a combined biophysical and structural analysis of human P450 2B6 interactions with the monoterpene (þ)-a-pinene was undertaken to elucidate the basis of the very high affinity binding of this pure hydrocarbon. The study indicates that the strong enthalpic contribution to binding of (þ)-a-pinene to 2B6 is tied closely to the degree of enzyme flexibility. Demonstration of the substantial conformational flexibility of P450 2B4 and 2B6 has important implications for using static X-ray crystal structures to predict P450-mediated substrate oxidation.

S21. APPLICATION OF NEXT GENERATION SEQUENCING TO PREDICTION OF SERIOUS ADVERSE DRUG REACTIONS Thomas J. Urban Center for Human Genome Variation, Duke University Medical Center, Durham, NC, USA Over the past five years, advances in genetic approaches to the study of drug response in humans, including genome-wide association studies (GWAS) of common genetic variants, have already yielded a tremendous amount of information regarding the genetic basis of variable drug responses, particularly for serious adverse drug reactions such as Stevens-Johnson Syndrome (SJS) and drug-induced liver injury (DILI). More recently, the development of massively parallel (or ‘‘next-generation’’) sequencing has started to allow for the comprehensive interrogation of nearly all genetic variation in the human genome, including rare variants that may even be unique to individual patients, and has been deployed with unparalleled success in the study of rare human diseases. Several large studies of rare, serious ADRs using next-generation sequencing are currently under way, with the goal of identifying positive predictors of these potentially life-threatening reactions. By providing biological insight into the causes of severe ADRs, these studies may also allow for more rational and specific toxicity screens to avoid such reactions in future drug development.

S22. GENETICS AND DRUG REPOSITIONING Philippe Sanseau Computational Biology, GlaxoSmithKline, Stevenage, United Kingdom In a drug discovery and development context human genetics could provide one of the strongest signals to support target validation. Over the last few years we have seen significant investments in genetics studies such as GWAS (Genome Wide Association Studies) but the translation into new drugs remains limited. To address this we investigated whether genetics data,

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especially, GWAS studies could point to unsuspected indications for marketed drugs or drugs in development. We built a simple computational pipeline to examine the relationship between the disease indications of drugs, the drug targets, the GWAS traits and the GWAS genetics signals. We demonstrated statistically that GWAS genes were significantly more likely to be tractable drug targets than a random set of genes. Next, we hypothesized that, when the disease indication of the drug matched the disease trait associated with the target gene, GWAS increased the confidence that the right indication is pursued and that, conversely, a mismatch would point to drug repositioning opportunities. We will present examples of GWAS genes with at least one associated drug that suggest potential drug repositioning opportunities. We will also discuss the challenges to progress these opportunities and how further investigations can provide additional evidence. Considered together, these analyzes suggest new and immediate translational applications for genetics.

S23. IMMUNOGENETIC RISK FACTORS FOR ADVERSE DRUG REACTIONS AND THE UNDERLYING MECHANISMS


Yuan-Tsong Chen Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Drug hypersensitivity, a type of adverse drug reactions (ADRs), remains a major problem for both clinical practice and pharmaceutical industry. Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), hypersensitivity syndrome (HSS) and drug-induced liver injury (DILI) are example of severe drug hypersensitivity, still carrying significant mortality and mobility. For pharmaceutical industry, occurrence of severe drug hypersensitivity can be detrimental; a few cases have prompted the withdrawal of newly released drugs from the market. Recently, certain class I HLA alleles (e.g., HLA-A and HLA-B alleles) and certain class II HLA alleles (e.g., HLA-DR alleles) have been found to be associated with drug hypersensitivity and in the most severe forms, such as SJS, TEN, HSS and DILI, the association is primarily with the HLA-B molecules; for example, HLA-B*5701 in abacavir hypersensitivity and flucoxacillin-induced DILI, HLA-B*1502 in carbamazepine-induced SJS and HLA-B*5801 in allopurinol-induced HSS/SJS/ TEN. The strong genetic association suggests a direct involvement of HLA in the pathogenesis of drug hypersensitivity and indeed HLA molecule presents an antigenic drug and resulted in clonal expansion and activation of CD8þ cytotoxic T cells having restricted and common TCR usage have been shown. Pharmacogenomic study identified an unusual form of granulysin secreted by these cytotoxic T lymphocytes and natural killer cells responsible for the rapid and disseminated keratinocyte death in SJS/TEN. The high sensitivity/specificity of some genetic markers provides a plausible basis for developing tests to identify individuals at risk for drug hypersensitivity and in a large prospective study, we have shown that HLA-B*1502 screening before carbamazepine treatment can effectively reduce the incidence of carbamazepine-induced SJS/TEN. As ‘‘proof-of concept’’, we have also shown that CBZ and other aromatic anti-epileptics bind directly to the HLA-B*1502, but not other tested HLA proteins. These translational researches demonstrated that preventing drug toxicity by screening people at risk before prescription of a drug is a clinical reality and suggest that a potential to use HLA library to guide the pre-clinical drug development.

S24. ADME/PK STUDIES OF HERBAL MEDICINES AND WHAT WE LEARNED FROM THEM Chuan Li Laboratory for DMPK Research of Herbal Medicines, Shanghai Institute of Meteria Medica, Chinese Academy of Sciences, Shanghai, China Botanical products are gaining growing popularity and increasing use worldwide. Botanical products are very diverse; some products are regarded and registered as herbal medicines but others come close to food. The scientific standards for accepting the value of a botanical product as a medicine should be comparable with the existing requirements for establishing the value of a synthetic drug. Unlike the synthetic drug that often contains only one pharmacologically active ingredient, the herbal medicine normally contains numerous chemical constituents. With regard to exerting drug responses, it is hypothesized that the herbal medicine relies on a few of key ingredients having favorable drug-like properties to exert its drug responses, rather than all the chemical constituents present. These key ingredients are referred to as medicinal principles of herbal medicines. An herbal constituent can be defined as drug-like when it possesses the desired pharmacological potency, a wide safety margin, and appropriate pharmacokinetic (PK) properties and exists in adequate abundance in the herbal medicine. For herbal medicines, an important scientific question from pharmaceutical scientists deals with the absorption, disposition (including distribution, metabolism, and excretion), and pharmacokinetics (ADME/PK) of their constituents, especially the pharmacologically active ones. ADME/PK studies aim to support making evidence-based decision about the therapeutic value and use of herbal medicines. These studies also contribute to ensuring the efficacy, safety, and quality of herbal medicines by bridging the gaps in knowledge between chemistry and pharmacology and between pharmacology and therapeutics. Recent ADME/PK studies of Salvia miltiorrhiza roots (Danshen), Panax notoginseng roots (Sanqi), and Ginkgo biloba leaves have characterized that large differences in systemic exposure level and PK profile exist among the chemically related herbal constituents detected in the

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bloodstream after dosing. This suggests that the PK profiles, like the pharmacological activities, can be used as a ‘‘sieve’’ to assess the importance and usefulness of the individual herbal constituents for an herbal therapy. The presentation will also include the effect of elimination kinetics, as well as that of oral bioavailability, on the systemic exposure levels of herbal constituents after dosing, the combinatorial metabolism of herbal compounds, dose-dependent and dose-independent PK markers substantiating body exposure to the administered herbal medicines, the molecular mechanism of elimination of the herbal compounds, and the associated interspecies PK differences. (Supported by NNSFC Grant 30925044 and Chinese 973 Program Grant 2012CB518403)

S25. ASSESSMENT AND MANAGEMENT OF ADVERSE EFFECTS AND HEPATOTOXICITY ASSOCIATED WITH THE METABOLISM OF HERBAL PRODUCTS


Ge Lin School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong Idiosyncratic adverse reactions, including the drug metabolism-induced adverse effects/toxicity, of the orthodox drugs are widely acknowledged and extensively investigated, however, there are less awareness and evidence of such adverse effects/toxicity induced by alternative medicines such as herbal preparations. With the increasing worldwide attraction for the use of herbal medicines for disease treatment and health care, over the last decades it has become apparent that safety problems arise due to the adverse effects/toxicity caused by herbal medicines alone and/or their combinational use with orthodox drugs. Herbal medicines consist of multi-ingredients acting on multi-targets, which make the investigation of the metabolism-mediated safety problems much more difficult and challenging. With the significant advancement of analytical technologies capable of qualitative and quantitative measurement of multi-ingredients, the knowledge on the metabolisms of multi-ingredients of herbal medicines and their metabolites interventions with cellular macromolecules has been remarkably improved in the recent decade. In this presentation, using our studies on the hepatotoxicity induced by herbal ingredients, pyrrolizidine alkaloids, as an example, the delineation of the toxic mechanism involving cytochrome P450-mediated metabolic activation to the reactive metabolites followed by their interactions with nucleophilic macromolecules leading to hepatotoxicity will be illustrated. In addition, the significance and contribution of metabolic study to the assessment and management of hepatotoxicity caused by pyrrolizidine alkaloids-containing herbal medicines and pyrrolizidine alkaloids-contaminated foodstuffs will be addressed. [Supported by Research Grant Council of Hong Kong (GRF Grants no. 471310 and 469712) and CUHK (Direct Grants no. 2041744 and 4054047)]

S26. INTERACTIONS BETWEEN MEDICINAL PHYTOCHEMICALS AND DRUG METABOLISM ENZYMES (PHASE I AND PHASE II) Ling Yang Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Drug metabolism plays a key role in determining the fate of the prospective drugs. The assignment whether a drug is or not metabolized and the involved drug-metabolizing enzyme (DME) are very important. Subsequently, the metabolism of a drug can be altered by co–administered drugs or natural products, leading to an unusually slow or fast clearance of said drug. Clinical studies have demonstrated that the combined use of herbs and drugs can enhance or attenuate the drug efficacy and toxicity. Herbs-drug interactions (HDIs) may reduce a drug efficacy and lead to treatment failure after long-term administration. This is especially true for drugs that have a narrow therapeutic index (Curr Drug Metab, 2012; 13: 599–614). In many instances, the multi-enzymes with overlapping substrate specificity participate in the metabolism of the same natural active ingredient. In addition, due to the lack of the specificities of enzyme inhibitors or inducers, the mixture of hybrid phytochemicals co-administrated can more or less change the enzyme activities. To determine the contribution of a given DME to the metabolism of a natural drug, a thorough understanding of the selectivity of substrates, inhibitors and inducers for a specific enzyme isoform is necessary. The qualitative and quantitative structure-metabolism relationships (SMRs) can thus be established for the interactions between medicinal phytochemicals and DMEs. Herein, we reported identification of several novel specific substrates and selective inhibitors and the establishment of a new method of the enzymatic assignment. After screening hundreds of herbal ingredients, we identified a number of specific substrates and inhibitors, including: 1) bufodienolides (such as bufalin, resibufogenin and cinobufagin) are identified to serve as isoform-specific probes for CYP3A4 (Chem. Comm. 2013; DOI: 10.1039/C3CC45250F & Drug Metab Dispos, 2011; 39:675–82); 2) Bavachinin serves as a specific UGT1A1 substrate; 3) Shikonin as a highly selective probe substrate of UGT1A9; 4) Bufalin serves as a selective inhibitor of CYP3A5; 5) Magnolol is a potent and selective inhibitor of UGT1A9 (Xenobiotica, 2012; 42:1001–8). Meanwhile, we also established a novel proteomic-based method for enzymatic assignment. Taken together, our findings will shed light on understanding SMRs between medicinal phytochemicals and DMEs in HDIs.

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S27. PROGRESS AND PROMISE OF CHINESE HERBAL MEDICINES: LESSONS FROM THE FIRST EU-CHINA FRAMEWORK PROGRAMME PROJECT GP-TCM


Olavi R. Pelkonen Pharmacology and Toxicology, University of Oulu, Oulu, Finland Progress and Promise of Chinese Herbal Medicines: lessons from the first EU-China Framework Programme Project GP-TCM Olavi Pelkonen, University of Oulu, Department of Pharmacology and Toxicology, Finland GP-TCM (Good Practice in Traditional Chinese Medicine Research in the Post-genomic Era) was the 1st EU-funded Coordination Action consortium (200 scientists from 24 countries and4100 institutions) dedicated to traditional Chinese medicine (TCM) research and running 2010–2012. The Action developed good practice guidelines and produced an open-access theme issue (1) consisting of 20 papers on various aspects of TCM research. The consortium emphasised high-quality effectiveness and mechanistic studies best served by omics and systems biology approaches and necessity of quality and pharmacovigilance of TCM products for the effective management of chronic diseases. The efficacy of complex herbal medicines in the prevention and treatment of complex diseases is thought to be due multiple active components exerting their synergistic effects on multiple targets in disease networks (2). However, there is insufficient knowledge to assess how the pharmacokinetic behaviour and efficacy and toxicity are determined in complex mixtures and whether there are so called matrix effects and interactions between components affecting the fate and effects of the total mixture. One possible scheme of ADMET studies is presented by Pelkonen et al in (1). Firstly, metabolism and metabolic interactions of complex mixtures and their main components are studied in in vitro systems, principally humanderived tissue preparations such as hepatocytes. Secondly, associations between the metabolic and kinetic behaviour and selected in vitro toxicity markers (e.g., cytotoxicity in hepatocytes, reactive intermediates, Nrf2 expression) are elucidated. Thirdly, the most relevant findings and predictions to in vivo investigations in humans and in selected cases to experimental animals are carried out. The goal is to develop a scheme on how to elucidate the ADMET properties of complex mixtures in in vitro/ex vivo systems so that the results can be reliably extrapolated to in vivo. Needless to say, the above scheme is still highly conjectural and based on current views of conventional drugs, but it could be a feasible starting point for investigations directed towards an evidence-based regulatory dossier for complex herbal products. (1)Journal of Ethnopharmacology, The GP-TCM Special Issue; 2012; 140 (3), pp. 455–642. (2) Xu Q, Qu F, Pelkonen O. Network Pharmacology and Traditional Chinese Medicine. In Sakagami H, ed. Alternative Medicine. 2012; pp277–297, Intech. Acknowledgements: Dr. Qihe Xu (KCL, UK), Dr. Tai-Ping Fan (Cambridge Univ, UK)

S28. IN VITRO APPROACHES TO DETECT DRUG INDUCED ORGAN TOXICITIES- LEARNINGS AND FUTURE PERSPECTIVES Michael Aleo Compound Safety Prediction, Pfizer, Groton, CT, USA The single most frequent reason for safety-related drug withdrawal is organ toxicity. Although drug-induced liver injury (DILI) tops the list of potential causes, cardiac and other organ toxicities are also of major concern for drug developers, particularly because they can have significant and serious impact when discovered in the post-marketing phase. Since late stage attrition is very costly, effort have been made in recent years to move safety testing earlier in the drug development process when chemical matter is still available for structure activity relationship work. To do so, in vitro assays have been and are being developed to assist with this approach. The challenge with building in vitro predictive assay platforms is that one needs to profile a large set of well annotated compounds that exhibit the toxicity to be predicted, other compounds that are not toxic as well as compounds with other toxicities than the one to be predicted, in order to differentiate. In this lecture I will share learning’s from reviewing literature reported studies and our own work. For example, we have shown that simple cytotoxicity measurements in organ specific cell lines is not sufficient to detect organ toxicity and neither is high content imaging of several mechanistic parameters such as cell loss, lipid accumulation, apoptosis, ER stress and DNA damage.

S29. REACTIVE METABOLITE FORMATION IN THE SKIN AND THE MECHANISM BY WHICH IT CAUSES A SKIN RASH Jack Uetrecht Department of Pharmacy, University of Toronto, Toronto, ON, Canada Idiosyncratic drug reactions can affect virtually any organ, but skin rashes are the most common. Most IDRs appear to be caused by reactive metabolites and are immune-mediated. The liver has a high concentration of metabolic enzymes that can form reactive metabolites; however, the default immune response in the liver is immune tolerance. In contrast, the skin has very low


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concentrations of enzymes, especially P450, that can metabolize drugs to reactive metabolites, but it is immunologically very active. One of the few metabolic enzymes with high activity in the skin is sulfotransferase. Nevirapine is used to treat HIV infections, and it is associated with a high incidence of skin rashes. We developed an animal model of nevirapine-induced skin rash in Brown Norway rats with characteristics very similar to the rash that occurs in humans. We found that the methyl group of nevirapine is oxidized in the liver by P450 to a radical intermediate that partitions between oxygen rebound to form a benzylic alcohol (12-OH-NVP) and loss of another hydrogen atom to form a reactive quinone methide. The quinone methide covalently binds to hepatic proteins, and this is presumably the cause of nevirapine-induced liver injury. The 12-OH-NVP reaches the skin where it forms a benzylic sulfate. The reaction of this sulfate with glutathione is very slow, but it readily reacts with proteins, and we found covalent binding of 12-OH-NVP sulfate in the skin of rats treated with nevirapine or 12-OH-NVP. When we inhibited sulfation in the liver it decreased the circulating levels of 12-OH-NVP sulfate, but it did not prevent covalent binding in the skin, and it did not prevent the rash. When we inhibited sulfation by topical application of a sulfotransferase inhibitor, it did prevent covalent binding in the skin and the rash. Therefore, 12-OH-NVP sulfate formed in the skin is the cause of nevirapine-induced skin rash. Incubation of human skin homogenate with 12-OH-NVP led to covalent binding that was dependent on PAPS cofactor. In contrast, incubation of mouse skin homogenate with 12-OH-NVP and PAPS did not lead to covalent binding, and we have not been able to induce a skin rash in mice with nevirapine. This model is somewhat related to contact hypersensitivity rashes. Contact hypersensitivity involves activation of inflammasomes, which produce IL-1b and IL-18. Treatment of rats with an antibody-against IL-1b markedly attenuated the rash caused by nevirapine, and incubation of human keratinocytes with 12-OH-NVP sulfate produced IL-1b. From these studies we conclude that nevirapine-induced skin rashes are caused by a reactive sulfate formed in the skin that activates inflammasomes. This is the first example in which a reactive metabolite formed by sulfotransferase in the skin has been shown to be the cause of a skin rash, but it is likely that there are other examples. In addition, other drugs that are weakly reactive may cause skin rashes by activation of inflammasomes. This research was funded by grants from CIHR.

S30. ROLE OF SPECIES- AND CELL TYPE-SPECIFIC EXPRESSION OF MEMBRANE TRANSPORTERS IN NEPHROTOXICITY Lawrence Harold Lash Department of Pharmacology, Wayne State University Schoolof Med, Detroit, MI, USA This presentation focuses on the role of membrane transport processes in determining susceptibility of the kidneys to chemically induced nephrotoxicity. Most clinically important drugs and environmental contaminants (or metabolites derived therefrom) are organic anions or organic cations. Of the various epithelial segments that comprise the nephron, the proximal tubules possess the highest expression of organic anion and cation transporters on the basolateral and brush-border plasma membranes. This fact, and the direct exposure of this nephron segment to blood-borne chemicals that undergo glomerular filtration make this cell type the primary target for nephrotoxicity. The existence of species-dependent differences in membrane transporter expression and drug metabolism makes experimental models that closely reflect events that occur in human kidney particularly valuable for drug development and design and other mechanistic investigations. Such an experimental model is primary cultures of human proximal tubular (hPT) cells, which are cultured in a serum-free, hormonally-defined medium. These hPT cells express a wide array of organic anion and cation transporters, including several members of the solute carrier 22A (SLC22A; organic anion transporters [OATs] and organic cation transporters [OCTs]), the solute carrier organic anion transporting polypeptide (SLCO; OATPs), the ABC cassette transporters (ABCC; multidrug resistance-associated protein [MRPs]), and multiple drug resistance proteins (ABCB; MDR or P-glycoprotein [P-gp]) gene families. Protein expression of these carriers is maintained throughout the time course of primary cell culture. However, due to intracellular recycling of these proteins, it is critical to correlate expression and transport function. Several of these carriers, in particular OAT1 (SLC22A6), OAT3 (SLC22A8), and MDR1 (P-gp; ABCB1), exhibit genetic polymorphisms or alternate splicing, leading to a marked degree of variation in transporter function among individuals. This variation has important pharmacological and toxicological implications. As an illustration of how genetic manipulation of transporter expression can result in altered susceptibility of renal proximal tubular (PT) cells from the rat to nephrotoxic chemicals, two examples are described involving the two membrane carriers that transport glutathione (GSH) into renal mitochondria, namely the dicarboxylate carrier (DIC; Slc25a10) and 2-oxoglutarate carrier (OGC; Slc25a11). The first example is overexpression of either wild-type DIC or OGC or a low-activity double-cysteine mutant of the OGC in a rat PT cell line (NRK-52E cells). The second example is overexpression of either wild-type DIC or OGC in primary cultures of PT cells from diabetic rats. Both examples illustrate the profound effect of variation in transporter function on cytotoxicity.

S31. MECHANISTIC BIOMARKERS OF DRUG-INDUCED LIVER AND KIDNEY INJURY B. Kevin Park MRC Centre for Drug Safety Science, University Of Liverpool, Liverpool, United Kingdom Drug-induced liver (DILI) and kidney injury (DIKI) represent a significant cause of patient morbidity, mortality and is a major contributor to attrition within drug development. Prediction of these adverse effects in the clinic remains difficult, particularly


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in cases characterised by marked inter-individual variation. A lack of sensitivity, specificity and an indirect mechanistic basis of currently used biomarkers of organ injury remains a factor for the delayed identification of drug toxicity. Currently, ‘Hy’s law’ represents the regulatory endorsed model to predict serious DILI and remains the standard for novel biomarkers to surpass. Pre-clinical biomarker identification and validation has been focused on molecular biomarkers such as cytokeratin-18, high mobility group box-1, glutamate dehydrogenase and micoRNA-122 from the perspective of acetaminophen hepatotoxicity [Antoine, 2009]. These biomarkers hold translational application to inform the sensitive identification of DILI and its mechanistic basis in man [Starkey-Lewis, 2011. Antoine, 2012]. A number of these biomarkers also provide enhanced prognostic information following acetaminophen overdose [Antoine, 2013]. Significant progress has been made regarding the qualification of renal biomarkers for DIKI, such as kidney injury molecule-1, in preclinical species but their clinical utility is still unclear. Here we discuss their clinical utility, specificity and sensitivity to report renal injury focusing on aminoglycoside-induced nephrotoxicity in neonatal infants and children with cystic fibrosis [McWilliam and Antoine 2012]. Significant challenges remain for new investigational biomarkers such as the characterization of inter- and intra-subject variability, as well as the impact of gender, age, and diurnal variation in healthy volunteers remain regarding these putative biomarkers, as well as the utility in idiosyncratic DLI and the prediction of serious toxicity from benign elevations in ALT activity for proposed liver markers. These challenges are the current focus of the Predictive Safety Testing Consortium (PSTC) and the IMI Safer and Faster Evidence based Translation (SAFE-T) consortium. The integrated use of these and qualification strategies will be discussed from a backdrop of imperfect current standards in the context of understanding fundamental hepatology and nephrology. DJ Antoine et al., 2009 Toxicol Sci. 112(2):521–31. DJ Antoine et al., 2012 J Hepatology. 56:1070–9. DJ Antoine et al., 2013 Hepatology. doi: 10.1002/hep.26294. P Starkey Lewis et al., 2011 Hepatology. 54(5):1767–76. S McWilliam and DJ Antoine et al., 2012 PLOS One e43809

S32. INSIGHTS FROM HEALTHY VOLUNTEER STUDIES Paul B. Watkins Hamner-University of North Carolina Institute for Drug Safety Sciences, Hamner, Research Triangle Park, NC, USA Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) have been used for over 40 years to monitor the liver safety of drugs and are sensitive measures of liver injury. However, there are drugs that, when administered at recommended doses, are quite safe for the liver yet cause marked elevations in serum ALT and AST. We have been exploring whether current mechanistic biomarkers can distinguish benign elevations in these enzymes from those that may lead to clinically important liver injury. We have analyzed serial serum samples obtained from cohorts of healthy adult volunteers treated with different therapeutic drug regimens that can cause marked elevations in serum ALT and AST but have low or no risk for serious hepatotoxicity (acetaminophen, various heparins, and cholestyramine). We and our collaborators have employed a variety of techniques, including transcriptomics, metabolomics and proteomics to identify biomarkers that may provide insight into the mechanisms underlying these benign and self-limited laboratory abnormalities. These studies indicate that recurrent therapeutic doses of acetaminophen, heparins and cholestyramine cause hepatocyte death, but the mechanisms involved in cell death differ between these treatments. The reason why these treatments are safe for the liver remains unclear but several theories have been proposed.

S33. DNA AND PROTEIN ADDUCTS AS BIOMARKERS OF EXPOSURE TO ENVIRONMENTAL AND DIETARY TOXICANTS Robert Turesky1, Lijuan Peng2, Dan Gu2, Kim M. Yonemori3, Kami K. White3, Lynne R. Wilkens3 and Loı¨c Le Marchand3 1 Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA, 2Environmental Health Sciences, Wadsworth Center, Albany, NY, USA, 3Epidemiology Program, University of Hawaii Cancer Center, Honolulu, HI, USA There is considerable interest in the role of gene-environment interactions in relation to the risk of cancer. For example, epidemiologic studies have implicated heterocyclic aromatic amines (HAAs), which are formed in well-done cooked meat, in human cancers of the digestive tract, prostate gland, and the mammary gland. However, some studies have shown no associations between well-done cooked meat and cancer risk. A critical limiting factor in most epidemiological studies is the uncertainty in quantitative estimates of chronic exposure to HAAs. For most studies, the extent of HAA exposure is inferred by a food frequency questionnaire (FFQ). However, the FFQ is problematic because the levels of HAAs formed are highly dependent on the type of meat cooked and the method of cooking, which can lead to differences of HAA concentrations by more than 100-fold. There is a critical need to establish chemical markers of HAAs that can be employed in molecular epidemiology studies to assess long-term exposure and the biologically effective dose. We established a rapid and sensitive tandem mass spectrometry method to biomonitor 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass-abundant carcinogenic HAA formed in well-done meat, by measurement of the accrual of PhIP in hair. PhIP non-covalently binds to protein and pigments in the hair follicle and it becomes entrapped in the


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cortex of the hair shaft during hair growth. The limit of quantification of PhIP is 50 parts-per-trillion, when 25 mg of hair is employed for assay. The amount of PhIP in hair increased, on average, 15-fold in light- and dark-haired individuals during consumption of cooked meat over 4 weeks in a semi-controlled feeding study. PhIP levels in hair were correlated to PhIP intake and the relationship was strengthened when PhIP levels were normalized for the melanin content of hair. However, PhIP accrual in hair was not correlated to cytochrome P450 1A2, the major enzyme involved in PhIP metabolism. N-Oxidized metabolites of PhIP form covalent adducts with protein and DNA. The reactivity of the genotoxic N-hydroxy (HONH-PhIP), and nitro (NO2-PhIP) derivatives of PhIP were examined with serum albumin (SA). LC/MS analysis of the tryptic/chymotryptic digests of SA modified with NO2-PhIP revealed that adduction occurred at Cys34, Lys195, Lys199, Lys351, Lys541, Tyr138, Tyr150, Tyr401, and Tyr411, whereas HONH-PhIP adduction occurred only at Cys34, and formed a labile sulfinamide adduct. Oxidation of PhIP-modified SA with m-chloroperoxybenzoic acid produced the arylsulfonamide adduct, which is stable to the conditions employed to denature SA. Methods are under development to biomonitor PhIP-SA adducts in humans. A novel multi-stage scan (MS3) method employing a linear quadrupole ion trap mass spectrometer was established to biomonitor DNA adducts of PhIP formed with deoxyguanosine. N-(Deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP) can be measured at levels as low as 3 adducts per 109 bases, when employing 10 mg DNA. We identified dG-C8-PhIP in the mammary gland of women with breast cancer, but at much lower frequency than adducts reported by immunohistochemistry methods. Mass spectrometric based methods are sensitive and can be employed in epidemiologic studies to assess factors involved in PhIP metabolism and HAA-related cancer risk

S34. THE EPR EFFECT AND BEYOND FOR CANCER SELECTIVE DRUG DELIVERY FOR TREATMENT AND IMAGING USING NANOMEDICINE Hiroshi Maeda Institute of Drug Delivery System, Sojo University, Kumamoto, Japan The enhanced permeability and retention (EPR) effect of macromolecular drugs or nanomedicine is now well recognized after 27 years of its initial finding (1). So far it was primarily demonstrated in the implanted tumor models in rodents, and not rigorously pursued for spontaneous or autochthonous primary tumors, nor for metastatic tumors. It was also reported that minimal molecular size of macromolecules to exhibit was above 50KDa, however, upper limit of molecular size for EPR effect was not clearly demonstrated above 800KDa(2). Another issue of criticism is heterogeneity of the EPR effect which exists in some tumors, or very weak EPR effect in other tumors such as pancreatic and prostatic tumors, both are known hypovascular type tumor by angiography. In completely different settings of human solid tumors, we could demonstrate the EPR effect by using LipiodolR (lipid contrast agent used as carrier for a polymer conjugated drug SMANCS)(2,3). Namely, when SMANCS/ LipiodolRwas infused into the tumor- feeding artery, i.e. the hepatic artery for hepatoma, we observed tumor selective drug delivery that could visualize liver tumor clearly by X-ray systems. This was also shown applicable to renal cancer, metastatic cancer of liver, etc (3,4). In this meeting, I will demonstrate tumor selective delivery of fluorescent nanoprobes such as HPMA-polymer conjugatedzinc protoporphyrin (PP) or -anthracycline (pirarubicin), and Rhodamine B conjugated bovine serum albumin (BSA-rhodamine). To solve heterogeneity problem of EPR effect, one can augment EPR effect by using potentiator of EPR effect such as nitroglycerin or ACE (angiotensin II converting enzyme) inhibitors (3). The new findings to be reported are that the EPR effect can be seen in spontaneous or autochthonous tumor (DMBA induced breast cancer in rats and azoxymethane induced colon cancer in mice). EPR effect is also seen in the metastatic lung cancer of colon 26 tumor implanted in the dorsal skin of syngeneic mice using above described fluorescent nanoprobes. Augmentation of EPR effect resulted significantly higher tumor delivery of even bacteria [Lactobacillus casei] by using nitroglycerin and other potentiator where bacteria having size as large as 2 mm were administered intravenously. The similarly augmentation of EPR was achieved by use of angiotensin II-induced high bloodpressure (eg 110 ! 160 mmHg) during arterial infusion of SMANCS/LipiodolR in the metastatic liver cancer, pancreatic cancer, and others in human (3,4). Retrospectively speaking Gaþþ-scintigraphy for the tumor selective imaging can be explained by the EPR effect as radioemitting Gaþþ is entrapped by transferrin (90KDa) in plasma; namely Gaþþbecome nanoparticle and exhibited EPR effect, as described above. With regards to the size limitation for EPR effect, even bacteria can be delivered much more selectively to tumor than to normal tissues with use of nitroglycerin, an EPR effect enhancer.

References (1) (2) (3) (4)

Y. Matsumura, H. Maeda, Cancer Res. (1986) 46, 6387–92. H. Maeda et al, J. Cont. Release (2000) 65, 271–84. H. Maeda, Proc. Jpn. Acad. Ser. B (2012) 88, 53–71. A. Nagamitsu et al, Jpn. J. Clin. Oncol. (2009) 39, 756–66.

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S35. ACCESSING THE POWERHOUSE OF THE MAMMALIAN CELL WITH MITOCHONDRIA-PENETRATING PEPTIDES Shana Kelley Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada


The mitochondria of human cells play a central role in the life and death of the cell due to the diverse processes and proteins, such as energy production and cell death regulators, that it houses. The role of mitochondria in cancer progression and tumorigenesis has been widely acknowledged. A major challenge to the study of mitochondrial processes and the development of mito-targeted therapies is presented by the impermeability of the innermost mitochondrial membrane and its highly negative membrane potential, which exclude most exogenous molecules from the organelle. We have developed a new class of peptidebased mitochondria-targeting vectors that can deliver various cargos to this previously impenetrable organelle. We have used these vectors to understand the chemical requirements for mitochondrial entry, to study oxidative stress in the organelle, and to deliver several different therapeutics. Insights into the unique chemical and biochemical features of this organelle gained from the use of these peptides will be presented.

S36. OPTIMIZING DELIVERY OF HER2-TARGETED LIPOSOMAL DOXORUBICIN (MM-302) THROUGH IMAGING, COMPUTATIONAL ANALYSES AND EXPERIMENTATION Bart Hendriks Merrimack Pharmaceuticals, Cambridge, MA, USA Liposomal therapeutics are very large on a molecular scale relative to the small molecule active pharmaceutical ingredients they contain. As a result, they have fundamentally different transport properties and biodistribution than small molecules. In both preclinical models and human patients, it has been shown that liposome deposition is highly variable. Furthermore, computational modeling efforts have identified liposome deposition in tumors as a critical determinant of liposomal doxorubicin delivery to tumor cell nuclei. We demonstrate two complementary strategies for optimizing liposome deposition. First, pre-treatment with cyclophosphamide was shown in preclinical models to dramatically increase overall liposome deposition and correspondingly boost the targeting effect of immunoliposomes. Second, we demonstrate the development of a 64Cu-liposome PET tracer in order to identify patients with ‘high deposition’ tumors. We demonstrate that, in preclinical models, the extent of deposition of a non-therapeutic liposome, as assessed by PET/CT imaging, is able to predict the extent of tumor shrinkage following treatment with HER2-targeted liposomal doxorubicin (MM-302). Finally, the clinical translation of these findings will be discussed.

S37. DRUG TARGETING WITH NANOPARTICLES Arto O. Urtti Centre for Drug Research, University of Helsinki, Helsinki, Finland Liposomes are lipid based nanoparticles that can be modified on the cell surface to achieve prolonged half-life in plasma and improved homing to the target cells and tissues. We have built liposomes with two types of cancer targeting moieties: peptides from phage display screening and antibodies. Liposomes with peptide targeting towards neovascular endothelia were built. The targeting did not work as expected in thorough studies: 1) neovascular cell cultures; 2) in vivo biodistibution in mice; 3) cell level distribution into the tumours. Then, molecular dynamics was used to investigate the molecular level orinetation of the peptides in the PEGylated surface of the liposomes. It appears that the hydrophobic peptides associate with PEG chains, and they are not properly exposed to the liposomal surface for proper interaction with the targets. More hydrophilic RGD type control peptide did not show similar PEG interactions. The antibody targeted doxorubicin liposomes were investigated in the cell and animal experiments using direct and pre-targeting approaches. Intraperitoneal pre-targeting experiments showed promising results in targeting. The study shows the importance of the targeting moiety orientation on the liposomal surface.

S38. TRANSLATIONAL MODELING AND SAFETY ASSESSMENT OF TRANSPORTER-MEDIATED PHARMACOKINETICS AND DRUG-DRUG INTERACTIONS Aleksandra Galetin School of Pharmacy and Pharmaceutical Sciences, Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, United Kingdom Investigation of potential transporter mediated drug-drug interactions (DDI) is becoming increasingly important considering the large number of drugs reported to be either substrates or inhibitors of active hepatic uptake processes. In the current talk,

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different in vitro models for the assessment of hepatic uptake, in isolation or combined with metabolism and biliary excretion, are discussed. Integration of the generated uptake kinetic parameters in physiologically-based pharmacokinetic (PBPK) models in order to simulate plasma and tissue exposure of selected drugs is illustrated. Simvastatin and repaglinide hybrid PBPK models are used to assess drug safety and susceptibility to DDIs in subjects with different OATP1B1 genotypes. In addition, quantitative prediction of transporter-mediated DDIs is exemplified by cyclosporine as a perpetrator drug, considering its potency towards a range of uptake and efflux transporters and a number of clinically significant DDIs. The cyclosporine PBPK model was extended to assess the contribution of its main metabolite AM1 to the DDI potential, considering that AM1 concentrations can exceed those of cyclosporine following single and multiple drug administration. This dynamic modeling approach was applied for the prediction of cyclosporine-repaglinide interaction; the changes in the inhibitor concentration, potential contribution of multiple transporters and simultaneous assessment of the modulation of transporter/enzyme activity in different organs (liver and intestine) were accounted for by the model. The current presentation highlights the application of PBPK modeling to guide the design of clinical transporter-mediated DDI studies.

S39. COUPLING IN VITRO STUDIES AND PBPK MODELING TO PREDICT HUMAN SAFETY AND EFFICACY Harvey J. Clewell The Hamner Institute for Health Sciences, Research Triangle Park, NC, USA The physiological structure of PBPK models facilitates the incorporation of in silico- and in vitro-derived chemical-specific parameters in order to predict in vivo absorption, distribution, metabolism and excretion. In particular, the combination of in silico- and in vitro parameter estimation with PBPK modeling can be used to predict the in vivo exposure conditions that would produce chemical concentrations in the target tissue equivalent to the concentrations at which effects were observed with in vitro assays of tissue/organ toxicity. This dosimetry can then be coupled with in vitro data and modeling of cellular response pathways to characterize the likely in vivo dose-response for toxicity. This presentation will describe two examples of biological models developed to provide quantitative descriptions of the key processes involved in drug efficacy and toxicity in vivo on the basis of in vitro data. In the first example, preclinical and clinical data generated during the development of pafuramidine, a pro-drug of the active metabolite, furamidine, were used together with rat isolated perfused liver data to develop a PBPK model for rats. Scaling to the human was performed using metabolic and transport clearance data generated from rat and human sandwich cultured hepatocytes. The human model was used to define a more effective dose regimen of pafuramidine (40 mg once daily) based on a predefined efficacy/safety index. The second example describes a biosimulation model developed to provide a quantitative description of the key processes involved in drug-induced liver injury. The initial development of the platform has focused on acetaminophen (APAP) and includes a whole-body physiologically based pharmacokinetic (PBPK) model for APAP and its metabolites in the mouse, rat, and human. The PBPK model is linked to a pharmacodynamic (PD) model describing the reaction of APAP-derived N-acetyl-p-benzoquinone imine (NAPQI) with glutathione and the resulting depletion and resynthesis of glutathione. The formation of NAPQI-adducts with cellular proteins and increased oxidative stress resulting from glutathione depletion are linked to cellular damage and response. This PBPK/PD model of APAP metabolism and glutathione depletion has been used to investigate the extent to which differences in metabolism and glutathione homeostasis explain interspecies differences in susceptibility to APAP toxicity, and to investigate optimal N-acetylcysteine (NAC) treatment after APAP overdose. The model was also combined with in vitro data for the hepatotoxicant methapyrilene to perform in vitro to in vivo extrapolation; simulations comparing DILI responses across species illustrated how modeling can aid in selecting the most appropriate pre-clinical species for safety testing results relevant to humans.

S40. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELING AS A TOOL TO SUPPORT RISK ASSESSMENT Ursula Gundert-Remy Clinical Pharmacology and Toxicology, Charite, Medical School Berlin, Berlin, Germany In the past twenty years there is growing interest in physiologically based pharmaco-/toxicokinetic (PBPK/PBTK) modeling as a technique to support defining the dose which is intended to be given in the first studies in humans based on preclinical in vitro and in vivo animal study results, the so-called first dose in man. Similarly in toxicology, interest is growing to apply this approach to be used in risk assessment. Here, the typical situation for a chemical is characterized by existence of data in animal species but only few, if any, experimental data in humans. For risk assessment or setting limit values (e.g. tolerated daily intake, accepted daily intake, occupational exposure limit the animal data have to be extrapolated to the human physiology and in addition, also to the physiology of the whole population including special subgroups. Typically, default factors are used to bridge species difference between animal to man (4 for toxicokinetic differences, 2.5 for toxicodynamic differences) and a


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second factor of ten is applied for the interspecies differences in the human population (3.2 for toxicokinetic differences, 3.2 for toxicodynamic differences). PBTK modeling may help to derive chemical-specific assessment factors at least for the kinetic portion of the chemical-specific assessment factors PBTK modeling may also help to better understand the mode of action by identifying which kinetic metric is really related to the toxic effect which is to be assessed. Three examples are given to demonstrate the power of this approach. Intra-human variability across ages: The chemical BPA was modeled for newborn and compared the steady state concentration in blood of this dose with the steady state concentration in blood in an adult. In the newborn the sulfation pathway is predominant and with increasing age (and maturation of the glucuronide pathway) excretion by sulfation pathway decreases. Bisphenol A – dermal exposure: We simulated the BPA concentration time profile in blood, in the target organs for toxicity which are liver and kidney using a PBTK human model after oral and dermal exposure. The simulation showed that dermal exposure leads to lower peak concentration in the target organ liver and to higher peak concentrations in blood and kidney as compared to the oral exposure. The AUC in blood and kidney is higher after dermal exposure as compared to dosing on the oral route. AUC in the liver is determined by the extent of absorption. Coumarin – dermal exposure: We used a human PBTK model to simulate the concentration time profile in blood and in the target organs for toxicity which is liver after oral and dermal exposure. It turned out that the crucial point was to identify the relevant dose metric for liver toxicity. We used the AUC - and Cmax – data from a rat PBTK model and combined them with rat toxicity data. The analysis revealed that the severity grade of hepatotoxicity increases systematically with increasing Cmax in the liver whereas for increasing AUC in the liver no systematic increase of the severity grade could be seen.

S41. SCIENTIFIC AND REGULATORY PERSPECTIVES ON DRUG INTERACTION EVALUATION DURING DRUG DEVELOPMENT Lei Zhang Office of Clinical Pharmacology, Office of Translational Sciences, CDER, U.S. Food and Drug Administration, Silver Spring, MD, USA Pharmacokinetic drug interactions can lead to serious adverse events or decreased drug efficacy. The evaluation of a new molecular entity’s (NME’s) drug–drug interaction potential is an integral part of risk assessment during drug development and regulatory review prior to market approval. Alteration of activities of enzymes or transporters involved in the absorption, distribution, metabolism, or excretion of an NME by concomitant drugs may alter drug exposure, which can impact response (safety or efficacy). In 2012, the Food and Drug Administration (FDA) published a revised draft drug interaction guidance1 highlighting the methodologies and criteria that may be used to guide drug interaction evaluation by industry and regulatory review staff and to construct informative labeling for health practitioners and patients. In addition, the FDA has established a ‘‘Drug Development and Drug Interactions’’ website2 to provide up-to-date information regarding evaluation of drug interactions. The presentation will provide an overview on key elements in the FDA’s draft drug interaction guidance including regulatory science considerations and new science that can guide the evaluation of drug–drug interactions during the drug development process. 1. FDA Draft Guidance for Industry: Drug Interaction Studies —Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations (February 2012) http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatory Information/Guidances/UCM292362.pdf 2. FDA Drug Development and Drug Interactions Website: http://www.fda.gov/Drugs/DevelopmentApprovalProcess/ DevelopmentResources/DrugInteractionsLabeling/ucm080499.htm

S42. THE 2012 EMA DDI GUIDELINE: THE MOST IMPORTANT UPDATES AND POST-ADOPTION EXPERIENCES Eva Gil Berglund ES2, Medical Products Agency, Uppsala, Sweden The European Drug Interaction guideline has gone through a major revision. The new, final document was adopted by the CHMP in June last year and the guideline came into force on the 1st of January 2013. The revised guideline contains new recommendations regarding investigations of the drug as a transporter substrate and inhibitor, recommendations on enzyme induction investigations, the evaluation of how a drug is eliminated, requirements for active metabolites, etc. The document provides detailed advice when possible attempting to illustrate what is assessed from a regulatory point of view. When detailed advice is not possible, the applicant is referred to following the scientific development in the literature. The approaches chosen need to be well justified. The lecture will address the most important updates and reflections on the application of the guideline over the first 6 months of use.

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S43. AN INDUSTRY PERSPECTIVE AND EXPERIENCE ON THE MOST RECENT REGULATORY GUIDELINES ON DRUG INTERACTION STUDIES Thomayant Prueksaritanont Department of Drug Metabolism/WP75A-203, Merck & Co, West Point, PA, USA


AN INDUSTRY PERSPECTIVE AND EXPERIENCE ON THE MOST RECENT REGULATORY GUIDELINES ON DRUG INTERACTION STUDIES THOMAYANT PRUEKSARITANONT, MERCK & CO. Recently, the US FDA (Food and Drug Administration) and EMA (European Medicines Agency) have issued new guidances on drug interaction studies designed to help pharmaceutical researchers characterize and better understand DDI potential for a new molecular entity. Both of the latest guidance documents contain extensive recommendations on in vitro and in vivo studies and approaches to evaluate drug-drug interaction (DDI) mediated via drug metabolizing enzymes and drug transporters. Here we will share an industry perspective and experience in applying the guidance to support drug development programs. In particular, case examples will be presented to highlight potential complications associated with certain aspects of the proposed approaches related to transporter-mediated DDIs and DDIs arising from mixed time-dependent inhibitors (TDI) and inducers. Also presented are results from our recent studies in an on-going effort to fill in knowledge gaps in the transporter area, where the science is rapidly evolving.

S44. STRATEGIES TO ASSESS HUMAN METABOLISM OF NCES - 5 YEARS AFTER THE MIST GUIDANCES J. Greg Slatter Early Development - Clinical Pharmacology, Amgen Inc., Seattle, WA, USA The 2008 CDER Safety Testing of Drug Metabolites, ICHM3(R2), M3(R2) Q&A, and ICH S9 regulatory guidances and guidelines (the ‘‘metabolites in safety testing’’ or ‘‘MIST’’ guidances), emphasized the importance of human radiochemical excretion and metabolism studies (‘‘human ADME studies’’). At the time the MIST guidances were written, the human ADME study was viewed as the definitive word on patient exposure to drug metabolites. These studies continue to be a standard aspect of most small molecule drug development programs. Coincident with the appearance of these guidances, new technologies such as accelerator mass spectrometry (AMS) and high resolution LCMS/MS were being adopted across the industry and may be changing how, when and why human radiochemical ADME studies are run. New study designs, made possible by these technologies, and sometimes involving very low doses of radioactivity, or no radioactive tracer, are now being used for the assessment of in vivo human drug metabolism. This talk will explore the past, present and future of how human metabolism is done during clinical development.

S45. INTEGRATED EXAMINATION OF AH RECEPTOR-MEDIATED HEPATIC FAT ACCUMULATION Abstract not available.

S46. PHARMACOMETABOLOMICS: GLOBAL BIOCHEMICAL APPROACH FOR MAPPING DRUG EFFECTS Rima Kaddurah-Daouk Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, NC, USA Metabolomics, the study of metabolism at an ‘‘omic’’ level, has the potential to transform our understanding of mechanisms of drug action and the molecular basis for variation in drug response. It is now possible to define metabolic signatures of drug exposure that can identify pathways involved in both drug efficacy and adverse drug reactions. Additionally the ‘‘metabotype’’, the metabolic ‘‘signatures’’ of a patient, is a unique identity that contains information about drug response and disease heterogeneity. The application of metabolomics for the study of drug effects and variation in drug response is creating ‘‘pharmacometabolomics’’, a discipline that will contribute to personalized drug therapy and will compliment pharmacogenomics by capturing environmental and microbiome influences on response to drug therapy. This field has the potential to transform pharmacology and clinical pharmacology in significant ways and will contribute to efforts for personalized therapy. The talk will highlight developments in the new discipline of pharmacometabolomics.

S47. MECHANISTIC UNDERSTANDING AND BIOMARKERS FOR DRUG TOXICITY USING METABOLOMICS Lining Guo Metabolon, Durham, NC, USA


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Global metabolomics is a new and powerful technology that can provide a relatively complete picture of metabolism in biological systems. We have developed an unbiased global metabolomics platform based on a combination of three independent systems: ultrahigh performance liquid chromatography/tandem mass spectrometry (UHLC/MS/MS2) optimized for basic species, UHLC/MS/MS2 optimized for acidic species, and gas chromatography/mass spectrometry (GC/MS). Following sample extraction, full scan mass spectra is carried out to record retention time, molecular weight (m/z) and MS/MS2 of all detectable ions presented in the samples. Rapid and confident identification of metabolites in the experimental samples is achieved by automated comparison of the ion features in the experimental samples to a comprehensive chemical reference library. After data generation, integrated tools including statistical analysis, pathway mapping, and data visualization can rapidly provide powerful insights for understanding biological systems. This technology is especially well suited to investigate mechanism of drug induced toxicity and to discover mechanistic based biomarkers. Data from two specific toxicology studies will be discussed. In the first study, we investigated the mode of action of ethylene glycol monomethyl ether (EGME) induced testicular injury in rats. Rats were administrated with multiple EGME doses, then serum, urine, liver and testes were collected at multiple time point for analysis. Statistical analysis and pathway mapping of nearly 1,900 metabolites revealed that the site of EGME action was the disruption of key biochemical pathways utilizing flavoprotein dehydrogenases. These metabolite perturbations in conjunction with downstream actions collectively resulted in the EGME-induced tissue damage. In the second study, we aimed to gain insights into drug-induced liver injury (DILI) pathogenesis and identify potential biomarkers for improved DILI detection in preclinical stage. Thirteen hepatotoxicants were administrated in two doses to rats, then plasma, urine, and liver tissues were collected at two time points for analysis. The most consistent change induced by the hepatotoxins, detectable even at the early time point/low dose, was the significant elevation of a panel of bile acids in the plasma and urine. In addition, bile acid biosynthesis in the hepatocytes was altered due to hepatotoxin-induced oxidative stress. Our data suggested that the perturbation of bile acid homeostasis was an early event of DILI and was apparent well before conventional biochemical and histological biomarkers. Selected bile acids could be potentially used as sensitive and early DILI preclinical biomarkers.

S48. PREGNANE X RECEPTOR AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR a HUMANIZED MICE Frank J. Gonzalez Laboratory of Metabolism, National Cancer Institute, Bethesda, MD, USA Pregnane X receptor (PXR) is a pivotal nuclear receptor modulating xenobiotic metabolism, primarily through its regulation of CYP3A4, the most important enzyme involved in drug metabolism in humans. Due to the marked species differences in ligand specificity exhibited by PXR, PXR-humanized mice were produced by inserting the complete human gene on the Pxr-null mouse background. Mice expressing human PXR and CYP3A4 (hPXR/CYP3A4) were also established. hPXR and hPXR/CYP3A4 mice are valuable models for investigating the role of PXR in xenobiotic metabolism and toxicity, and in lipid, bile acid and steroid hormone homeostasis. These mouse lines have been of great value in determining the role of intestinal PXR as a drug target for rifaximin, a gut-specific antibiotic under clinical trials for the treatment of inflammatory bowel syndrome, due to the role of PXR in modulation of inflammation. The PXR-humanized mice were also pivotal in establishing the mechanism of the isoniazid-rifampicin drug interaction that causes hepatic toxicity in some tuberculosis patients. Other studies revealed that PXR is a contributing factor in metabolic and inflammatory diseases such as type 2 diabetes and colitis. Peroxisome proliferatoractivated receptor a(PPARa) controls the expression of genes involved in fatty acid, cholesterol and glucose homeostasis through regulating fatty acid transporters and enzymes involved in fatty acid B-oxidation. PPARa is the target for the lipid lowering fibrate drugs that are widely prescribed in humans. The mouse and human PPARa have different ligand specificities and affinities, different expression levels, and differential gene targets. Thus, the PPARa-humanized mouse line was generated by inserting the complete human gene on the Ppara-null mouse background. This mouse line was used to establish the mechanism for the differential sensitivities of mice and humans to the hepatocarcinogenic effects of activators, including the fibrate drugs. Recent studies on PXR- and PPARa-humanized mice and other humanized models will be discussed.

S49. THE ROLE OF INTESTINAL AND HEPATIC GLUCURONIDATION IN CHEMOTHERAPY INDUCED TOXICITY BY IRINOTECAN (CPT-11) Shujuan Chen1, Mei-Fei Yueh1, Olivier Barbier2, Cyril Bigo2, Kepeng Wang3, Michael Karin3, Nghia Nguyen1 and Robert H. Tukey1 1 Laboratory of Environmental Toxicology, Department of Chemistry & Biochemistry and Pharmacology, University of California, San Diego, La Jolla, CA, USA, 2Laboratory of Molecular Pharmacology, CHUQ Research Center and Faculty of Pharmacy, University Laval, Quebec, QC, Canada, 3Laboratory of Gene Regulation and Signal Transduction, University of California - San Diego, La Jolla, CA, USA Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world. Irinotecan (CPT-11) is an analog of the alkaloid camptothecin, and is used alone or in combination with other drugs as the first line therapeutic agent for CRC.


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CPT-11 is a prodrug that is hydrolyzed by carboxyesterase (CE) activity to the active topoisomerase 1 inhibitor, SN-38. Inactivation and detoxification occur by UGT1A family catalyzed glucuronidation to form the SN-38 glucuronide (SN-38G). CPT-11 is metabolized in both the liver and intestinal tissue by CEs and UGTs, but its efficacy and safety are compromised because of severe late diarrhea, thought to be initiated by the toxic side effects of SN-38. Clinical data has indicated more severe toxicity in patients with Gilbert’s Syndrome, indicating that the reduced hepatic UGT1A1 expression may play a role in SN-38 induced intestinal toxicity. However, the mechanism leading to CPT-11 induced diarrhea and GI track damage is not understood. We elected to examine the impact of tissue specific glucuronidation on CPT-11 induced toxicity. To undertake these experiments, the Cre/loxP recombination system was used for the generation of cell-type specific gene targeting of the mouse Ugt1 locus. Cre-recombinase specific loxP sites were positioned in the intronic regions to delete the common regions of the Ugt1 locus. When Ugt1 floxed (Ugt1F/F) mice were crossed with transgenic mice that express albumin-Cre or villin-Cre, Cre-mediated recombination led to the deletion of the Ugt1 locus in either liver hepatocytes (Ugt1DHep) or intestinal enterocytes (Ugt1DGI), respectively. Glucuronidation capacity towards bilirubin and SN-38 was completely eliminated in microsomes prepared from liver tissue from Ugt1DHep mice and intestines from Ugt1DGI mice. When Ugt1DHep and Ugt1DGI mice were treated with increasing concentrations of CPT-11 by i.p. injection, the survival rate of Ugt1DHep mice was similar to that of the Ugt1F/F mice. This result indicates that hepatic UGT1A enzyme activity may not be responsible CPT-11 detoxification, which contradicts conventional thinking. To our surprise, Ugt1DGI mice treated with CPT-11 were extremely sensitive in response to CPT-11 treatment. Weight loss, intestinal damage and lethality were closely linked to deletion of the Ugt1 locus in Ugt1DGI mice. Thus, expression of UGT1A proteins in intestinal tissue dictates both the efficacy and toxicity of CPT-11 chemotherapy. When we examined colon tumor samples driven by loss of the Apc genes in CPC-APC mice, Ugt1a1 gene expression in tumor samples was reduced to less than 10% compared to normal tissue. Thus, regulation of intestinal UGT1A1 in normal tissue may serve as a therapeutic target for improving the therapeutic index and efficacy associated with CTP-11 treatment. This work was supported in part by USPHS Grants R21CA171008 (SC), ES010337 (RHT and MK), GM086713 and GM10048 (RHT), AI043477 (MK), and CIHR MOP-84338 & MSH95330 and CFI 17745 (OB).

S50. OPTIMAL SURVIVAL OF N-MYC MYELOID LEUKEMIAS REQUIRE A PORPHYRIN EXPORTER Yu Fukada1, Yao Wang1, Shinjiro Nagai1, Bruce Fanshawe1, Ayten Kandilci2, Brian P. Sorrentino3, Geoff Neale4, Yiping Fan4, Gerard Grosveld2 and John D. Schuetz1 1 Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, TN, USA, 2Genetics and Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA, 3Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA, 4Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital, Memphis, TN, USA Identifying pathways facilitating the survival of certain leukemias may reveal unique vulnerabilities. Here we determined that pediatric AML with a unique NMYC gene signature strongly upregulated genes in the porphyrin biosynthetic pathway. However, the significance and relative importance of porphyrin biosynthesis and homeostasis in myeloid leukemias is unknown. We show NMYC strongly activates de novo porphyrin biosynthesis. Using a murine model of N-MYC myeloid leukemia we demonstrated that absence of the porphyrin exporter, Abcg2, impaired N-MYC leukemogenesis and blocked self-renewal of N-MYC expressing hematopoietic progenitors. Consistent with this, in N-MYC hematopoietic progenitors, Abcg2 absence in vivo strongly reduced the number of leukemic stem and myeloid progenitors. Inhibition of porphyrin biosynthesis or re-introduction of ABCG2 in N-MYC expressing hematopoietic progenitors mostly restored self-renewal. In human myeloid leukemias expressing ABCG2, inhibition of ABCG2 produced porphyrin elevation that was accompanied by mitochondrial ROS as well as loss of both mitochondrial membrane potential and viability. In total, there results reveal a critical dependence of N-MYC myeloid leukemias upon the porphyrin exporter, Abcg2, in mice and suggest that modulating porphyrin levels in patients with these AMLs may be a promising approach to improve therapeutic efficacy.

S51. MOLECULAR IMAGING FOR DRUG DEVELOPMENT Yasuyoshi Watanabe RIKEN Center for Life Science Technologies, Hyogo, Japan In vivo molecular imaging has become a key technology for medical innovation, i.e., integrated live science, patho-physiological science, molecular evidence-based diagnosis, innovative drug development, and preemptive medicine. Rapid progress in life science brought the stage up to the living objects or subjects, even functioning human beings. The invention of a variety of imaging techniques including Positron Emission Tomography (PET) accelerated the paradigm shift in life science from patho-physiological science in disease model animals to that realized in patients. Especially, the molecular imaging techniques could promote the bridging between findings in gene-manipulated animals and those in healthy volunteers and patients. When the biomarkers for early detection of signs toward diseases and also those for surrogate end-point were established and their changes could be followed by molecular imaging, it could be quite beneficial for preemptive medicine and evaluation of

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Drug Metab Rev, 2013; 45(S1): 1–25

therapeutic outcome. For example, beta-amyloid imaging and pancreatic beta-cell mass imaging have been highlighted for prediction of Alzheimer’s disease and diabetes mellitus, respectively. Such types of valuable biomarkers related to the cause or influencing factors are also really important to develop the new drugs. In this sense, mutual collaboration among the research consortia in biomarker exploration, in gene manipulation, and in molecular imaging would be really important. In this context, in 2005, our Center in RIKEN was selected as the key hub center of the Molecular Imaging Research Program under MEXT, Japanese Government, for development of All-Japan research network to further promote mutual international and multi-disciplinary collaboration on in vivo molecular imaging. To realize the molecular probing concept, we have so far developed a variety of novel chemical methods for labeling the low and higher molecular weight compounds with due positron emitters, such as C-11, F-18, Ga-68, and Cu-64, and the number of different types of the molecular probes to be used in PET study is more than 221 so far developed in our Center and the repertoire being increasing every day. Direct application of this molecular probing concept is to pharmacokinetics and efficient DDS development. The concept, outline of our activities, PK/PD studies, and preemptive medicine studies with efficient application of molecular imaging will be presented.


S52. MOLECULAR IMAGING FOR PHARMACOKINETICS Tomotaka Shingaki1, Tadayuki Takashima2 and Yasuyoshi Watanabe3 1 Molecular Dynamics Imaging Unit, RIKEN Center for Life Science Technologies, Hyogo, Japan, 2Molecular Probe Dynamics Laboratory, RIKEN Center for Molecular Imaging Science, Hyogo, Japan, 3RIKEN Center for Life Science Technologies, Hyogo, Japan Molecular imaging technology using positron emission tomography (PET) is a powerful method for pharmacokinetic research in living systems. The non-invasiveness, high sensitivity and exceptional spatiotemporal resolution of PET studies enable repeated estimation of the in vivo distribution of PET probes from the same individual. PET studies also allow the pharmacokinetic evaluation of drug candidates without changing their chemical structure, substituting their molecular structure with short-lived positron-emitting radionuclides, such as 11C and 18F, under micro-dose levels. Hence, it is expected that PET is applicable throughout all drug development stages from early drug discovery to the clinical phase. In this presentation, we introduce how we apply PET imaging analysis to pharmacokinetic investigations for bridging the gap between preclinical and clinical research. Investigation of membrane transporter activities using PET Membrane transporters play key roles in the pharmacokinetics of tissue distribution and excretion. In terms of genetic polymorphisms and drug-drug interactions, changes of these functions may impact tissue or systemic blood concentrations that lead to the adverse side-effects. Although the difficulty of measuring drug concentrations in human organs was a major obstacle in estimating tissue pharmacokinetics in humans, recent advances in molecular imaging technology using PET may resolve these problems and provide new breakthroughs by combination with pharmacokinetic methods, such as integration plot analysis. In this presentation, separate estimations of transporter activity in hepatic uptake and canalicular efflux are introduced for the hepatobiliary transport of [11C]TIC-methyl ester, [11C]dehydropravastatin, [11C]telmisartan, and so on, as PET probes. The effect of co-administered rifampicin on hepatobiliary transport is also introduced as a model of investigation of drug-drug interaction. Transporter activities on the renal excretion process and drug interaction are also introduced with [11C]metformin as a PET probe and pyrimethamine as an inhibitor. Investigation of absorption process in gastrointestinal tract and nasal cavity using PET The detail of drug behavior in the gastrointestinal tract after oral administration is currently poorly understood, even though the oral dosage form is the first choice in drug development. To develop potent drugs for oral use, information about what occurs in the gastrointestinal tract after oral administration is very important. Simplifying the theory of gastrointestinal absorbability, total amount of absorption (FaD) can be expressed as the product of membrane permeability clearance (CLperm) by area under the drug concentration dissolved in the intestinal tract time curve (AUCintestine, 0–t), which can be estimated by non-invasive PET imaging in humans. We introduce the results of clinical and non-clinical investigations of PET study with [18F]FDG and [18F]FPEG as PET probes. In addition, considering the lifecycle management of drug products, nasal administration has attracted attention as an alternative dosage form. However, lack of valid evaluation methods makes it difficult to prove the effectiveness of nasal formulations. We introduce the latest nasal absorption research using PET imaging analysis with pharmacokinetic model analysis to overcome these problems.

S53. MOLECULAR IMAGING OF DRUG TARGET MOLECULES Tetsuya Suhara Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan Positron emission tomography (PET) techniques have enabled the visualization of target molecules of various psychotropic drugs, such as receptors and transporters in the living human brain. The concept of occupancy has been used as a reliable index


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for therapeutic drug monitoring at specific binding sites. Previous PET studies on antipsychotics have suggested that 70%–80% of central dopamine D2 receptor occupancy provides the desired therapeutic effects without any extrapyramidal symptom. Regarding the antidepressant therapy, serotonin transporter occupancy is also used as one of the indices for the evaluation of antidepressants such as selective serotonin reuptake inhibitors. Clinical doses of clomipramine and fluvoxamine occupied about 80% of serotonin transporter, and dose escalation would have minimal effect on serotonin transporter blockade. Norepinephrine transporter (NET) is another target of antidepressants. The mean NET occupancies by nortriptyline doses were 16.4% at 10 mg, 33.2% at 25 mg, and 41.1% at 75 mg, respectively. The application of PET method on drug evaluation can provide us useful information about the characteristics of psychotropics, including an optimal clinical dose and the kinetic profile at the sites of action as well as their ability to penetrate into the brain. However, in psychiatric disorders, reliable diagnostic biomarkers are still awaited. Nonetheless, in the case of Alzheimer disease (AD), distinctive pathological changes such as deposition of b-amyloid (Ab) and tau protein have been identified. Several amyloid ligands, such as [11C]PIB and [11C]BF227, have been developed for imaging Ab deposition. On the other hand, tau pathology is considered to be closely related with neural death in AD and non-AD tauopathy, and could accordingly be an important target for both therapeutic intervention and diagnostic imaging. [11C]PBB3 has been recently developed as a tau imaging PET ligand, and showed high affinity and selectivity for tau deposits in our preclinical studies. [11C]PBB3-PET demonstrated high accumulation in the medial temporal cortex of all AD patients, in which binding of [11C]PIB was minimal. Distribution of [11C]PBB3 accumulation observed in AD patients extended to the entire limbic system and subsequently to the neocortex as a function of the disease severity.

S54. MALDI-IMAGING MS: A GATEWAY TO TRANSLATIONAL DRUG DEVELOPMENT Stephen Castellino and M. Reid Groseclose PTS Dmpk, GlaxoSmithKline, Rtp, NC, USA Interest in the emerging technology of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) stems from the ability to determine the tissue distribution of drugs and their metabolites as well as endogenous compounds without the need for labeling. With high spatial resolution (pixel size), distributions within tissue subcompartments can be mapped and correlated with histology images. This capability to view the molecular histology opens new options for integrating biology and chemistry in drug discovery and development. As such, more detailed chemical and biological information can be obtained from target engagement experiments, PK/PD studies, as well as safety investigations. This presentation highlights our application of MALDI IMS to a variety of studies in discovery and drug development.

S55. MODELING HEPATIC DEVELOPMENT, METABOLIC DISORDERS AND DRUG REACTIVITY USING HEPATIC-LIKE CELLS Nicholas RF Hannan Surgery, University of Cambridge, Cambridge, United Kingdom Human embryonic stem cells (hESCs) as well as induced pluripotent stem cells (IPSCs) represent a unique opportunity to study human development, model diseases in vitro, optimise drug screening platforms and develop cell replacement therapies for degenerative diseases. One of the main challenges in achieving these promises is the production of populations of progenitor cells that are of high purity, retain their full spectrum of differentiation capacity and are able to expand into numbers relevant for therapeutic needs. Following a normal path of development remains the best approach to generate such cells as well as the development of a universal differentiation protocol applicable to a wide variety of pluripotent cell lines. Our lab has developed a defined culture system to drive differentiation of hESCs cells through the early developmental stages of endoderm specification and then into hepatic endoderm. We were able to demonstrate a near homogeneous population of cells that have morphological characteristics and gene expression profiles of hepatic progenitors including HHEX, HNF4a, PROX1, HNF3B, HNF1a, HNF1B and cytokeratin 18 and 19. Further maturation of these cells produced cells positive for more mature hepatocyte markers such as alpha-fetoprotein and albumin. These cells also display functional characteristics such as albumin and alpha-1-antitrypsin secretion, cholesterol uptake, glycogen storage and inducible cytochrome P450 activity. More recently we have demonstrated the capability of these cells to model inherited metabolic disorders such as alpha-1-antitrypsin deficiency, familial hypercholesterolemia and glycogen storage disease type 1. These cells presents a unique advantage for development of drug screening platforms, disease modeling and transplant therapy applications and also represent a first toward the generation of hepatic cells fully compatible with in vivo applications.

S56. INDUSTRIAL PRODUCTION OF FUNCTIONAL HUMAN PLURIPOTENT STEM CELL DERIVED HEPATOCYTES: APPLICATIONS AND COMPARISON TO OTHER HEPATOCYTE MODELS Petter Bjo¨rquist Cellectis AB, Gothenburg, Sweden


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The failure to predict drug metabolism, efficacy and toxicity is a major cause of compound attrition during the development of new drugs. This is in part due to the lack of availability of human cells of high quality and the interspecies problems associated with animal models. Altogether this highlights the need to front-load physiologically relevant human models in pre-clinical testing. Harnessing the capacity of pluripotent stems cells to differentiate into various phenotypes provides access to multiple types of human cells. Moreover, recent technological advancements have enabled the reprogramming of adult human somatic cells into induced pluripotent stem cells (hiPSC), which theoretically offers a continuous supply of human cell material from genetically diverse backgrounds. However, to date the genetic diversity of current commercially available stem cell based derivatives has been limited. Robust protocols for maintaining cells in their undifferentiated state and for differentiation to desired cell types are essential for producing this material in industrial quantities. Both human embryonic stem cells (hESC) and hiPSC have previously been shown to vary in their propensity to differentiate to a given cell type. Thus, there is a great need for robust differentiation protocols that can be universally applicable over multiple cells lines without the need for adaptation. Here, we demonstrate how a panel of hESC and hiPSC lines generated from healthy donors, can be expanded using the novel highly efficient culturing system DEF-CSÔ to provide sufficient cell material for further differentiation and toxicity studies. Robust differentiation protocols were applied to drive the panel of cell lines into hepatocytes. We show proof-of-principle of how the variability of the general population can be represented in vitro, using well established tests, including Cytochrome P450 and transporter activity as functional assay end-points. Panels of human pluripotent stem cell derived hepatocytes are expected to be powerful tools in the prediction of compound toxicity and efficacy, and can aid in the modeling of human diversity in drug responses. Understanding the impact of an individual’s genetic background on well-established pharmaceutical targets is critical for the development of safe and effective drugs, and forms the foundation for future personalized medicine. In conclusion, we will show important aspects of routine mass production of hPSC-derived hepatocyte-like cells, and how these cells can be used in various industrial in vitro applications.

S57. GENERATION OF METABOLICALLY FUNCTIONING HEPATOCYTES FROM HUMAN PLURIPOTENT STEM CELLS BY TRANSDUCTION OF FOXA2 AND HNF1a Hiroyuki Mizuguchi Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are expected to be utilized as a tool for screening for hepatotoxicity in the early phase of pharmaceutical development. However, the drug enzyme activities of the hepatocyte-like cells were lower than those of primary human hepatocytes. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors (such as FOXA2 and HNF1a) but also a three-dimensional (3D) spheroid culture systems using a Nanopillar Plate (Hitachi High-Technologies Corporation). The levels of many hepatocyte functions, such as albumin secretion ability, urea secretion ability, hepatocyte-related gene expressions, drug metabolism capacity, and cytochrome P450 induction potency, of the hepatocyte-like cells differentiated in 3D culture condition by using Nanopillar Plate were higher than those of the hepatocyte-like cells differentiated in monolayer culture condition. We also developed the method of long-term culture of hepatoblast-like cells derived from hESCs and hiPSCs. Hepatoblasts have ability to differentiate into both hepatic and biliary lineages and to self-replicate. The establishment of selfrenewing hepatoblast-like cells from hESCs and hiPSCs would lead to a stable supply of the hepatocyte-like cells for medical application such as drug screening and liver cell transplantation. In this symposium, I will show the recent data of our study about the generation of hepatocyte-like cells from hESCs and hiPSCs and the application for drug toxicity testing.

S58. IMPACT OF ENVIRONMENTAL CHEMICALS ON THE DIFFERENTIATION OF HUMAN ADULT HEPATIC PROGENITORS Irena Iankova1, Wafa Hassen2, Cedric Duret1, Sabine Gerbal-Chaloin1, Philippe Briolotti1, Jean-Michel Fabre3, Jeanne Ramos4, Jamila Faivre5, Patrick Maurel1 and Martine Daujat-Chavanieu1 1 INSERM U1040, Universite´ Montpellier 1, UMR-1040, CHU Montpellier, Montpellier, France, 2Laboratory of Research of Cellular and Molecular Toxicology, LRSBC, Faculty of Medical Dentistry, Universite´ de Monastir, Monastir, Tunisia, 3Chirurgie digestive A, CHU Montpellier - Service de Chirurgie Digestive II, Montpellier, France, 4CHU St Eloi Montpellier, Montpellier, France, 5Hoˆpital Paul Brousse, Centre He´patobiliaire, Inserm, U785/UPS, Villejuif, France Human liver stem cells localize in the adult liver within the canals of Hering, the most extreme parts of the biliary tree. They represent a facultative stem cell compartment that is activated during liver injury when hepatocytes are nolonger able to support liver regeneration. Their recruitment, which is demonstrated histologically by the ductular reaction, is triggered during chronic liver diseases including hepatitis and cirrhosis, and its extent correlates with the severity of the disease. Liver progenitor cells (LPC) are bipotent cells able to give rise to hepatocytes and to cholangiocytes. We previously isolated a population of

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non-parenchymal epithelial (NPE) cells from the liver of patients exhibiting no signs of liver failure. These cells were suspected to represent LPC because they express typical progenitor markers, exhibit a marked proliferative potential, differentiate into hepatocyte-like cells that express an intermediate hepato-biliary phenotype when cultured under appropriate conditions, and are able to engraft and differentiate in vivo after transplantation in mice liver in a regenerative context. Recent data suggest that certain hepatocellular carcinomas (HCC) with a high recurrence rate and poor survival could arise from hepatic progenitors. This presentation will address the effects of certain environmental compounds described as etiologic agents of HCC on the behavior of human hepatic progenitor cells.


ABSTRACTS

Poster Awards Finalist Absracts


A1. CYP2B6*6 ALLELE ALTERS KETAMINE’S PHARMACOKINETICS IN CHRONIC PAIN PATIENTS AND METABOLISM IN VITRO Yibai Li1, Janet K. Coller1, Mark R. Hutchinson2, Kathrin Klein3, Ulrich M. Zanger3, David Currow4, Barry Slon5, Kate Jackson5 and Andrew A. Somogyi1 1 Pharmacology, University of Adelaide, Adelaide, Australia, 2Physiology, University of Adelaide, Adelaide, Australia, 3 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany, 4Department of Palliative and Supportive Services, Flinders University, Adelaide, Australia, 5Monash Medical Centre, Melbourne, Australia Ketamine [2-(2-chlorophenyl)-2-(methylamino)cyclohexanone hydrochloride] is being increasingly used as an adjuvant analgesic in chronic pain management. Ketamine is primarily metabolized to norketamine (weakly active) by hepatic CYP2B6 and CYP3A41. The CYP2B6*6 allele is the most prevalent and clinically important variant of CYP2B6, which affects plasma concentrations and in vitro metabolism of many CYP2B6 substrates2. However, little is known regarding its effect on ketamine metabolism. The hypothesis of this study is that the CYP2B6*6 allele alters ketamine metabolism to norketamine. We examined the impact of the CYP2B6*6 allele on ketamine pharmacokinetics using steady-state blood samples obtained from 23 chronic cancer pain patients who had received 100 mg racemic ketamine by continous 24 hour subcutaneous infusion, and on metabolism of ketamine enantiomers in cDNA-expressed CYP2B6.1 and CYP2B6.6 variant and human liver microsomes (HLMs) from 11 donors that were selected based on CYP2B6 genotype. Plasma concentrations of ketamine and norketamine and the kinetics of norketamine formation from separate ketamine enantiomers were determined and compared between CYP2B6 genotypes. The two-enzyme Michaelis-Menten model best fitted the HLM kinetic data. The intrinsic clearance rate for both ketamine enantiomers by the high affinity enzyme (CYP2B6) in HLMs with CYP2B6*1/*1 genotype (n = 4) were at least 2- and 6-fold higher, respectively, than those with CYP2B6*1/*6 (n = 4) and CYP2B6*6/*6 genotypes (n = 3) (Jonckheere-Terpstra p = 0.008 and 0.001 for S- and R-ketamine, respectively). The intrinsic ketamine clearance rates for CYP2B6.1 were approximately 2-fold higher than those for CYP2B6.6. At 100 mg dose, the plasma ketamine clearance rate was significantly higher in patients with CYP2B6*1/*1 genotype (861 290 [mean SD] ml/min, n = 10) compared with those with CYP2B6*1/ *6 (637 135 ml/min, n = 10) and CYP2B6*6/*7 genotypes (350 ml/min, n = 1) [Jonckheere-Terpstra p = 0.016]. A significant gene-dose effect in plasma norketamine/ketamine concentration ratio was also found (1.2 0.4 for *1/*1, 0.6 0.2 for *1/*6, and 0.22 for *6/*7, Jonckheere-Terpstra p = 0.001). The human liver microsomal results indicate an impact of the CYP2B6*6 allele on ketamine metabolism, which supports the clinical data. These results suggest that variability in ketamine efficacy and adverse effects may be significantly related to the CYP2B6*6genotype.

References 1 Yanagihara, Y. et al. Involvement of CYP2B6 in n-demethylation of ketamine in human liver microsomes. Drug Metab Dispos 29, 887–890 (2001). 2 Turpeinen, M. & Zanger, U. M. Cytochrome P450 2B6: function, genetics, and clinical relevance. Drug Metabol Drug Interact 12, 1–13 (2012).

A2. GLUICAT: A NOVEL LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY-BASED METHOD FOR PROFILING PROTEIN GLUTATHIONYLATION Chun Yip Chan1, Brook L. Nunn2, David R. Goodlett3, Siew Kwan Koh4, Lei Zhou4 and Eric Chun Yong Chan5 1 Department of Pharmacy, National University of Singapore, Singapore, Singapore, 2University of Washington, Seattle, WA, Singapore, 3Medicinal Chemistry, University of Washington, Seattle, WA, 4Singapore Eye Research Institute, Singapore, Singapore, 5Pharmacy, National University of Singapore, Singapore, Singapore


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Proteins can be modified post-translationally in response to cellular stress, via oxidation, nitrosylation and glutathionylation of cysteine thiols. Protein glutathionylation induced by reactive drug metabolites lead to perturbations in protein function and cellular processes, giving rise to drug-induced toxicity. While several approaches have been developed to detect protein glutathionylation, involving either metabolic labelling using 35S-cysteine, chemical labelling using biotinylated-N-ethyl maleimide, or Western blotting using anti-glutathione antibody, these gel-based proteomics methods are unsatisfactory as they are inherently limited in terms of separation, sensitivity, dynamic range and reproducibility. In this work, we aimed to develop a gel-free, liquid chromatography-mass spectrometry (LC/MS)-based proteomics method, termed GluICAT, to profile protein glutathionylation. The GluICAT method was based on modifications of the method by Leichert et al.1 to allow differential labelling of reduced and glutathionylated cysteine thiols with light (12C) and heavy (13C) isotope-coded affinity tags (ICAT) respectively. Reduced thiols were directly labelled with light ICAT, while glutathionylated thiols were specifically deglutathionylated using an enzyme, glutaredoxin-1, before labelling with heavy ICAT. Being an enzymatic reducing agent, glutaredoxin-1 does not tolerate denaturing protein solubilizing buffers with high SDS and urea content. An important innovation was the optimization of the filter-aided sample preparation (FASP) method to substitute these buffers with glutaredoxin-1-friendly ammonium bicarbonate, permitting gentle, non-denaturing conditions during the deglutathionylation step. Differentially labelled ICAT-tagged peptides were detected and quantified using LC/MS, where the glutathionylation status of proteins was determined from the heavy to light (H:L) ICAT ratio, with a high H:L ratio indicating extensive glutathionylation. The results indicated that GluICAT successfully detected glutathionylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Native GAPDH yielded a H:L ratio of 0.38, suggesting that the native protein was in an unglutathionylated state, while glutathionylated GAPDH had a H:L ratio of 8.84, 23-fold higher than that of native GAPDH. When glutaredoxin-1 was omitted, glutathionylated GAPDH yielded a lower H:L ratio of 3.57, indicating that in the absence of the enzyme no deglutathionylation and labelling with heavy ICAT took place. Resolution, identification and quantification of proteins were achieved using liquid chromatography-mass spectrometry (LC/MS), circumventing the need for gel electrophoresis, allowing us to exploit the high reproducibility, large dynamic range and unbiased detection of hydrophobic or hydrophilic proteins by LC/MS. In summary, the GluICAT method is the first in-solution, LC/MS based approach to allow profiling of glutathionylated cysteine proteins. The application of this method to profile global protein glutathionylation in response to reactive drug metabolites will enhance our mechanistic understanding of adverse drug reactions. (405 words) References: 1. Leichert, L. I. et al. Quantifying changes in the thiol redox proteome upon oxidative stress in vivo. PNAS 105, 8197–8202, (2008).

A3. THE IMPORTANCE OF 5A-REDUCTASE GENE POLYMORPHISMS ON CIRCULATING AND INTRAPROSTATIC ANDROGENS IN PROSTATE CANCER Isabelle Laverdiere1, E´ric Le´vesque2, Louis Lacombe2, Patrick Caron1, Me´lanie Rouleau1, Ve´ronique Turcotte1, Bernard Teˆtu2, Yves Fradet2 and Chantal Guillemette1 1 Centre Hospitalier Universitaire de Que´bec Research Center, Faculty of pharmacy, Laval University, Que´bec, QC, Canada, 2 Centre Hospitalier Universitaire de Que´bec Research Center, Faculty of medecine, Laval University, Que´bec, QC, Canada Prostate cancer (PCa) is a heterogeneous disease at the molecular and clinical levels reinforcing the need to individualize the patient care. The importance of androgen in cancer initiation and progression is clearly established at all disease stages. Moreover, 5a-reductase inhibitors have shown potential clinical efficacy as chemopreventive medications in previous studies. The 5a-reductase types 1 and 2 convert testosterone into DHT, a potent androgen agonist. We previously identified polymorphisms in SRD5A1 and SRD5A2 genes associated with an altered risk of biochemical recurrence after radical prostatectomy in localized prostate cancer (PCa).1 To gain insights into SRD5A biological effects, we examined the relationship between SRD5A prognostic markers and endogenous sex-steroid levels measured by mass spectrometry methods in 500 plasmas and corresponding prostatic tissues from Caucasians diagnosed with PCa. The plasma samples were obtained the morning of the prostatectomy and prostatic tissues collected during the surgery. We report that five of the seven SRD5A markers differentially affect sex-steroid profiles of dihydrotestosterone (DHT) and its metabolites in both circulation and prostatic tissues of PCa patients. Remarkably, a 32% increase in intraprostatic testosterone levels was observed in the presence of the high-risk SRD5A rs2208532 polymorphism. Besides, SRD5A2 markers are associated predominantly with circulating levels of inactive glucuronides. Indeed, the rs12470143 SRD5A2 protective allele is associated with high circulating 3a-diol-17G levels as opposed to lower levels of both 3a-diol-17G and ADT-G observed with the rs2208532 SRD5A2 risk allele. Moreover, SRD5A2 rs676033 and rs523349 (V89L) risk variants, in strong linkage disequilibrium, are related to greater circulating levels of 3a-diol-3G. The SRD5A2 rs676033 variant further correlates with enhanced intraprostatic exposure to 5a-reduced steroids (DHT and its metabolite 3b-diol). Similarly, the SRD5A1 rs166050C risk variant is associated with greater prostatic exposure to DHT while no association is noted with circulating steroids. In PCa patients, data support the influence of 5a-reductase germline polymorphisms on the hormonal milieu that likely explain their connection to prognostic. The utility of understanding the impact of SRD5A germline determinants on androgen exposure is that it could potentially be used to identify subgroups of patients, with a particular androgen microenvironment profile, that would most benefit from 5-ARI. Therefore, protective and high-risk germline SRD5A alleles associated with changes in hormone levels could possibly lead to a more personalized approach to

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refine 5-ARI therapy, especially early in disease course. Supported by The Canada Research Chair Program, Cancer Research Society and Fonds de recherche du Quebec - Sante (FRQ-S).

Reference 1. Audet-Walsh E, Bellemare J, Nadeau G, et al: SRD5A Polymorphisms and Biochemical Failure After Radical Prostatectomy. Eur Urol 60:1226–34, 2011

A4. PHARMACOGENOMIC PREDICTORS OF GRAFT-VS-HOST DISEASE AND SURVIVAL AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION IN CANCER PATIENTS


Isabelle Laverdiere1, Ryad Tamouza2, Pascale Loiseau3, Fe´lix Couture1, Alain Filion1, Marc Lalancette1, Dominique Charron2, Chantal Guillemette1, Ge´rard Socie´3 and E´ric Le´vesque1 1 Research Centre of the Centre Hospitalier Universitaire de Que´bec and Laval University, Que´bec, QC, Canada, 2INSERM U940 Hoˆpital Saint-Louis and University Paris Diderot, Paris, France, 3INSERM U728 Hoˆpital Saint-Louis and University Paris Diderot, Paris, France Graft-vs-host disease (GvHD) is one of the main obstacles limiting the success of allogeneic hematopoietic stem cell transplantation (HSCT) and is associated with high mortality rate. Following transplantation procedures, effective immunosuppression is mandatory to prevent GvHD and mortality. We tested the prognostic value of inherited genetic variations in genes related to metabolism, transport and action of immunosuppressive drugs commonly used to prevent GvHD. This hypothesis was tested in a cohort of 104 sibling donorrecipient pairs totalling 208 genomes. A total of 219 polymorphic markers across 20 candidate genes related to pharmacokinetics and pharmacodynamics pathways of methotrexate and cyclosporine immunosuppressive drugs were studied. Genetic variations in 14 of the 20 candidate genes studied were significantly associated with patients’ clinical outcomes after the HSCT. Among all markers tested, 28 SNPs in the recipients and 25 SNPs in the donors were associated with at least one of the outcomes. The genetic status in ABCC1, ATIC, CYP3A4/5, MTHFR and NFAT exhibit a remarkable influence on GvHD occurrence. Indeed, an increased risk of acute GvHD is reported with genetic markers in ABCC1 (HR = 4.2, p = 0.02), ATIC (HR = 6.8, p50.01) and CYP3A4/5 (HR = 3.9–8.4, p50.05). Donor MTHFR SNPs are associated with a reduced risk of acute GvHD (HR = 0.2–0.3, p50.02), while recipient MTHFR genotypes influence transplant-related mortality (HR = 3.4–8.4, p50.02). In addition, variations in NFAT1/2 genes are inversely correlated with the risk of death (HR = 3.7–35.4 and HR = 0.2–0.4, p50.05, respectively). By integrating biologically relevant genes, this work emphasizes the potential role of pharmacogenomic markers in determining outcomes of HSCT patients. A replication study is ongoing for the most promising markers in a much larger cohort of over 900 donor-recipient pairs. Identification of such biomarkers assessed before transplantation can help personalized patient care in order to prevent GvHD and improve survival. This work was supported by Canadian Institutes of Health Research and Canada Research Chair program.

A5. ENHANCED SYNTHESIS OF CHOLESTEROL AND BILE ACID IN THE LIVER OF CYP3AKNOCKOUT MICE Mari Hashimoto1, Kaoru Kobayashi1, Mio Watanabe1, Yasuhiro Kazuki2, Shoko Takehara3, Asumi Inaba1, Shin-ichiro Nitta4, Naoto Senda4, Mitsuo Oshimura2 and Kan Chiba1 1 Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, 2 Graduate School of Medical Science, Tottori University, Yonago, Japan, 3Chromocenter Inc., Japan, 4Analytical Technology Research Department, Mitsubishi Chemical Medience Corporation, Tsukuba, Japan CYP3A enzymes metabolize not only xenobiotics but also many endogenous compounds. It has been reported that CYP3A enzymes metabolize cholesterol to 25-hydroxy-cholesterol (25-HC). 25-HC is not only a precursor of bile acids but also a physiologically active oxysterol that suppresses sterol regulatory element binding protein-2 (SREBP-2) and down-regulates cholesterol biosynthesis. These findings imply that CYP3A enzymes play a role in cholesterol homeostasis. In order to clarify this possibility, we studied the effect of CYP3A-deficiency on cholesterol and bile acid synthesis using Cyp3a-knockout (Cyp3a/) mice. LC-MS/MS analysis demonstrated that 25-HC levels in the livers of Cyp3a/ mice were lower than those of wild-type (WT) mice. Western blot analysis showed nuclear SREBP-2 protein levels in the livers of Cyp3a/ mice were higher than those of WT mice. Results of ChIP assay showed that binding of SREBP-2 to sterol regulatory element located in a promoter region of the Hmgcs1 gene was more abundant in the livers of Cyp3a/ mice. Quantitative real-time PCR analysis also showed mRNA expression levels of SREBP-2 target gene (i.e., HMG-CoA synthase 1 and Squalene epoxidase) in the livers of Cyp3a/ mice were higher than those of WT mice. In addition, plasma lathosterol levels, an indicator of cholesterol biosynthesis, in Cyp3a/ mice showed a trend toward higher than those in WT mice. On the other hand, total cholesterol levels

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in the livers of Cyp3a/ mice were lower than those of WT mice. To investigate the mechanism which caused reduction of hepatic cholesterol in Cyp3a/ mice, we examined effect of CYP3A-deficiency on bile acid synthesis from cholesterol. The mRNA expression levels of enzymes involved in bile acid synthesis (i.e. CYP7A1, CYP8B1 and CYP7B1) in the livers of Cyp3a/ mice were higher than those of WT mice. Plasma concentration of 7a-hydroxy-4-cholesten-3-one, an indicator of bile acid synthesis, was also higher in Cyp3a/ mice than in WT mice. Furthermore, total bile acid levels in the livers of Cyp3a/ mice were higher than those of WT mice. In conclusion, deficiency of CYP3A enhanced cholesterol synthesis through the activation of SREBP-2, which appeared to be triggered by the reduction of 25-HC and cholesterol in the liver of Cyp3a/ mice. In addition, synthesis of bile acid from cholesterol was enhanced in the liver of Cyp3a/ mice, which appeared to contribute to the reduction of cholesterol in the liver.

A6. DUAL ROLE FOR UDP GLUCURONOSYLTRANSFERASE (UGT) 1A SPLICE VARIANT FORMS 2 (I2) IN DEFINING PHARMACOLOGICAL RESPONSE AND IN THE OXIDATIVE STRESS PATHWAY


Me´lanie Rouleau, Joannie Roberge, Judith Bellemare and Chantal Guillemette Pharmacogenomics Laboratory, CHU of Quebec Research Center and Faculty of Pharmacy, Laval University, Quebec, QC, Canada Interindividual variation in the conjugative drug metabolism by UDP-glucuronosyltransferase enzymes (UGTs) has a clear influence on drug bioavailability and biological responses to drugs. Alternative splicing is a source of high variability at the posttranscriptional level for human UGTs. The UGT1A gene is alternatively spliced resulting in active UGT1A enzymes named isoforms i1 and regulatory proteins (isoforms i2) that negatively modulate cellular glucuronidation activity by direct proteinprotein interaction with i1 enzymes. In this study, we initially defined the potential pharmacological influence of endogenous i2 using short hairpin shRNA-mediated stable knockdown (KD) and the HT115 colon cancer cell line expressing both classes of i1 and i2 isoforms. Depletion of i2 (49%; p = 0.005) resulted in an enhanced formation of drug-glucuronide (126%; p = 0.008) and a significant improved cell viability in response to the anti-cancer agent SN-38 (IC50 0.45 mM vs. 0.22 mM; p = 0.006); demonstrating for the first time a clear pharmacological impact of variation in endogenous levels of i2. We then applied nonbiased quantitative approaches to evaluate global changes associated with i2 depletion for gene expression (using microarray) and at the protein level (using stable isotope labeling of amino acids in cell culture coupled to mass spectrometry (SILAC-MS)). We observed a significant impact on key cellular processes such as those related to oxidative stress. For instance, i2 depleted cells displayed an increased expression of antioxidant enzymes namely hemoglobin alpha (HBA) and superoxide dismutase (SOD1) which is further associated with a reduced reactive oxygen species (ROS) production (30%; p50.05). This previously unknown capacity of i2 to affect oxidative stress pathways evokes its potential property as regulator of antioxidant enzymes. To test this hypothesis, we conducted unbiased interaction proteomics experiments to portray UGT protein complexes (IP-MS/MS) with endogenous proteins derived from human intestine and kidney tissues as the target. Indeed, while most protein complexes are usually isolated through the over-expression of an exogenous tagged protein within cells, instead we studied human tissues and specific antibodies against i1 or i2 proteins to establish their associated interactomes. We exposed for the first time specific protein partners of i2 such as the antioxidant proteins catalase (CAT) and peroxiredoxin-1 (Prdx1) that do not interact with UGT1A enzymes (i1 isoforms). Our work provides unprecedented evidence of a new biological function for UGT1A_i2 splice forms in the modulation of oxidative stress and involving protein interaction networks comprising antioxidant enzymes, thus potentially regulating a variety of biological phenomena. Supported by The Canada Research Program, CIHR and NSERC.

A7. INTERINDIVIDUAL VARIABILITY OF CYP2C19-CATALYZED DRUG METABOLISM DUE TO DIFFERENCES IN THE DIPLOTYPES Yoshiyuki Shirasaka1, Amarjit S. Chaudhry2, Timothy Wong1, Justina C. Calamia1, Nina Isoherranen1, Allan E. Rettie3, Erin G. Schuetz2 and Kenneth E. Thummel1 1 Department of Pharmaceutics, University of Washington, Seattle, WA, 2Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, TN, 3Departments of Medicinal Chemistry, University of Washington, Seattle, WA CYP2C19 is a drug-metabolizing enzyme in the cytochrome CYP450 family that is encoded by the CYP2C19 gene. It is involved in the metabolism and the drug-drug interactions for several important drugs including proton pump inhibitors, antiepileptics, anti-platelet drugs, and anti-depressants. Genetic polymorphism affects CYP2C19 expression, with poor metabolizers exhibiting no CYP2C19 protein accumulation and function as a result, for example, of homozygosity for the CYP2C19*2 (19154G4A) allele. In contrast, the CYP2C19*17 (806C4T) allele is associated with an increase in transcriptional activity, resulting in ultra-rapid metabolism of CYP2C19 substrates. These polymorphisms contribute to interindividual variability in the disposition of CYP2C19 substrates in humans. Interestingly, the CYP2C19*2 and CYP2C19*17 loci are not in linkage disequilibrium. Moreover, prospective studies aimed at understanding the impact of a resulting CYP2C19 diplotype on the CYP2C19 metabolic activity in individuals is still not well characterized. Hence, the purpose of this investigation was to determine the interindividual variability of CYP2C19-catalyzed drug metabolism that is due to differences in the gene


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diplotypes. Studying a panel of 347 human liver samples, the frequencies of the respective CYP2C19*1/*1, *1/*2, *2/*2, *1/*17, *17/*17, and *2/*17 diplotypes were 41.8% (n = 145), 15.9% (n = 55), 3.2% n = 11), 22.5% (n = 78), 7.2% (n = 25) and 9.5% (n = 33), respectively. To test whether the diplotype was associated with enzyme activity, CYP2C19 activity was measured using human liver microsomes (HLM) isolated from these genotyped liver samples and S-mephenytoin as a prototypical substrate. The Mann-Whitney test was performed for between-group comparisons. As expected, in the group of HLMs with a CYP2C19*2/*2 diplotype, the mean activity was only 7% of that found for the wild-type HLMs with a CYP2C19*1/*1 diplotype (p50.0001). The mean activity for the CYP2C19*1/*2 diplotype was 26% lower than that of the CYP2C19*1/*1 diplotype, although the difference did not reach statistical significance. Similarly, there was a trend for higher CYP2C19 activity in livers with a CYP2C19*1/*17 (1.2-fold) and *17/*17 (1.3-fold) diplotype, compared to the CYP2C19*1/*1 diplotype, but again the differences were not significant. Also, the metabolic activity of the CYP2C19*2/*17 diplotype livers was on average 26% lower than that of the CYP2C19*1/*1 diplotype livers, suggesting that there is not an equal offset of effects from the *2 and *17 alleles, but once again the difference was not significant. However, using a linear trend analysis, the rank order for enzyme activity (*17/*174*1/*174*1/*14*2/*174*1/*24*2/*2) was significant (p50.005) and consistent with a priori expectations. Inspection of the activity data in aggregate revealed considerable within-diplotype group variability, except for the CYP2C19*2/ *2 diplotype. Presence of less frequent gene variation [i.e., CYP2C19*4B (1A4G)] did not explain the extreme activity differences. Contributions to phenotypic variability from novel, rare CYP2C19 gene variation or common and rare variation in CYP2C19 regulatory genes should be considered, as well as differences in endogenous and exogenous activators of transcription. This work was supported in part by grants from NIH [U01 GM092676 and GM092666 and R01 GM094418] and JSPS Postdoctoral Fellowship for Research Abroad [H23-694].

A8. DETERMINATION OF SPECIES-DIFFERENCE IN MICROSOMAL METABOLISM OF AMITRIPTYLINE USING A PREDICTIVE MRM-IDA-EPI METHOD Ji-Yoon Lee1, Sang Yoon Lee1, KiHo Lee2, Soo Jin Oh3 and Sang Kyum Kim1 1 College of Pharmacy, Chungnam National University, Daejeon, South Korea, 2College of Pharmacy, Korea University, Sejong, South Korea, 3Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, South Korea To compare species differences in amitriptyline (AMI) metabolism among mouse, rat, dog, and human liver microsomes, we developed a method for simultaneous determination of metabolic stability and metabolite profiling using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) scanning. In the cofactordependent microsomal metabolism study, AMI was metabolized more rapidly in rat and human liver microsomes incubated with NADPH than UDPGA. In the simultaneous metabolic stability and metabolite profiling study, AMI incubated with NADPH þ UDPGA in rat, dog, or mouse liver microsomes disappeared rapidly with a half-life of 3.5, 8.4, or 9.2 min, respectively, but slowly in human liver microsomes with a half-life of 96 min. In total, 9, 10, 11, and 6 putative metabolites of AMI were detected in mouse, rat, dog, and human liver microsomes, respectively, based on mass spectrometric analyses. Kinetic analysis of metabolites in liver microsomes from each species over 120 min showed common metabolic routes of AMI, such as N-demethylation, hydroxylation, and glucuronidation, and subtle interspecies differences in AMI metabolism. The main metabolic routes in mouse, rat, dog, and human liver microsomes were hydroxylation followed by glucuronide conjugation, methyl hydroxylation, and N-demethylation, respectively. The MRM-IDA-EPI method can provide quantitative and qualitative information about metabolic stability and metabolite profiling simultaneously. Moreover, time course analysis of metabolites can not only eliminate false identification of metabolites, but also provide a rationale for proposed metabolic pathways. The MRM-IDA-EPI method combined with time course analysis of metabolites is useful for investigating drug metabolism at the early drug discovery stage.

A9. ABCC11/MRP8 POLYMORPHISMS AFFECT 5-FLUOROURACIL-INDUCED SEVERE LEUCOPENIA AND HEPATIC EXPRESSION Tarek M. S. Mohamed Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, Stuttgart, Germany The ABCC11 gene encodes multidrug resistance-associated protein (MRP) 8, a member of the ATP-binding cassette (ABC) superfamily of transporters. Since MRP8 acts as an efflux pump for 5-fluorodeoxyuridine monophosphate (5-FdUMP), the main active intracellular metabolite of 5-flourouracil (5-FU), we investigated whether ABCC11 functional polymorphisms play a role in severe toxicity of 5-FU. Genomic DNA from 672 cancer patients treated by 5-FU monotherapy and with documented toxicity according to World Health Organization criteria was genotyped for 12 tag SNPs capturing 111 SNPs spread over 65-kb encompassing the ABCC11 region. Tag SNPs included 5 coding synonymous SNPs, 2 coding non-synonymous SNPs and 5 intronic SNPs. Univariate and multivariate logistic regression analysis was applied to identify significant associations. Functional impact of ABCC11 polymorphisms on mRNA and protein expression was assessed in a Caucasian human liver cohort (n = 150) and by recombinant expression of MRP8 protein variants. Univariate and multivariate analysis considering sex, mode

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of administration, folinic acid administration, and polymorphisms in Dihydropyrimidine dehydrogenase (DPYD), Thymidylate synthase (TYMS) and Methylenetetrahydrofolate reductase (MTHFR) genes as covariates identified rs17822471 (G4A, T546M) as risk factor of severe leucopenia (p = 0.021, OR [95%CI] = 3.31 [1.26, 8.66]) but not of other major toxicity types. We validated the functionality of SNP T546M by showing its association with 1.7-fold lower protein expression in a human liver donor cohort. Recombinant expression of the protein variant in comparison to the wild type protein confirmed the deleterious effect of the T546M variant on protein expression. As leucopenia is an abnormal reduction in the count of circulating white blood cells, we propose that genetic MRP8 low expressors accumulate 5-FdUMP, the anabolic active 5-FU metabolite, in normal leucocytes and normal bone marrow blasts, leading to highly cytotoxic effects to bone marrow blasts and leucocytes and eventually to severe leucopenia. Our data indicates that the well known but still poorly characterized transporter MRP8 is polymorphically expressed in human liver and is associated with 5-FU induced severe leucopenia. Our explorative findings require further validation in an appropriate clinical 5-FU replication cohort. The study was supported by the German Federal Ministry of Education and Science (BMBF Virtual Liver Network grant 0315755, and grant 03 IS 2061C of the GANI_MED program), the 7FP EU Initial Training network program ‘‘FightingDrugFailure’’ (GA-2009-238132), and by the Robert-Bosch Foundation, Stuttgart, Germany.

A10. ABCC5 MODULATES SYSTEMIC EXPOSURE OF SN-38-GLUCORONIDE IN METASTATIC COLORECTAL CANCER PATIENTS TREATED WITH IRINOTECAN Wendy A. Teft1, John Lenehan2, Stephen Welch3 and Richard B. Kim4 1 Medicine, Division of Clinical Pharmacology, The University of Western Ontario, London, ON, Canada, 2Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON, Canada, 3Oncology, The University of Western Ontario, London, ON, Canada, 4Division of Clinical Pharmacology, The University of Western Ontario, London, ON, Canada Background: Irinotecan, a chemotherapy agent commonly used to treat metastatic colorectal cancer, is a prodrug that is bioactivated to SN-38 and functions as a topoisomerase I inhibitor. It undergoes detoxification via glucuronidation to form SN-38-glucuronide (SN-38G). Severe dose-limiting toxicities including neutropenia and diarrhea are a major barrier to its use. Studies have investigated genetic and non-genetic contributors to these toxicities and determined that interpatient variability in attained SN-38 plasma levels and gastro-intestinal (GI) exposure plays an important role. Studies have shown that polymorphisms in UGT1A1 (*28) and the uptake transporter OATP1B1 (*5) are associated with increased systemic SN-38 exposure. However, many patients experiencing toxicity, especially GI toxicity, do not harbor these SNPs. Therefore, we hypothesized that additional SNPs in OATP1B1 and OATP1B3, and efflux transporters ABCB1 (MDR1), ABCC2 (MRP2) and ABCC5 (MRP5) contribute to the observed interpatient variability in SN-38 and SN-38G plasma levels. Methods: Patients (n = 126) receiving irinotecan for the treatment of metastatic colorectal cancer were enrolled from the London Regional Cancer Program (London, Ontario). A single-blood sample was obtained immediately following the end of the irinotecan infusion and plasma levels of irinotecan, SN-38 and SN-38G were measured directly by LC-MS/MS. DNA was extracted for genotyping by TaqMan assays. Stepwise linear regression modeling was performed to determine significant associations between transporter polymorphisms and dose-normalized plasma concentrations. Results: Following adjustment for age and gender, we observed plasma levels of SN-38 were significantly associated with UGT1A1*28 and OATP1B1*5. Indeed, patients with an increasing number of combined variant alleles had significantly higher SN-38 plasma levels, suggesting an increased risk of toxicity. Additionally, we observed for the first time, that patients carrying the ABCC5 (rs562) C allele had significantly lower SN-38G plasma levels, suggesting a likely increase in SN-38G retention in the liver and intestine. Conversion of SN-38G to SN-38 via intestinal b-glucuronidases may place these patients at an elevated risk for GI toxicity. ABCC5 was also significantly associated with lower SN-38G/Irinotecan and SN-38G/SN-38 plasma ratios. Polymorphisms in OATP1B3, ABCB1 and ABCC2 were not associated with irinotecan, SN-38 or SN-38G levels. Conclusions: In addition to UGT1A1*28 polymorphism, it is clear that polymorphisms in drug uptake and efflux transporters significantly contribute to toxicities observed during irinotecan-based chemotherapy. We outline new data which link genetic variation in ABCC5 to modulation of in vivo exposure to SN-38G. Our findings may prove to be clinically useful for predicting patients at risk for GI toxicity during irinotecan therapy.

A11. ROLE OF KRU¨PPEL-LIKE FACTOR 9 (KLF9) IN CYTOCHROME P450 2D6 (CYP2D6) INDUCTION DURING PREGNANCY Kwi Hye Koh1, Xian Pan2, Ai-Ming Yu3 and Hyunyoung Jeong4 1 Department of Pharmacy Practice, University of Illinois at Chicago, Chicago, IL, 2Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, 3Department of Pharmaceutical Sciences, University at Buffalo, SUNY, Buffalo, NY, 4 Pharmacy Practice and Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL CYP2D6, a major drug-metabolizing enzyme, is responsible for metabolism of 25% of marketed drugs. Clinical data indicate that pregnancy increases metabolism of CYP2D6 substrates by 20–50%. However, the underlying mechanisms of CYP2D6 induction during pregnancy remain completely unknown. The objective of this study is to identify transcription factors


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responsible for CYP2D6 induction during pregnancy and characterize the underlying regulatory mechanism. In CYP2D6humanized transgenic mice (tg-CYP2D6), pregnancy induced mRNA and protein expressions, indicating that tg-CYP2D6 may serve as a potential model to study CYP2D6 induction during pregnancy. To elucidate the mechanisms underlying CYP2D6 regulation during pregnancy, a list of genes differentially expressed in livers of pregnant vs. nonpregnant tg-CYP2D6 mice was compiled through microarray experiments followed by verification of the results by qRT-PCR. This led to identification of the following 7 transcription factors upregulated during pregnancy: Atf5, Egr1, Foxa3, Junb, Klf9, Klf10 and Rev-erba. Although individual transcription factors did not increase promoter activity of CYP2D6, KLF9 significantly increased CYP2D6 promoter activity by potentiating transactivation of HNF4a, a known transcriptional activator of basal CYP2D6 expression. Using a series of sequential deletion luciferase constructs of CYP2D6 promoter, we found that 100/þ90 region of CYP2D6 promoter is responsible for potentiation of HNF4a activity on CYP2D6 by KLF9. Results from mutation assays of CYP2D6 promoter indicated that KLF putative binding motif at 17/14 was critical in the potentiation of HNF4a transactivation by KLF9. Results from electrophoretic mobility shift assay demonstrated binding of KLF9 to the putative KLF binding motif on CYP2D6 promoter. Results from chromatin immunoprecipitation assay showed that recruitment of KLF9 and HNF4a to CYP2D6 proximal promoter in livers of tg-CYP2D6 mice was increased during pregnancy. Taken together, our data suggest that increased expression of KLF9 may be in part responsible for CYP2D6 induction during pregnancy via potentiation of HNF4a transactivation of CYP2D6. For this, direct binding of KLF9 to the CYP2D6 promoter appears necessary. These results provide potential mechanisms underlying enhanced metabolism of CYP2D6 substrates during pregnancy and may contribute in better understanding of CYP2D6 regulation and thus improving drug therapy of CYP2D6 substrates in pregnant women. This study was supported by NIH (R01HD065532).

A12. ADME-RELATED MICRORNAS IN HUMAN LIVER: ASSOCIATION WITH NON-GENETIC FACTORS AND EFFECTS ON DRUG METABOLISM Jessica K. Rieger1, Benjamin Kandel1, Kathrin Klein1, Stefan Winter1, Georg Damm2 and Ulrich M. Zanger1 1 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany, 2Department of General-, Visceral- and Transplantation surgery, Charite´ University Medicine Berlin, Berlin, Germany MicroRNAs (miRNA) are small non-coding RNA molecules which regulate genes at the post-transcriptional level. They are believed to regulate about 60% of all human genes and play important roles in nearly all cellular processes including drug metabolism. Several ADME (absorption, distribution, metabolism, excretion) genes e.g. CYP3A4, CYP2E1, HNF4a, have been shown to be regulated by miRNAs. However, the extent of regulation by miRNAs in the context of other genetic and non-genetic influencing factors remains unclear. To investigate the impact of non-genetic factors on miRNA expression and to elucidate a possible connection to drug metabolism, we selected a set of 56 microRNAs predicted to target the 30 -UTR of ADME genes to assess their hepatic expression levels by stem-loop real-time RT-PCR in a well-documented human liver tissue cohort (n = 92). Statistical analysis considering liver donor meta-data was performed to elucidate the impact of these factors on miRNA expression. Furthermore, we implemented functional studies in primary human hepatocytes (PHH) to examine the effect of altered miRNA expression on ADME genes. Therefore PHH were transfected either with miRNA mimics, antagomirs or siRNAs. Our data suggest that expression of certain miRNAs is strongly affected by hepatic pathophysiological conditions and is linked to the expression of certain ADME genes. In particular, several miRNAs including miR-21, miR-34a, and miR-130b were pronouncedly increased (52.5 fold) in cholestatic livers as well as during acute phase response. Functional studies of miR34a in PHH confirmed HNF4a as a target and indicate direct or indirect regulation of other nuclear receptors including PXR and CAR and their target genes. Furthermore, comparable expression changes of PHH transfected with either a siRNA targeting HNF4a or a miR-34a mimic were observed. This study identified several miRNAs including miR-34a that are associated with cholestasis and inflammation suggesting their potential usefulness as liver disease biomarkers. Moreover, miR-34a may also be involved in the pathophysiological process of liver diseases and interfere with drug metabolism by targeting crucial regulating factors such as HNF4a, PXR and CAR. The study was supported by the German Federal Ministry of Education and Science (BMBF Virtual Liver Network) and by the Robert Bosch Foundation, Stuttgart, Germany.


ABSTRACTS

Poster Abstracts


P1. DEVELOPMENT OF A NOVEL QUANTITATIVE LC-MS/MS BASED ASSAY FOR ALBUMIN-ACETAMINOPHEN ADDUCTS Andre´ LeBlanc, Souade Ben Haddou, Tze Chieh Shiao, Rene´ Roy and Lekha Sleno Chemistry, Universite´ du Que´bec a` Montreál, Montreal, QC, Canada Acetaminophen toxicity is the most common cause of acute liver failure in the United States. The hepatotoxicity of acetaminophen is caused by an electrophilic reactive metabolite of acetaminophen, N-acetyl p-benzoquinone imine or NAPQI, that forms covalent adducts with nucleophilic thiol groups in liver protein, causing hepatic necrosis. Serum albumin is known to be covalently modified by NAPQI and is present at high concentrations in the blood stream. Serum albumin is therefore a potential biomarker to assess the levels of protein modification by NAPQI. To date, no quantitative method exists for NAPQI modified serum albumin. We have developed a novel method for the absolute quantitation of modified serum albumin from in vivo samples. This method was developed for assessing levels of albumin adducts in rat plasma. RSA is digested with pepsin to liberate the active site peptide LLQCKYEE. The quantitation of this peptide in its modified forms was performed by microflow LC-MRM on a 5500 QTRAP quadrupole linear ion trap mass spectrometer. In order to design an appropriate standard, we have synthesized an iodo-analog of acetaminophen (iodo-APAP), an efficient cysteine modifier. This analog is used to quantitatively modify known quantities of RSA to be used as the standard for an external calibration curve. The resulting peptides are positional isomers of NAPQI modified HSA peptides, assuring that the target peptides’ chromatographic, ionization and fragmentation behavior are as similar as possible. Albumin is semi-purified from plasma using a molecular weight cut-off filter and then combined with a known amount of internal standard albumin (methylated iodo-IPAP-labeled RSA). Following digestion, peptides were fractionated to enrich the sample with the peptide of interest prior to LC-MRM analysis. The method was also adapted to human serum albumin (HSA), with a slightly different peptide being produced following pepsin digestion. The sensitivity and linear dynamic range of the calibration curve was assessed in presence of background albumin digest. During method development, we tested different LC-MS/MS platforms including the 5600 TripleTOF in IDA-MS/MS as well as MRMHR modes was compared to the 5500 QTRAP system in MRM mode. Furthermore, the sensitivity of the method using conventional LC flow rates on a Shimadzu Nexera UHPLC was being compared to micro-flow on an Eksigent microLC system.

P2. APPLICABILITY OF DIFFERENT SOFT NUCLEOPHILES FOR SCREENING REACTIVE METABOLITES RESULTING FROM MICROSOMAL ACTIVATION OF FURAN MIXTURES AND CIGARETTE SMOKE Arno Knorr, Audrey Debrick, Quentin Dutertre, Martin Almstetter, Elyette Martin and Mark C. Bentley Philip Morris International R&D, Philip Morris Products S.A., Neuchatel, Switzerland The lung is the predominant target organ for numerous types of chemically-induced damage related to smoking, due to its direct exposure to aerosol constituents. Although the lung has significant capability for biotransforming such compounds with the aim of reducing any potential toxicity, in some instances this biotransformation can result in the generation of more reactive, and often more toxic, metabolites. Knowledge regarding these reactive metabolites will enable the establishment of a link between potentially harmful smoke constituents and disease relevant mechanisms or pathways (i.e., oxidative stress, inflammation, apoptosis. . .). Due to the instability and therefore short lifetime of reactive electrophilic intermediates, a widely accepted approach for their analysis is by trapping with nucleophiles to generate more stable adducts and subsequent measurement by liquid chromatography coupled to mass spectrometry (LC/MS). Well established procedures using glutathione as a nucleophile for trapping soft electrophiles are often chosen due to a direct relevance for biological systems. However, the use of glutathione has significant limitations associated with the structural elucidation of the conjugated electrophilic compounds, which thereby limits its applicability for generic screening approaches for phase I activation of xenobiotics in complex mixtures.

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In order to define the most suitable approach for screening, identifying and characterizing reactive intermediates generated by microsomal activation of smoke constituents, four different nucleophiles were evaluated for use within the test system, namely glutathione, cysteine, mercaptoethanol and 3-aminopropan-1-thiol. Reaction kinetics towards acrolein, applicability for microsomal incubations for phase I oxidation of furan and 2-methylfuran, and the ability to provide structural information using mass-spectrometry were considered for each nucleophile. The nucleophile 3-aminopropan-1-thiol was also assessed for reaction selectivity, demonstrating its ability to distinguish between nonreactive and reactive electrophiles, since there was no information available in the public domain regarding its strength as a nucleophile for this purpose. The ability to perform structural elucidation of reactive intermediates using LC/MS was highly dependent upon the nucleophile used. The structural information that could be derived from each specific type of nucleophile-adduct using high resolution mass spectrometric data was compared. Mass-spectrometric fragmentation patterns, diversity of conjugation reactions, including intramolecular rearrangement reactions, and the feasibility to predict these different nucleophilic conjugates using the in silico metabolite prediction tool Meteor Nexus were considered.


P3. COMPARISON OF DRIED BLOOD SPOT AND PLASMA CONCENTRATIONS OF NIFEDIPINE IN HEALTHY VOLUNTEERS Sara K. Quinney1, Ahmed N. Mohamed2, Andrea R. Masters3 and David R. Jones3 1 Department of OB/GYN, Indiana University, Indianapolis, IN, 2Pharmaceutical Sciences, College of Pharmacy Qatar University, Doha, Qatar, 3Department of Medicine, Indiana University, Indianapolis, IN Dried blood spot (DBS) assays provide advantages over traditional venous plasma collection, e.g. small sample volume, ease of storage and shipment, and drug stability. We developed a DBS assay for nifedipine (and oxidized nifedipine) and conducted a clinical study to determine if the DBS could be used in place of plasma for pharmacokinetic studies. The primary objectives were to determine the relationship between 1) DBS from venous blood and venous plasma and 2) venous DBS and capillary DBS. This study was conducted as a sub-aim of a study evaluating the effect of fluid volume on nifedipine absorption. The study was approved by the IUPUI IRB and all participants gave written informed consent. Six healthy volunteers took 10 mg immediate release nifedipine by mouth on two occasions (with 50 mL or 250 mL of water) separated by at least three days. DBS samples were collected via venous puncture or finger stick into heparinized tubes, and 15 mL aliquouts transferred to Whatman 903 paper. A 6 mm punch of the DBS was extracted in ethyl acetate, with nitrenpidine as internal standard. Plasma was isolated from the remaining venous blood and extracted with ethyl acetate. Appropriate standards were prepared for DBS and plasma and samples were protected from light. Quantification was conducted on a Thermo TSQ Quantum Ultra MS/MS linked to a Leap CTC Autosampler. Mobile phase consisted of acetonitrile:5 mM ammonium acetate with gradient infusion from 10% to 80% acetonitrile. Nifedipine (347.1/315.1) and oxidized nifedipine (345.1/284.0) had limits of quantification of 1 ng/mL from the DBS and 0.01 and 0.1 ng/mL, respectively, from plasma. Pharmacokinetic parameters did not differ between the two phases of the study, so data were combined for this analysis. Venous DBS concentrations of nifedipine were correlated with plasma concentrations, although there was a large degree of scatter at higher concentrations (r2 ¼ 0.62, p50.001). The median ratio of venous DBS to plasma nifedipine concentration was 0.44 (range 0.06–2.31l). Capillary DBS concentrations of nifedipine were similar to venous DBS concentrations, with a median capillary to venous ratio of 0.91 (range 0.26–2.7) and r2 of 0.89 (p50.001) after exclusion of one outlier. Plasma concentrations were higher than DBS concentrations. Mean sd plasma AUC(0–1) was 151 30 ng h/ml (CV ¼ 20%) from plasma and 72 36 ng (CV ¼ 50%) h/ml for venous DBS (p50.01). Median DBS:Plasma ratio of oxidized nifedipine was 0.72 (range 0.30–14), r2 ¼ 0.82 (p50.001). Despite using a validated assay to determine nifedipine and oxidized nifedipine concentrations, when applied to clinical samples the DBS method was associated with greater variability.

P4. MULTIPLE-DOSE PHARMACOKINETICS OF PIPERPHENTONAMINE HYDROCHLORIDE, A NOVEL CALCIUM SENSITIZER, IN HEALTHY VOLUNTEERS Min Li1, Aixin Shi1, Wei Xue1, Yang Li1, Kexin Li1, Xin Hu1, Huayin Wan2 and Rubin Li3 1 Department of Clinical Pharmacology, Beijing Hospital, the Ministry of Health, Beijing, China, 2Zhongwei Biotechnology Limited Company, Guangzhou, China, 3Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, China Objective: Calcium sensitizers comprise a novel class of positive inotropic agents that may possess potential advantages for the treatment of acute decompensated heart failure, compared with conventional cardiotonic drugs. Piperphentonamine is a novel calcium sensitizer for the treatment of congestive heart failure. The present study is to investigate the pharmacokinetics of piperphentonamine and its 2 metabolites, 2H-piperphentonamine (M1) and 2H-3-hydroxyl-piperphentonamine (M6) after multiple dosing in healthy Chinese volunteers. Methods: This study was a randomized, single-blind, placebo-controlled, multiple-dose phase I clinical trial. All volunteers signed a written informed consent before initiation of the screening procedure. Twenty-four subjects were enrolled and randomly assigned into two dosage groups (group 1/ 2, 12 subjects for each group). Piperphentonamine was given as an intravenous infusion of 1.00 (group 1) or 1.32 (group2) mg/kg for 3 hours following a


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loading dose of 0.40 mg/kg over 10 min. Blood samples were collected on day 1–3 and were analyzed for piperphentonamine, M1 and M6. Results: The pharmacokinetic parameters were derived by non-compartmental analysis of measured plasma concentrations on day 1 and day 3. The main pharmacokinetics parameters of two different dosages (group 1/2) were as follows: Cmax of piperphentonamine were (91.49 39.93), (45.76 22.76) ng/mL, respectively in day1 and (92.04 40.17), (75.93 56.70) ng/mL, respectively in day3; Cmax of M1 were (227.71 63.45), (215.41 67.38) ng/mL, respectively in day1 and (252.25 38.34), (240.75 90.35) ng/mL, respectively in day3; Cmax of M6 were (6.27 0.92), (8.55 1.72) ng/mL, respectively in day1 and (6.22 0.90), (8.71 1.47) ng/mL, respectively in day3. T1/2 of piperphentonamine were (6.24 4.36), (5.47 3.36) min, respectively in day1 and (7.61 3.94), (5.05 3.87) min, respectively in day3; T1/2 of M1 were (70.62 8.70), (57.62 8.74) min, respectively in day1 and (71.34 13.07), (67.87 8.29) min, respectively in day3; T1/2 of M6 were (273.80 187.63), (229.37 38.76) min, respectively in day1 and (215.24 41.86), (189.15 16.73) min, respectively in day3. AUC(0,t) of piperphentonamine were (2713.96 951.92), (2330.71 821.95) ng/mL, respectively in day1 and (2748.64 968.93), (2597.65 995.38) ng/mL, respectively in day3; AUC(0,t) of M1 were (24565.02 4404.34), (31744.34 5885.81) ng/mL, respectively in day1 and (25067.16 3359.72), (32924.13 609.41) ng/mL, respectively in day3; AUC(0,t) of M6 were (1994.82 279.12), (2647.97 507.70) ng/mL, respectively in day1 and (1987.74 279.90), (2852.80 511.33) ng/mL, respectively in day3. No significant dose effect was observed in Cmax, t1/2, and AUC(0,t). The steady state was not achieved after daily dosing for three days since the t1/2 of piperphentonamine was very short. After repeated dosing, there was no significant difference of mean AUC(0,t) values, or Cmax between day 1 and day3 (p40.05) for piperphentonamine, M1 and M6. Conclusion: The results showed that piperphentonamine was transformed to its metabolites M1/M6 and eliminated rapidly from plasma. The PK profiles after repeated doses were similar to those following the initial dose. No drug accumulation was observed for piperphentonamine, M1 or M6.

P5. CONTRIBUTION OF QUANTITATIVE MASS SPECTROMETRY IMAGING (MSI) IN PHARMACEUTICAL FIELD: APPLICATION TO DRUG AND PEPTIDE ANALYSIS IN TISSUE Gregory Hamm, Alain Heron, Raphael Legouffe, Fabien Pamelard, David Bonnel and Jonathan Stauber Imabiotech, Loos, France MSI permits the label-free study of several compounds of interest simultaneously on the surface of biological tissues. Quantitative development is a key success in the application of MSI for drug discovery as it is compared to gold standard quantitative techniques (such as autoradiography or LC-MSMS). This application may play a significant role in early phases of pharmaceutical discovery to evaluate small molecule concentration, notably drugs. This presentation will describe the possibility of quantifying a drug and a peptide using MSI and will show the main advantages of this new analytical technique in pharmaceutical field. Rat were dosed with diazepam at 5 mg/Kg and sacrificed at 15 minutes post-injection according to the kinetics of the compounds. Snap frozen brain and pancreas cryosections were carried out with a Microm cryostat HM560 (Thermo Scientific) at 10 mm thick and mounted on several conductive ITO glass slides before matrix automated deposition. Insulin analogue and a labeled diazepam were used to perform the quantification as internal standards. Mass spectra were acquired using a MALDI-TOF Mass Spectrometer (Bruker Daltonics) and data were generated by proprietary Quantinetix software (ImaBiotech). The quantification of a small and a large molecule, respectively the diazepam and the mouse, insulin was performed using MSI and internal standard approach. Insulin level was determined in whole pancreas tissue (approximately 200 mg/g of tissue) and the results were in agreement with previously published data (LC-MS/MS). Moreover, we have access to its content in small histological regions such as the Langerhans islets. Concerning the diazepam quantification, QMSI were used on adjacent sections and with the same dilution range. A higher amount of Diazepam (mg/g of tissue level) has been in the grey matter of the brain than in white matter which is cross-validated by pharmacokinetics data. A good reproducibility between quantitative data has been observed with an intra and inter-variability of 10 and 20% respectively. In conclusion, this study is the first example of multiple quantitative approaches on small and large molecules by MSI.

P6. DETERMINATION OF GDC-0068 AND ITS METABOLITE IN HUMAN PLASMA USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY COUPLED TO MASS SPECTROMETRY Yuzhong Deng1, Bianca Liederer1, Luna Musib2, Brian Dean1 and John Perkins3 1 Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA, 2Clinical Pharmacology, Genentech, Inc., South San Francisco, CA, 3Advion Bioanalytical Labs, Ithaca, NY Akt is a central node in cell signalling downstream of growth factors, cytokines, and other cellular stimuli. It plays an important role in cancer development, progression, and therapeutic resistance and is activated in most human cancers. GDC-0068 is a potent, highly selective small molecule inhibitor of all three isoforms of the serine/threonine kinase Akt, and it is being developed by Genentech as a treatment for a broad range of human cancers. To support clinical studies, a liquid chromatographytandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of GDC-0068 and its N-dealkylated metabolite in human plasma. In this assay, GDC-0068 and its metabolite were isolated from the plasma samples

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using a liquid-liquid extraction (LLE) procedure. Chromatographic separation was achieved on a Thermo BetaBasic C18 (2.1 20 mm, 5 mm) column using a pair of Shimadzu LC-10AD pumps with a SCL-10A controller. The aqueous mobile phase was 10 mM ammonium formate in water and the organic mobile phase was 50:50 methanol/acetonitrile. The detection was accomplished using an API 4000 tandem mass spectrometer in positive ion electrospray mode with multiple reaction monitoring (MRM). The standard curves, which ranged from 0.5 to 400 ng/mL for GDC-0068 and its metabolite, were fitted to a 1/x2 weighted linear regression model. The extraction recovery of GDC-0068 and its metabolite were 58.5 and 47.1% in human plasma, respectively. The internal standard normalized matrix factor of GDC-0068 and its metabolite were approximately 1.0 in human plasma. The intra-assay precision (%CV) and inter-assay precision (%CV) were 11.2%, and the assay accuracy (%RE) was within 12.0% for human K2EDTA plasma. This accurate, precise, and selective LLE/LC-MS/MS assay has been successfully applied to analyse clinical study samples.


P7. APPLICATION OF DRIED PLASMA SPOT SAMPLING IN COMBINATION WITH LC-MS/MS METHOD FOR PHARMACOKINETIC STUDIES OF FOUR IRIDOID GLYCOSIDES IN DOGS Yuan Gu, Youyou Cai, Miaoyun Zhang, Guangli Wei and Duanyun Si State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, China Dried plasma spot (DPS) is a prominent sampling technique for preclinical pharmacokinetic studies, as it provides some benefits in terms of sample collection, preparation, storage, and transport. In this study, DPS sampling in combination with a rapid, sensitive, and specific LC-MS/MS method were developed and validated for the simultaneous quantification of four iridoid glycosides including geniposide, deacetyl asperulosidic acid methyl ester, shanzhiside, and genipin geniobioside to support pharmacokinetic studies in dogs. After solvent extraction and sonication, DPS samples were chromatographed on an Ecosil C18 column (150 mm 4.6 mm, i.d., 5 mm) eluted with a gradient mobile phase consisting of methanol and 20 mM ammonium formate (containing 0.1% formic acid) at a flow rate of 0.5 mL/min for 15 min. The quantitation analysis was performed using multiple reaction monitoring in the positive ion mode with electrospray ionization source. Good linearity was achieved over the concentration ranges of 0.02 25 mg/mL with the LLOQ of 0.02 mg/mL for geniposide and 0.01 12.5 mg/mL with the LLOQ of 0.01 mg/mL for the other three analytes. The intra- and inter-day precisions were less than 13.3%, and the intra- and inter-day accuracies were within 10.0% for all analytes. The absolute recoveries of all analytes were between 78.2% 91.4%, and the matrix effects of all analytes were between 98.6% 105%. All analytes were stable during different storage, pretreatment and analytical conditions. After intravenous infusion of Naoluotai injection (containing 547.4 mg geniposide, 51.8 mg deacetyl asperulosidic acid methyl ester, 21.8 mg shanzhiside, and 93.0 mg genipin geniobioside) to six healthy dogs, DPS specimens were collected and quantified by the validated LC-MS/MS method. The main pharmacokinetic parameters of t1/2, AUC0–t, and Cmax calculated by WinNonlin 6.3 software were 6.86 h, 158 hmg/mL, and 158 mg/mL for geniposide, 2.11 h, 34.0 hmg/mL, and 30.5 mg/mL for genipin geniobioside, 1.11 h, 23.8 hmg/mL, and 17.8 mg/mL for deacetyl asperulosidic acid methyl ester, and 0.687 h, 9.16 hmg/mL, 7.49 mg/mL for shanzhiside, respectively. The systematic exposure levels of four iridoid glycosides appeared to be proportional to the content of the injection. In conclusion, DPS sampling combined with LC-MS/MS method seems to be a promising and advantageous technique, especially when applied to pharmacokinetic assessment.

P8. OPTIMIZATION OF SAMPLE PREPARATION METHOD FOR PROTEIN DRUG QUANTIFICATION IN HUMAN SERUM BY LC-MS/MS Katsuaki Ito, Kumiko Wakamiya, Daisuke Ishii, Masanobu Mukoyoshi, Tsutomu Mochizuki and Yoshinori Kasahara Pharmaceutical Development Research Laboratories, Teijin pharma limited, Hino, Japan Purpose: LC-MS/MS is useful tool for bioanalysis of protein drug development. In the drug development, the high accuracy is required for the bioanalytical method. For the high accuracy quantification, large interference peaks and low recoveries become critical problems. The purpose of this study is to investigate sample preparation condition such as protein denaturing, trypsin digestion, and the depletion of abundant serum protein to improve the interference peaks and the recovery. Method: In the LC-MS/MS analysis, the model protein was applied to human serum and denatured by different denaturants and incubation temperatures. After the alkylation and trypsin digestion, the stable isotope labeled peptide was spiked to the digested sample as internal standard, and the sample was injected to LC-MS/MS (4000 QTRAP). The recovery of the model protein from denaturing to trypsin digestion step was calculated by the peak area ratio to the stable isotope labeled peptide. To assess the recovery of the model protein on the albumin depletion column, the radio-labeled model protein was spiked to human serum, and the recovery was calculated from radio-activity before and after the depletion treatment. To examine the performance of the established method, the accuracy of the calibration curve was investigated. Result: The large interfering peak was observed by denaturation with some commercial available denaturants. The interfering peak was dramatically decreased by denaturation using 6 M urea with 95 C incubation. On the room temperature incubation, the large interfering peak was observed by using 6 M urea. The recovery after trypsin digestion was increased from 28% to 78% by sample dilution before trypsin digestion for decreasing

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the urea concentration from 1 M to 0.1 M. The recovery at the depletion was 85% by using radio-labeled model protein. By using the established method, the accuracy of calibration curve was within 15% and the correlation coefficient was more than 0.99. Conclusion: We found that the protein denaturing condition is key step for the decrease of the interference peaks, and the sample dilution before trypsin digestion is key step for the increase of the recovery. The established sample preparation method has the satisfactory accuracy for bioanalysis (15%) by LC-MS/MS.

P9. IN VITRO METABOLISM OF CAPSAICIN AND DIHYDROCAPSAICIN: POSSIBLE BIOMARKERS AFTER PEPPER SPRAY EXPOSURE


Mia Halme1, Maija Pesonen2, Heta Salo2, Tapio Kuitunen2, Markku Pasanen2, Kirsi Va¨ha¨kangas2 and Paula Vanninen1 1 VERIFIN, Institute for Verification of the Chemical Weapon Convention, Helsinki, Finland, 2Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, Finland Pepper spray products are widely used all over the world for the protection of policemen, security workers and solders. The active components of pepper sprays are capsaicinoids, the secondary metabolites of chili peppers, which are mostly known in spicy foods. The main components of the extract (oleoresin capsicum, OC) are capsaicin and dihydrocapsaicin in variable concentrations [1]. Exposure to capsaicinoids via pepper sprays occurs mainly through eyes, skin and a respiratory tract. Severe cardiovascular and pulmonary toxicities have also been observed in subjects exposed to pepper sprays. In this study, our goal was to concentrate on phase II metabolites (e.g. conjugates) to find a possible biomarker for the external exposure through eyes and airways. A biomarker is considered as a distinctive biological or biologically derived indicator (e.g. a metabolite) of a process, event or condition [2]. To verify exposure and to link the exposure with the putative health effects of harmful agent, the good validated biomarkers of exposure are essential. In vitro experiments were performed using human and pig hepatic microsomal and cytosol fractions, human small lung epithelial cells (A549) and human primary bronchial epithelial cells (HBEpC). Based on the earlier studies, the acute metabolism of capsaicin includes hydrolysis, oxidation, and conjugation with glutathione [3–4]. As there are no reference chemicals for the metabolites, the structural characterization were made using different LC-MS techniques. LC-MS analyses of in vitro samples were performed using two different MS analyzer: ion trap and triple quadrupole. These provided wide range of data using various acquisition methods e.g. full scan MS/MS, MS3, parent ion scan and neutral loss scan for the screening and identification of the metabolites of capsaicin and dihydrocapsaicin. It was observed that the phase I metabolism of capsaicinoids occurs in all studied sample types but different glutathione conjugates (phase II metabolism) were detected only in hepatic cell fractions. However, at least one new conjugated metabolite was found also from exposed cell lines. Results and conclusions will be presented.

References [1] Reilly C. A., Croucha D. J., Yosta G. S., Fatah A. A., Determination of capsaicin, dihydrocapsaicin, and nonivamide in selfdefense weapons by liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry, Journal of Chromatography A, 912, 259–267, 2001. [2] Pesonen M., Va¨ha¨kangas K., Halme M., Vanninen P., Seulanto H., Hemmila¨ M., Pasanen M., Kuitunen T., Capsaicinoids, chloropicrin and sulfur mustard: possibilities for exposure biomarkers, Frontiers in Predictive Toxicity, 1 (140), 1–12, 2010. [3] Reilly C. A., Ehlhardt W., Jackson D., Kulanthaivel P., Mutlib A. E., Espina R. J., Moody D. E., Crouch D. J. and Yost G. S., Metabolism of Capsaicin by Cytochrome P450 Produces Novel Dehydrogenated Metabolites and Decreases Cytotoxicity to Lung and Liver Cells, Chemical Research in Toxicology, 16 (3), 336–349, 2003. [4] Reilly C. A., Henion F., Bugni T. S., Ethirajan M., Stockmann C., Pramanik K. C., Srivastava S. K., and Yost G. S., Reactive Intermediates Produced from the Metabolism of the Vanilloid Ring of Capsaicinoids by P450 Enzymes, Chemical Research in Toxicology, 26 (1), 55–66, 2013.

P10. A SIMULTANEOUS CHROMATOGRAPHIC ANALYSIS OF TYPICAL DRUGS AND METABOLITES FOR PSYCHOTIC DISORDERS Maho Okubo1, Norie Murayama1, Satoshi Yamaori2, Shigeru Ohmori2 and Hiroshi Yamazaki1 1 Showa Pharma Univ, Machida, Japan, 2Shinshu Univ Hospital, Matsumoto, Japan Numbers of patients with mental disorders are administered with many medicines for their hospital cure. Pharmacological treatments of mental disorders are often carried out with simultaneous uses of two or more clinical drugs to achieve full therapeutic effects. Therefore, it is necessary to analyze each drug in plasma samples of patients simultaneously in a simple chromatographic system. The aim of this study was to develop a chromatographic analysis of frequently used drugs for mental disorders, namely atypical antipsychotic drugs (risperidone, olanzapine, quetiapine, and aripiprazole), antidepressants

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(mirtazapine and duloxetine), and anti-Alzheimer’s drug (donepezil), and their active metabolites (9-hydroxirisperidone and dehydroaripiprazole) simultaneously to evaluate plasma concentrations and drug interactions. These medicines and their metabolites were separated on an analytical octadecyl-silica column, eluted with a mobile phase of acetonitrile-phosphate buffer (pH 3.0), and detected by UV with inter-day and intra-day coefficients of variations of less than 15%. Among seven medicines, risperidone and several drugs, which would be metabolized commonly by cytochrome P450 2D6 and 3A4/5, were co-incubated in the presence of human liver microsomes or recombinantly expressed P450 enzymes to determine the drug interactions. The substrate depletion rates were inhibited, not affected or enhanced differently by the co-incubated medicines. These results suggest that the new simultaneous chromatographic analysis system developed in this study could be of use for evaluation of a variety of drug interactions in vitro and would be expected to adapt for rapid and reliable clinical evaluation. Plasma concentrations of co-administrated medicines in patients of psychotic disorders will be reported by this simple chromatographic system.


P11. IONIC LIQUID GCXGC-TOF-MS APPROACH FOR GENERIC SCREENING OF AEROSOL CONSTITUENTS IN AQUEOUS TOBACCO AEROSOL FRACTIONS AND ANALYSIS OF MICROSOMAL INCUBATES Quentin Dutertre, Arno Knorr, Audrey Debrick, Martin Almstetter, Elyette Martin and Mark Charles Bentley Philip Morris International R&D, Philip Morris Products S.A., Neuchatel, Switzerland The lung is the predominant target organ for chemically induced damage by tobacco aerosol constituents. While metabolism by oxidative enzymes and subsequent conjugation with endogenous compounds generally results in their detoxification and excretion from the body, it can also lead to the production of more toxic metabolites. In vitro metabolic investigations using microsomes, a well-established test system, were used to investigate the potential toxification of tobacco aerosol related xenobiotics. In addition, parent compound kinetics were also investigated using comprehensive two-dimensional gas chromatography coupled to fast acquisition time-of-flight mass spectrometry (GCxGC-TOF-MS). GC GC-TOF-MS is a widely established method for non-targeted analyses, suitable for the analysis of highly complex matrices such as tobacco aerosols. Aqueous trapping solutions perfused with tobacco aerosol as well as microsomal incubate were injected directly on-column and analyzed by GC GC-TOF-MS using a combination of water resistant ionic liquid based analytical columns. In the absence of a solvent front, this setup enables the detection of highly volatile constituents, including furans. This method represents a novel approach, which defies the common belief that aqueous media are incompatible with gas chromatography. Furan compounds are naturally present in tobacco leaves and are further generated during the smoking of tobacco products by thermal degradation. Furan and alkylfurans are rapidly metabolized by microsomes as a substrate of cytochrome P450 2E1-catalyzed oxidation, which leads to the generation of reactive intermediates (like cis-1,4-butendial by furan oxidative CYP activation), whereas furfural and furfuryl alcohol are predominantly detoxified. A mixture of furan, alkylfurans, furfural, furfuryl alcohol and 5-methylfurfuryl alcohol was investigated for phase I metabolism using human liver and lung microsomes. The kinetics for parent compound transformation rates were determined by GCxGC-TOF-MS measurements in parallel with a LC high resolution MS approach for gaining complementary information regarding any reactive metabolites. In addition to that, we analysed reactive intermediate metabolites using nucleophile trapping. Potentially unknown furan metabolites were investigated using non-targeted data analysis complemented by in silico metabolism prediction using Meteor NexusÕ . The impact of microsomal activation upon aqueous tobacco aerosol fractions was also investigated using a non-targeted workflow for parent compound kinetics.

P12. DETERMINATION OF BEPOTASTINE BESILATE IN HUMAN PLASMA AND URINE SAMPLES BY LC/MS/MS AND ITS APPLICATION TO CLINICAL PHARMACOKINETIC STUDY OF CHINESE VOLUNTEERS Aixin Shi1, Kexin Li2 and Xin Hu2 1 Department of Clinical Pharmacology, Beijing Hospital, the Ministry of Health, Beijing, China, 2Beijing Hospital, the Ministry of Health, Beijing, China Objective: Bepotastine besilate is a selective histamine H1-receptor antagonist and a second-generation, non-sedating antihistamine that has recently been used for the treatment of allergic disorders. The purpose of the present study is to develop a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for determination of bepotastine and apply it to a pharmacokinetics study. Methods: Solid phase extraction method was used for the pre-treatment of plasma and urine samples. The chromatographic separation was achieved on a C18 column using mobile phase of acetonitrile: water: 200 mM ammonium formate (75:20:5, v/v/v). The detection was performed using a triple quadurpole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via a positive electrospray ionization source. A total of 36 healthy Chinese subjects participated in the pharmacokinetics study of single oral administration of bepotastine besilate tablets. They were divided into three dosage groups and given 5 mg, 10 mg, 20 mg bepotastine besilate tablets, respectively. After drug administration, the concentration of bepotastine in human plasma and urine samples were determined to evaluation the pharmacokinetic characteristics of

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bepotastine. Results: The m/z ratios of monitored precursor ions and product ions of bepotastine and internal standard (IS) were 388.8 ! 201.7 and 299.9 ! 226.7. The concentration ranges of standard curve for bepotastine in plasma and urine samples were both 1–400 ng/mL. The main pharmacokinetics parameters of three different dosages (5 mg, 10 mg, 20 mg) were as follows: Cmax of bepotastine were (54.41 9.83), (116.01 37.80) and (233.40 35.35) ng/mL, respectively; t1/2b of bepotastine were (2.35 0.34), (2.73 0.85) and (2.77 1.37) min, respectively. Urinary excretion test results showed that bepotastine excretion mainly from the urine, urinary excretion rate within 24 h of bepotastine is approximately 60%. In addition, bepotastine besilate was generally well tolerated in this study, and none of adverse events associated with bepotastine besilate were occurred. Conclusion: This method is accurate, sensitive and suitable for the determination of the concentration of bepotastine in human plasma and urine. The pharmacokinetic characteristic of bepotastine in Chinese healthy volunteers within the dosage range (5 mg, 10 mg, 20 mg) was fitted with linear kinetic model.


P13. ENANTIOMERIC DETERMINATION OF LANSOPRAZOLE ENANTIOMERS IN DOG PLASMA BY LC-MS/MS WITH ACHIRAL-CHIRAL COLUMN -SWITCHING Hao Wang1, Jingkai Gu1, Wei Guo2, Jingrui Liu1, Yantong Sun3 and Yan Yang1 1 College of life sciences, Research Centre for Drug Metabolism, Jilin University, Changchun, China, 2College of Chemistry, Jilin University, Chuangchun, China, 3School of Pharmaceutical Sciences, Jilin University, Chuangchun, China Purpose: Lansoprazole is a proton-pump inhibitor (PPI), which decreases the amount of gastric acids and is used to treat and prevent stomach and intestinal ulcers, erosive esophagitis and other conditions involving excessive stomach acid. The purpose of this work was to develop a stereoselective and sensitive LC-MS/MS method with achiral-chiral column-switching for simultaneous quantification of lansoprazole enantiomers in dog plasma. Methods: The use of C18 column coupled to chiral column was successfully employed for quantification of lansoprazole in dog plasma to protect chiral column from clogging with macromolecules. After protein precipitation with acetonitrile, lansoprazole enantiomers and internal standard (pantoprazole enantiomers) were firstly purified online with a C18 column and then separated on a CHIRALCEL OZ-RH column with a column-switching procedure. The mobile phase was 0.1% formic acid-acetonitrile with a chromatographic gradient at a flow rate of 0.8 mL/min. Positive ions m/z 370.1 ! 252.0 for lansoprazole and m/z 384.1 ! 200.0 for pantoprazole were used for quantitation in multiple-reaction monitoring model. Analyses were performed on an AB Sciex API 4000 mass spectrometer with an ESI source. Results: The lower limit of quantitation of lansoprazole enantiomers were both 0.5 ng/mL, and the calibration curve of R(þ)- and S()- lansoprazole was linear over the concentration range of 0.5–500 ng/mL in dog plasma with correlation coefficient greater than 0.995 for both enantiomers. The recoveries of lansoprazole enantiomers were 101.08 4.91% for R(þ)lansoprazole and 97.81 2.42% for S()-lansoprazole from dog plasma. The matrix effects of lansoprazole enantiomers were 96.09 6.44% for R(þ)-lansoprazole and 94.40 4.35% for S()-lansoprazole. The intra-day precision was 4.1%–6.7% for R(þ)-lansoprazole and 4.0%–5.7% for S()-lansoprazole, and the inter-day precision was 7.3%–14.0% for R(þ)-lansoprazole and 2.6%–12.4% for S()-lansoprazole, respectively. Lansoprazole enantiomers were found to be stable in dog plasma after 4 h at room temperature for short term stability, after one month at 80 C for long term stability and after 3 freeze and thaw cycles at 80 C. No chiral inversion of lansoprazole was observed during storage and extraction. Conclusion: The method provided good sensitivity and selectivity of detection lansoprazole enantiomers. A good resolution of lansoprazole enantiomers was achieved with this method, and the analysis time was only 10 min. The developed LC-MS/MS method with achiral-chiral column switching was successfully applied to pharmacokinetic study and chiral inversion of lansoprazole enantiomers in dog.

Reference [1] Juliana Cristina Barreiro, et al. Direct injection of native aqueous matrices by achiral–chiral chromatography ion trap mass spectrometry for simultaneous quantification of pantoprazole and lansoprazole enantiomers fractions. J CHROMATOGR A, 2011, Vol 1218, 2865–2870.

P14. SIMULTANEOUS DETERMINATION OF CYCLOPHOSPHAMIDE AND DEXAMETHASONE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH UV WAVELENGTH SWITCHING Makhotso Rose Lekhooa1, Andrew Walubo1, J.B. du Plessis1 and M.G. Matsabisa2 1 Pharmacology department, University of the Free state, Bloemfontein, South Africa, 2Indigenous-Knowledge-system, Medical Research council, Cape town, South Africa Cyclophosphamide and dexamethasone are often used for cancer therapy and during in vivo experimental pharmacology. Hence, a need to develop and validate an high performance liquid chromatography (HPLC) method with UV wavelength switching detection for rat plasma analysis. Plasma spiked with cyclophosphamide and dexamethasone and surprofen as the internal standard was extracted with ethyl acetate. Centrifugation was 10 min long at 6 705 g and the supernatant was evaporated under nitrogen at 47 C. The residue was reconstituted with 100 ml of mobile phase and 50 ml was injected on an isocratic HPLC

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system. The sample was eluted with 45% of acetonitrile in 0.05 M sodium phosphate buffer (pH 3.7; containing 0.5 ml triethylamine) at a flow rate of 1 ml/min and 10 minutes runtime. Separation was achieved on a RP-Luna C18 (250 mm 4.6 mm 5 mm) column coupled to a guard column. The UV wavelength was set at 190 nm for cyclophosphamide and switched to 240 nm for dexamethasone and internal standard detection. Linearity was evaluated with a 6-point calibration curve over five days. Accuracy was tested with a repeated sampling of low, medium and high concentrations. Short-term stability was evaluated at different temperature settings over 24 and 48 hours. Plasma from rats treated with the two drugs was analyzed with the validated method. Cyclophosphamide eluted at 4.2 minutes while dexamethasone and internal standard eluted at 5.7 and 8.1 minutes respectively. Cyclophosphamide linear regression was y ¼ 0.04x þ 0.11 and y ¼ 0.32x–1.52 for dexamethasone. The correlation coefficient was 0.999 and 0.998 for cyclophosphamide and dexamethasone respectively. The coefficient of variance was less than 15% for both drugs. Recovery was 101–108% and 99–107% for cyclophosphamide and dexamethasone respectively after accuracy testing. Both drugs were stable in the fridge and ultra freezer over 24 and 48 hours. The method was successfully used to measure the two drugs after administration to rats. The validated high performance liquid chromatography method with UV wavelength switching is simple, rapid, specific and accurate.


P15. EFFECT OF FOOD INTAKE ON THE PHARMACOKINETICS OF CATALPOL IN BEAGLE DOGS Rong Lu, Yuan Gu, Linan Wu, Guangli Wei, Changxiao Liu and Duanyun Si State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, China Catalpol, an iridoid glycoside found in the radix of R. glutinosa Libosch, is the most abundant and bioactive component, which is proved to have important and extensive pharmacological action including hypoglycemic, diuretic, anticancer, antispasmodic and antiinflammatory effects. Preliminary pharmacokinetic and toxicokinetic studies showed that there is significant difference in systematic exposure of catalpol in dogs, which is likely caused by food intake. The objective of this study is to verify the interaction of food intake and the in vivo disposition of catalpol in Beagle dogs. An oral dose of catalpol 100 mg/kg dissolved in aqueous solution was administered to six healthy dogs, under fasting and fed conditions, according to a single-dose, randomized, two-period crossover design. Serial blood samples were collected in heparin containing tubes before drug administration (0 h) and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h postdose. Plasma catalpol concentrations were determined by a validated LCESI-MS/MS method. Pharmacokinetic parameters were calculated using non-compartmental analysis. Under both fasting and fed conditions, catalpol was rapidly absorbed with Tmax at 1.67 h, indicating that food intake had little effect on the rate of catalpol oral absorption. In the meanwhile, no statistically significant differences were observed for t1/2 and MRT0–t under fasting and fed conditions, which means that the presence of food had negligible effect on the in vivo elimination of catalpol in dogs. Whereas, under the fed condition, the Cmax (3.82 mg/mL) and AUC0–t (17.7 hmg/mL) of catalpol was significantly decreased compared to that under the fasting condition (Cmax 10.4 mg/mL, AUC0–t 45.8 hmg/mL), demonstrating that the intake of food substantially reduced the extent of absorption. In conclusion, food had little effect on the absorption rate and elimination of catalpol, but significantly reduced the absorption extent of catalpol. These findings explained why there were different systematic exposure levels between pharmacokinetics and toxicokinetics in dogs, and suggested that the pharmacological effect of catalpol can differ distinctly attributed to food intake. Therefore, it is necessary to investigate the mechanism of food effect on pharmacokinetics of catalpol in the future study to reduce the undesirable variability when dosed with food in clinical uses.

P16. IDENTIFICATION OF ALTERED PROTEIN BINDING OF DEXAMETHASONE IN THE PRESENCE OF SUGAMMADEX Ian S. Westley1, Giovanni Licari2, Richard W. Watts3 and Benedetta C. Sallustio4 1 Dept of Clin Phamacol, The Queen Elizabeth Hospital, Woodville, Australia, 2Vascular Disease and Therapeutics Research Group, The Queen Elizabeth Hospital, Woodville, Australia, 3Dept of Anesthesia, The Queen Elizabeth Hospital, Woodville, Australia, 4Dept of Clin Pharmacol, Queen Elizabeth Hosp, Woodville, Australia Introduction: Sugammadex (Sug) (BridionÕ , Schering-Plough Pty Ltd), a modified g-cyclodextrin, exerts its effects as a reversing agent by binding steroidal neuromuscular blocking agents (ie rocuronium (Roc), EsmeronÕ , Merck Sharp and Dohme Ltd) forming tight water-soluble complexes creating a concentration gradient. This favours the movement of the drug from the junction back into plasma that allows binding and clearance.(1) The glucocorticoid dexamethasone (Dex) is frequently used perioperatively to reduce postoperative pain and nausea that may result in delayed functional recovery.(2) Dandona et al previously described that Dex exerts its anti-inflammatory effects in part by inhibiting reactive oxygen species (ROS) generation.(3) As Roc and Dex are both steroids we hypothesise that Sug may inadvertently bind Dex. Aim: To determine the extent of Dex:Sug binding and its effect on inhibition of ROS by Dex. Methods: The formation of Dex:Sug complex was determined by incubating Dex (10 mg/L) þ/ Roc (10 mg/L) was incubated in 1 mL of drug free plasma (n ¼ 6) at 37 C for 2 hr followed by addition of Sug (2 mg or 20 mg) to the plasma containing Dex or Dex þ Roc and incubated for a further 1 hr ` at 37 C. Volumes were adjusted with PBS. Samples were then filtered using AmiconO Ultra–0.5 Centrifugal Filter Devices

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(Merck KGaA, Germany) according to manufacturers instructions and Dex concentrations were measured in both plasma and ultrafiltrate by HPLC-MS/MS. Dex efficacy was investigated in whole blood samples (n ¼ 4) incubated with vehicle, Dex (10 mg/L), Sug (2 mg) or Dex þ Sug for 10 min and formation of ROS was measured by Electro Paramagnetic Resonance (Bruker Biospin GmbH, Germany). Results: There was no difference in total Dex concentrations in all samples however Sug significantly increased the ultrafiltrate fraction of Dex in samples incubated with Sug þ/ Roc (17.4% (control) vs 44.7% (2 mg Sug); vs 81.3% (20 mg Sug) with no significant effect of Roc. Dex reduced ROS by 10.5% compared to controls however Sug also reduced ROS by 25.4%. The combination Dex/Sug reduced ROS by a total of 37.3%. Discussion: This study demonstrates that although Sug has a higher binding affinity for Roc (Kass 1.79 107 mol/L) over Dex (Kass51.00 103 mol/ L)(4), Sug binds Dex in vitro, with the increased fraction of Dex in ultrafiltrate representing transfer of the Sug:Dex complex, which dissociates during sample preparation/analysis and is detected as Dex. The binding of Dex by Sug did not appear to alter net inhibition of ROS in whole blood. However, further work is required clinically to determine how this interaction impacts patient care.


References (1) Naguib M. Sugammadex: Another milestone in clinical neuromuscular pharmacology. Anethesia and Analgesia. 2007;104(3):575–581. (2) De Oliveira Jr G et al. Perioperative single dose systemic dexamethasone for postoperative pain. A meta-analysis of randomized controlled trials. Anesthesiology.2011;115(3):575–588. (3) Dandona P et al. Effect of dexamethasone on reactive oxygen species generation by leukocyte and plasma interleukin-10 concentrations: a pharmacodynamic study. Clin Pharmacol Ther.1999;66(1):58–65 (4) Zwiers A et al. Assessment of the potential for displacement interactions with sugammadex. A pharmacokineticpharmacodynamic modeling approach. Clin Drug Invest. 2011;31(2):101–111

P17. EXTENSIVE METABOLISM AND P-GLYCOPROTEIN-MEDIATED EFFLUX OF Y101, AN ANTI-HBV AGENT, MAY CONTRIBUTE TO UNDERSTANDING THE INCOMPLETE BIOAVAILABILITY IN RATS Huirong Fan1, Xiaoyan CI1, Wei Li1, Xiulin Yi1, Quansheng Li1, Guangli Wei1, Yazhuo Li1, Duanyun Si1, Guangyi Liang2 and Chang-xiao Liu1 1 State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, China, 2The Key Laboratory of Chemistry for Natural Products of Guizhou Province, Chinese Academy of Sciences Y101, a novel phenyalanine dipeptide derivative, is an investigational new drug in china developed for treatment of patients with Hepatitis B virus (HBV)-infected hepatitis. During the process of drug development, oral bioavailability is often one of the key considerations for further development. The oral bioavailability of Y101 was approximately 46.0% in rats. From the perspective of some industrial scientists, factors that contribute to poor oral bioavailability have been divided into two interrelated categories: metabolism and absorption [1]. The purpose of the study is to investigate whether the metabolism and absorption are associated with the incomplete oral bioavailability of Y101. Initially, a quadrupole linear ion trap mass spectrometer system(QTRAP LC/MS/MS) was applied to identify the metabolites in bile, urine, feces and plasma after a single oral administration of 100 mgkg1 to rats, Of 33 metabolites detected, 4 were identified by comparing the chromatographic and mass spectrometric behaviors with the authentic standards. The predominant metabolite was believed to be amide hydrolysis to form M8, accounting for approximately 40% of the metabolites excreted in urine, feces and plasma. The other routes of metabolism involved might be hydroxylation, N-demethylation, dehydrogenation, N-oxydization and further converted to glucuronide and glucose conjugates. These results demonstrated that Y101 underwent extensive metabolism in rats. With respect to absorption, efflux transporters function as an absorptive barrier that limits oral bioavailability of xenobiotics. In order to identify whether efflux transporters were involved in absorption of Y101, in vitro models of the intestinal epithelium, Caco-2 cells and MDCKMDR1 cell line were therefore used. The apparent permeability coefficients (Papp) of Y101 in Caco-2 and MDCK-MDR1 cell monolayers from apical side (AP) to basolateral side (BL) or from BL to AP were assayed. The efflux ratio was42.0 from the bidirectional transportation studies and was significantly reduced in the presence of verapamil, a classical inhibitor of Pglycoprotein(P-gp). Meanwhile, the efflux ratio (22.1) in MDCK-MDR1 cell lines is greater than that obtained in Caco-2 cell monolayer (4.3). All results suggested that Y101 is likely to be the substrate of P-gp, indicating that P-gp-mediated efflux may play a key role in limiting intestinal absorption of Y101. In summary, extensive metabolism and efflux by P-gp may contribute to the incomplete bioavailability of Y101.

Reference 1. Thomas VH, Bhattachar S, Hitchingham L, et al. The road map to oral bioavailability: an industrial perspective. Expert Opin Drug Metab oxicol. 2006, 2(4):591–608.

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P18. EVALUATION OF INTESTINAL PERMEABILITY OF PROTOPANAXATRIOL AND PROTOPANAXADIOL USING CACO-2 CELL MONOLAYER


Ling-Ti Kong, Qian Wang, Bin-Xin Xiao, Yong-Hong Liao, Xiao-Xi He, Lin-Hu Ye, Xin-Min Liu, Rui-Le Pan and Qi Chang Institute of Medicinal Plant Development, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China Background: Protopanaxadiol (PPD) and protopanaxatriol (PPT), the two main metabolites of ginsenosides, are more easily absorbed into the body and displayed more potent activities than their original saponins. However, their pharmacokinetic properties and oral bioavailability were found to be quite different in our previous studies [1]. As absorption is one important step that modulates drug oral bioavailability [2], therefore, the intestinal permeability and the transport mechanisms of PPT and PPD would be important for understanding their pharmacokinetic properties. The present study is aimed to investigate their intestinal transport profiles and potential interaction during the intestinal absorption using the Caco-2 cell monolayer model. Methods: Caco-2 cells were seeded onto six-well transwell membrane inserts at a density of 1*105 cells/cm2 and cultured in DMEM cell culture medium, the medium was changed every two days. After being cultured for 18 and 21 days, the cell monolayers were used for the transport experiment. The transport buffer solution containing PPT or PPD was added to either the apical (AP, 1.5 mL) or basolateral side (BL, 2.6 mL) of the cell monolayer. Aliquots of 0.2 mL of transport medium in BL or AP side were taken at time intervals. The concentrations of PPT and PPD in the transport samples were measured by a LC-MS method. To explore the influence of P-gp inhibitor on the transport of PPT and PPD, their transport in both directions were also performed in the presence of verapamil, a known P-gp inhibitor. Results: In the present study, it was found that the average apparent permeability (Papp) values of PPD and PPT were both at a level of 105 cm/s, this indicated that PPD and PPT are two well-absorbed compounds. Their transport in small intestine were not involved in P-gp efflux and other active transport because the ratios of Papp BL– AB/Papp AB– BL for PPD and PPT were around 0.66–1.41, and less than 1.5. Futhermore, the Papp values for PPD and PPT in the presence of verapamil were not significantly different compared with that in the absence of verapamil. Conclusion: The in vitropermeability study indicated that both PPD and PPT were well-absorbed compounds, and their transport mechanism were passive diffusion. [The work was supported by the Ministry of Science and Technology of China (1108, 2011DFA32730, and 2012ZX09301002-001)].

References [1] Kong, LT., Wang, Q., Xiao, B.X., Liao, Y.H., He, X.X., Ye, L.H., et al., Different pharmacokinetics of the two structurally similar dammarane sapogenins, protopanaxatriol and protopanaxadiol, in rats, Fitoterapia. 86 (2013) 48–53. [2] Yu, H., Huang, Q., Investigation of the absorption mechanism of solubilized curcumin using Caco-2 cell monolayers, J. Agric. Food Chem. 59 (2011) 9120–6.

P19. SCALING FACTORS FOR INTESTINAL METABOLISM IN DOGS: EXAMINING INTERINDIVIDUAL AND REGIONAL VARIABILITY, AND CORRELATIONS TO HEPATIC SCALING FACTORS Oliver J. D. Hatley1, Christopher Jones2, Aleksandra Galetin1 and Amin Rostami-Hodjegan1 1 School of Pharmacy and Pharmaceutical Sciences, Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, United Kingdom, 2R&D, AstraZeneca, Macclesfield, United Kingdom The main focus of this project was to characterise intestinal metabolism in dog as a preclinical model in order to aid prediction of in vivo intestinal extraction and its contribution to oral bioavailability. Understanding whether regional differences in intestinal metabolism exist is important for assessing potential impact of formulations which release drug in different segments of the gut. Therefore, generation of zonal microsomal protein per gram mucosa (MPPGM) scaling factors are required for in vitro-in vivo extrapolation (IVIVE) within physiologically-based pharmacokinetic models (PBPK). The entire small intestine was obtained from beagle dogs (male and female), and then split into circa six segments. Intestinal microsomes were prepared via elution method from freshly isolated sections of intestinal tissue (n ¼ 3), as described previously [1]. Microsomal activity was used to characterise segments 1,2, 3 from the proximal region and also segment 6 from the distal region of the intestine; taken from female donors. In addition, liver and kidneys were processed from the same dogs. Markers, to correct for enzyme losses during microsome preparation, were measured in each sample, including cytochrome P450 content, CYP activity (testosterone 6b-hydroxylation), and UGT activity (4-nitrophenol-glucuronide formation). Values (mean 1 Stdev) of MPPGM from dog proximal intestine were 39 12 and 23 11 mg protein/ g mucosa for male and female dogs (p50.05), respectively. Regional scaling factors in female dogs increased gradually to the third segment (37 16), whereas distal microsomal scalars decreased 19 6. Values for the corresponding hepatic scalars were 43 8 and 37 8 mg protein/g liver in male and female dogs, respectively. Microsomal recovery was higher in the liver vs. intestine (61 8% vs. 20 4%), but showed low coefficient of variation (CV 8% and 18%, respectively), highlighting the reproducibility of the method. Matched liver and proximal intestine microsomal scalars showed good correlation (r2 ¼ 0.8, p50.02) for both male and female dogs. Regional differences in intestinal

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activity were observed according to 4-nitrophenol-glucuronide formation and decreased in the proximal to distal direction. Activity was significantly lower in distal compared to proximal segment 1 (p50.05). Testosterone 6b-hydroxylation was highest in proximal segments and lowest in the distal segment, analogous to gradients in dog intestinal phase I microsomal activity recently reported [2]. Application of these scaling factors for the IVIVE of intestinal metabolic clearance for a broad range of compounds is currently under investigation.

References 1. Hatley, O., et al., Methodology optimization in intestinal microsome preparation: implications for scaling approaches?, in 17th North American Regional ISSX Meeting; Oct 16–20, 2011: Atlanta, USA. 2. Heikkinen, A.T., et al., Mass spectrometry-based quantification of CYP enzymes to establish in vitro/in vivo scaling factors for intestinal and hepatic metabolism in beagle dog. Pharm Res, 2012. 29(7): p. 1832–42.


P20. COMPARISON OF THE PHARMACOKINETICS BETWEEN TWO VORICONAZOLE FORMULATIONS AND THE EFFECT OF CYP2C19 POLYMORPHISMS ON VORICONAZOLE EXPOSURE HyeKyung Han, Jieon Lee, Kwang-Hee Shin, Sang Chun Ji, Joo-Youn Cho, Kyoung Soo Lim, In-Jin Jang and Kyung-Sang Yu Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, Seoul, South Korea Voriconazole is an antifungal agent used in the treatment of invasive aspergillosis, candidiasis, and other fungal infections. A voriconazole loaded PNP (polynanomeric particle), SYP-1018, has been under development by Samyang Co., Ltd., Seoul, Korea. This study evaluated the safety and pharmacokinetics of SYP-1018 compared with a marketed form of voriconazole, VfendÕ , and evaluated the effect of CYP2C19 polymorphisms on the pharmacokinetics of the two voriconazole formulations. A randomized, open-label, two-treatment, two-period, two-sequence crossover study was conducted with 52 healthy volunteers receiving intravenous infusions of 200 mg of voriconazole, SYP-1018 and VfendÕ . To determine the pharmacokinetics and genotypes of the volunteers, blood samples were collected before and up to 24 hours after drug administration. The plasma concentrations of voriconazole were measured by liquid chromatography with tandem mass spectrometry, and the pharmacokinetic parameters were determined using noncompartmental methods. The pharmacokinetic parameters were compared using the geometric mean ratios and 90% confidence intervals (CI). Fifty-one subjects were divided into 3 CYP2C19 subgroups according to their genotypes: *1/*1 for homozygous extensive metabolizers (EMs, N ¼ 20), *1/*2, *1/*3 for intermediate metabolizers (IMs, N ¼ 21), and *2/*2, *2/*3, *3/*3 for poor metabolizers (PMs, N ¼ 10). One subject was not genotyped. After a single intravenous infusion of 200 mg of SYP-1018 or VfendÕ , the maximum concentrations (Cmax) (mean standard deviation) were 2120.7 274.3 ng/mL and 2141.8 464.1 ng/mL, and the concentration-time curves from time point 0 to the last measurable time point (AUClast) were 7848.0 3445.2 h*ng/mL and 8125.4 3539.1 h*ng/mL for SYP1018 and VfendÕ , respectively. The geometric mean ratios (GMR) of SYP-1018 to VfendÕ (90% CI) were 0.99 (0.93–1.04) for the Cmax and 0.97 (0.92–1.01) for the AUClast. The SYP-1018 exposures (GMR and 90% CI) in the IMs and PMs were 1.32 (1.14–1.52) and 2.30 (1.98–2.66) fold greater than in the EMs. The VfendÕ exposures in the IMs and PMs were 1.28 (1.11–1.48) and 2.37 (2.10–2.68) fold greater than in the EMs after a single intravenous infusion. In each of the genotype groups, the GMRs and 90% CIs of SYP-1018 to VfendÕ for the AUClast were within an acceptable range (0.8–1.25). There were no serious adverse events, and both treatments were well tolerated. SYP-1018 was well tolerated in healthy volunteers and showed comparable pharmacokinetic profiles with VfendÕ . Genetic polymorphisms of CYP2C19 influenced the pharmacokinetics of both SYP-1018 and VfendÕ .

P21. AN INTEGRATED APPROACH OF IN VITRO PHYSIOLOGICALLY RELEVANT INTESTINAL MODELS AND PBPK MODELING TO PREDICT ORAL BIOAVAILABILITY IN HUMANS Heleen M. Wortelboer, Joost Westerhout, Bart Anneveld, Dimitri Grossouw, Evita van de Steeg, Evelijn E Zeijdner, Mans Minekus, Jeroen DeGroot and Miriam Verwei Kinetics Research for Food & Pharma, TNO, Zeist, Netherlands A reliable assessment of oral bioavailability is essential in the development of pharmaceutical and nutritional products, as the size of the systemically available fraction co-determines the efficacy and possible toxicity of the active compound. To reveal predictive information on the fraction absorbed in humans, currently in silico tools, in vitro models (mainly cell lines) and animal models are performed pre-clinically. However, due to the complexity of the human gastrointestinal tract and due to large differences in physiology between animals and humans, the predictive value of an in silico tool or a single in vitro model and an animal model to assess oral bioavailability is often insufficient, especially when an effect of food on the bioavailability is involved. The aim was to set-up an integrated approach to predict the fraction absorbed in humans using a combination of physiologically relevant in vitro gastrointestinal (GI) models and a physiologically based pharmacokinetic (PBPK) model.

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Three model compounds were selected, ranitidine, fenofibrate and ciprofloxacin, and tested in a combination of GI models. As a first step, the three compounds were tested in TNO’s gastro-Intestinal Model (TIM-1), simulating the dynamic physiological processes in the human upper GI tract, under fasted state conditions (with the intake of a glass of water) or fed state conditions (with a FDA standardized high-fat meal). Jejunal and ileal filtrate samples were taken over time and analyzed to generate information on the availability for absorption (i.e. bioaccessibility) of the compounds. In addition, jejunal filtrate samples with fractions of the test compounds were combined with TNO’s newly developed InTESTineÔ model to assess the fraction absorbed across porcine ex-vivo intestinal tissue. Finally, these data will be integrated using PBPK models describing in vivo kinetics of the three model compounds. The results of these studies demonstrate the predictive value of the integrative strategy to assess the fraction absorbed in humans combining physiologically relevant in vitro tools, i.e. a multi-compartmental model of the GI tract (TIM-1 system) with intestinal absorption studies using ex vivo intestinal tissue in InTESTineÔ. With these systems two main processes on the overall bioavailability can be studied, namely 1) release and dissolution in the GI tract and 2) absorption from the intestinal lumen. This approach enables unraveling of critical factors in the bioavailability of food and drug compounds, therewith support innovations to control and optimize the bioavailability of compounds in drug and formulation development, as well as in the development of (functional) foods.

P22. COMPARISON OF LOCAL PHARMACOKINETIC MODELS OF GASTROINTESTINAL ABSORPTION: TRANSLOCATION MODEL AND ACAT MODEL (GASTROPLUSä) Hirotaka Ando1, Akihiro Hisaka2 and Hiroshi Suzuki3 1 Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd., Tochigi, Japan, 2Pharmacology and Pharmacokinetics, The University of Tokyo Hospital, Tokyo, Japan, 3Department of Pharmacy, The University of Tokyo Hospital, Tokyo, Japan Background: We previously developed the translocation model (TLM) to analyze various nonlinear phenomena during the absorption process caused by metabolism, transport, and the blood-flow limitation1). The TLM has only one absorption site, which is translocatable along the length of the intestinal tract, with the associated properties of the absorption site being locationdependent. Translocation of a drug is defined by an arbitrary mathematical equation in the TLM. The Advanced Compartmental Absorption and Transit (ACAT) model is a sophisticated drug absorption model, and it is implemented in the GastroPlusÔ (GP) that has been widely used in the pharmaceutical industries and also in various studies. In this model, the gastrointestinal tract is segmented into several compartments, and the movement of a drug from one compartment to the other follows first-order kinetics. In this study, predictions of the absorption profiles of drugs with varying properties were compared between the TLM and the ACAT model. Because it is difficult to understand all mathematical equations used in GP, we developed a simpler version of the ACAT model to compare the simulation results of the TLM, GP, and ACAT model. Methods: Various physicochemical parameters (e.g. pKa, logP, MW), in vitro experimental values (e.g., Papp, Vmax, and Km), default values of GP and its parameter prediction models (e.g., Opt logD Model SA/V 6.1) were used without any modification. The TLM and the simple ACAT model were implemented in Napp2) by entering their differential equations. The relationship between apparent permeability and absorption ratio (FA) or bioavailability (F), and time course of the intestinal tissue concentration were compared in these models for various values of surface area microvilli expansion (ME) and intestinal blood flow rate. Results: By adjusting the translocation function of the TLM, similar results were obtained from all models for the movement of a nonabsorbable drug across the gastrointestinal tract, from the stomach to the duodenum, followed by the jejunum to the ileum. Furthermore, the relationship between apparent permeability (hypothetically determined by Caco-2 experiments) and FA or F was very similar in all models for drugs not metabolized or transported actively when back flux from the blood to the enterocytes was ignored. When back flux was included in the analysis, blood-flow limited absorption was observed in the TLM. In the TLM, drug concentration in the enterocytes was affected by ME. Conclusion: Predictions of the absorption profile by TLM were comparable with those obtained from the ACAT models for various situations. Since all parameters and mathematical equations are transparent and adjustable for the TLM, it would be suitable for special research needs. In order to improve the simulation technique, it is important to devise multiple ways to validate the model. [References] 1) Ando H et al., 17th ISSX meeting, 2011, Atlanta, GA. 2) Hisaka A and Sugiyama Y, J Pharmacokinet Biopharm, 1998, 26:495–519 (Napp is downloadable from http:// plaza.umin.ac.jp/todaiyak/download.php).

P23. BIOTRANSFORMATION OF POLYPHENOLS AND THEIR ANTIOXIDANT ACTIVITY AFTER DIGESTION IN THE COMPUTER CONTROLLED DYNAMIC HUMAN GASTROINTESTINAL MODEL Shima Sadeghi Ekbatan1, Joelle Khairallah1, Kebba Sabally1, Behnam Azadi1, Lekha Sleno2, Laetitia Rodes3, Satya Prakash3, Danielle J. Donnelly4 and Stan Kubow1 1 School of Dietetics and Human Nutrition, McGill University, Montreal, QC, Canada, 2Chemistry Department, Universite´ du Que´bec a` Montreál, QC, Canada, 3Department of Biomedical Engineering, McGill University, QC, Canada,4Plant Science Department, McGill University, QC, Canada


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Background: The health benefits of dietary polyphenols depend on their metabolism within the gastrointestinal tract1,2. As polyphenols are poorly bioavailable, large amounts reach the colon and get metabolized by the gut microflora to generate metabolites that can exert antioxidant effects both in the gut and systemically3,4,5,6. The nature and antioxidant capacity of metabolites formed during polyphenol digestion is relatively unexplored as most in vitroinvestigations have used digestive enzymes but have not included gut microflora. A few studies have incubated polyphenols in basic fermentors containing fecal suspensions without consideration of the dynamics of transit during the digestion process or the varying microbial or digestive conditions in different segments of the gastrointestinal tract7,8. The Computer Controlled Dynamic Human Gastrointestinal Model that simulates human digestion via enzymatic digestion and human gut microflora metabolism was used in this study to identify the metabolites and the antioxidant activity generated from digestion of a mixture of polyphenols commonly consumed in plant foods, including chlorogenic acid, caffeic acid, ferulic acid and rutin. Methodology: The GI model was composed of five double-jacketed vessels representing the stomach, the small intestine, the ascending colon, the transverse colon, and the descending colon. The model was fully computer-controlled and pH levels were controlled within each vessel according to the GI portion it represents.The difference in pH in the last three vessels can result in different microbial populations in each segment leading to different biotransformation activity and post-digest metabolites. The polyphenol mixture was subjected to the in vitro digestion by the GI model for 24 h. Samples were collected after 8, 16 and 24 h from all vessels, centrifuged at 1000 g for 20 min and stored at 80 C for later analysis. Post-digest metabolites were analyzed by HPLC and LC-MS and antioxidant activity was measured by the ABTS assay. Results: All parent polyphenols were completely biotransformed following 24 h digestion to form new metabolites within the colonic vessels. The first major biotransformation occurred in the ascending colon leading to a mixture of parent compounds and metabolites. Further extensive metabolism of the parent polyphenols occurred in the transverse and descending colon leading to the total disappearance of the parent polyphenols and the appearance of new metabolites including propionic, acetic and benzoic acids derivatives. The metabolite profiles differed among the three colonic segments. The ascending colon that contained both the parent compounds and their microbial metabolites showed the earliest increase in antioxidant capacity following polyphenol administration as antioxidant capacity increased after 8 h (p50.05) whereas the transverse and descending colon showed an increased antioxidant capacity after 16 h (p50.05).Conclusion: Polyphenols were extensively biotransformed by colonic microflora leading to differing metabolite profiles in the different colonic segments, which has implications regarding their health impact on different areas of the colon. The Computer Controlled Dynamic Human Gastrointestinal Model is a useful approach to study the wide variety of metabolites generated during microbial metabolism of polyphenols during their transit in the different segments of the colon.

References 1. Manach C, Scalbert A, Morand C, Re´me´sy C, and Jimeńez L. Polyphenols: food sources and bioavailability. American Journal of Clinical Nutrition, 2004. 79: p. 727–47 2. Scalbert A, Morand C, Manach C, and Re´me´sy C. Absorption and metabolism of polyphenols in the gut and impact on health. Biomedicine & Pharmacotherapy, 2002. 56(6): p. 276–82. 3. D’Archivio M, Filesi C, Di Benedetto R, Gargiulo R, Giovannini C, and Masella R. Polyphenols, dietary sources and bioavailability. Annali dell’Istituto Superiore di Sanita`, 2007. 43(4): p. 348–61. 4. Gonthier MP, Verny MA, Besson C, Remesy C, and Scalbert A. Chlorogenic acid bioavailability largely depends on its metabolism by the gut microflora in rats. Journal of Nutrition, 2003. 133: p. 1853–59. 5. Lafay S, Morand C, Manach C, Besson C, and Scalbert A. Absorption and metabolism of caffeic acid and chlorogenic acid in the small intestine of rats. British Journal of Nutrition, 2006. 96: p. 39–46. 6. Stalmach A, Mullen W, Barron D, Uchida K, Yokota T, Cavin C, Steiling H, Williamson G, and Crozier A. Metabolite profiling of hydroxycinnamate derivatives in plasma and urine after the ingestion of coffee by Humans: identification of biomarkers of coffee consumption. Drug Metabolism and Disposition, 2009. 37(8): p. 1749–58. 7. Gonthier MP, Remesy C, Scalbert A, Cheynier V, Souquet JM, Poutanen K, and Aura AM. Microbial metabolism of caffeic acid and its esters chlorogenic and caftaric acids by human faecal microbiota in vitro. Biomedicine & Pharmacotherapy, 2006. 60(9): p. 536–40. 8. Tagliazucchi D, Verzelloni E, Bertolini D and Conte A. In vitro bio-accessibility and antioxidant activity of grape polyphenols. Food Chemistry, 2010. 120(2):p. 599–606.

P24. REACTOME KNOWLEDGEBASE: A PLATFORM FOR PATHWAY AND NETWORK ANALYSIS Robin Haw1, Guanming Wu1, Marija Milacic1, Karen Rothfels1, David Croft2, Henning Hermjakob2, Peter D’Eustachio3 and Lincoln Stein1 1 Informatics and Bio-computing, OICR, Toronto, ON, Canada, 2Proteomics Services, EBI, Cambridge, United Kingdom, 3 Biochemistry and Molecular Pharmacology, NYUMC, New York, NY Background: Reactome is a open access, open-source, manually curated and peer-reviewed biological pathway knowledgebase, which serves as a platform for data visualization and analysis. The Reactome Functional Interaction (FI) plug-in is a Cytoscape

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extension that allows researchers to perform network module-based data analysis. The curation of cancer-related pathways, coupled with our efforts to create other disease-specific pathways, will interoperate with our existing pathway and network analysis tools. Observations: Biological differences between different cancer mutants have important implications for the design and application of targeted therapy, as already demonstrated in clinical practice by different sensitivity of EGFR mutants to gefitinib and erlotinib. Reactome has expanded its content and data model to provide users with cancer-specific information, capturing and displaying the mechanistic differences between protein variants in detail, including their interaction with drugs. Reactome has so far published EGFR, FGFR, Notch1 and PI3K/AKT signaling pathways in cancer. A recent extension of Reactome FI network, with updated protein-protein interactions and other pair-wise protein or gene relationships, genome-wide screening data sets has increased the FI network to covering just over half of human proteins in SwissProt and almost 274,000 interactions. We have also implemented the HotNet algorithm, for finding significantly altered subnetworks by using a graph heat-kernel model. The HotNet algorithm, combined with the survival analysis function and other previously implemented features of the Reactome FI plugin should provide a powerful framework for molecular signature discovery. Conclusions: Reactome annotations include information on the molecular function of over 200 cancer variants and approximately 50 anticancer drugs. Future updates to the Reactome website and FI plugin will promote efficient data analysis of cancer and other disease datasets.

P25. ADDRESSING TOXICITY RISK WHEN DESIGNING AND SELECTING COMPOUNDS IN EARLY DRUG DISCOVERY Matthew D. Segall and Nicholas W. Foster Optibrium Ltd., Cambridge, United Kingdom It has been estimated that toxicity accounts for approximately 30% of expensive, late stage failures in clinical development. Therefore, identifying and prioritising chemistries with a lower risk of toxicity, as early as possible in the drug discovery process, would help to address the high attrition rate in pharmaceutical R&D. We will describe how expert knowledge-based prediction of toxicity can alert chemists if their proposed chemistry is likely to have an increased risk of causing toxicity, based on precedence for similar compounds where experimental data are available. However, an alert for potential should be given appropriate weight in the selection of compounds. It is important to avoid missing opportunities as well as late stage failures toxicity and an alert may not be sufficient to ‘kill’ a compound or chemical series. If a chemistry achieves good outcomes for other requirements, it may be appropriate to progress selected compounds and generate experimental data to confirm or refute a prediction of potential toxicity. We will discuss how multi-parameter optimisation approaches can be used to balance the potential for toxicity with other properties that are required in a high quality candidate drug, such as potency and appropriate absorption, distribution, metabolism and elimination (ADME). Furthermore, it may be possible to modify a compound to reduce its likelihood of toxicity and we will describe how information on the region of a compound that triggers a toxicity alert can be interactively visualised to guide this redesign.

P26. CYP2E1 EXPRESSION AND ACTIVITY IN THE PERIPHERAL BLOOD MONONUCLEAR CELLS OF CIGARETTE SMOKERS Catherine A. Wassenaar1, Maria Novalen1, Adel Aziziyeh2, Caryn Lerman3, Tony P. George2 and Rachel F. Tyndale4 1 University of Toronto, Toronto, ON, Canada, 2Centre for Addiction and Mental Health, Toronto, ON, Canada, 3Center for Interdisciplinary Research on Nicotine Addiction, Philadelphia, PA, 4Centre for Addiction and Mental Health, University of Toronto, Toronto, ON, Canada CYP2E1 metabolically activates toxins and carcinogens from cigarette smoke, and variation in CYP2E1 has been associated with tobacco-related cancers. Nicotine treatment induces CYP2E1, and cigarette smoking is associated with faster CYP2E1 metabolism. CYP2E1 also contributes to alcohol metabolism, and alcohol consumption, which is common among smokers, may further contribute to CYP2E1 induction. Previous studies have found a relationship between CYP2E1 expression within peripheral blood mononuclear cells (PBMC) and systemic clearance of the CYP2E1 probe drug, chlorzoxazone, suggesting that CYP2E1 within PBMC could serve as a surrogate for hepatic CYP2E1. To further investigate CYP2E1 within PBMC, and to characterize the relationship between the level of smoking and CYP2E1 induction, we isolated PBMC from forty-five current smokers at intake sessions for a smoking cessation clinical trial in Toronto, Ontario and measured CYP2E1 RNA expression and protein activity. Plasma nicotine metabolites were also measured in smokers (over 10 cigarettes per day eligibility) and data was collected on alcohol consumption (less than 25 standard drinks per week eligibility). CYP2E1 RNA was detectable within 44 of 45 participants and was on average five orders of magnitude less abundant relative to the internal control, GAPDH. Normalizing CYP2E1 expression to the study participant with the lowest relative CYP2E1 abundance resulted in a range of CYP2E1 expression from 1 (reference) to 60 with a median of 16. Correlation analyses did not support a relationship between the relative CYP2E1 expression and either plasma cotinine, operating as a biomarker for nicotine exposure (spearman rho ¼ 0.16, p ¼ 0.3) or the ratio of 3-hydroxycotinine to cotinine, operating as a phenotypic marker for the rate of nicotine metabolism (spearman

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rho ¼ 0.03, p ¼ 0.9). Efforts to develop an assay to quantify CYP2E1 protein activity within limited PBMC tissue are ongoing in order to assess whether CYP2E1 RNA expression is correlated with protein activity. Additional analyses are also underway to investigate the association of CYP2E1 RNA expression and activity with self-reported measures of alcohol and cigarette consumption. Our preliminary findings suggest that PBMC can serve as an accessible biological source for the assessment of CYP2E1 expression and could have utility as a biomarker within epidemiological studies. Our preliminary findings do not support nicotine exposure as an important predictor of differential CYP2E1 induction among smokers. However, differential CYP2E1 activity within smokers may still contribute to differences in the susceptibility to tobacco-related cancers.

P27. THE DIFFERENCE IN THE METABOLITE PROFILES BETWEEN PLASMA AND SERUM, AGES OR SEXES, AND THEIR INTER-INDIVIDUAL VARIATIONS IN HUMAN SUBJECTS


Kosuke Saito1, Keiko Maekawa1, Kirk Pappan2, Masayo Urata1, Masaki Ishikawa1, Yuji Kumagai3 and Yoshiro Saito1 1 Medicinal Safety Science, National Institute of Health Sciences, Tokyo, Japan, 2Metabolon, Inc., Durham, NC, 3Clinical Trial Center, Kitasato University East Hospital, Kanagawa, Japan Endobiotic metabolites are associated with the biological processes in the body and thus suitable biomarkers to evaluate disease state, therapeutic efficacy, and toxicity of drugs. However, information regarding how differences among matrices, subject backgrounds, and sample handling affect the metabolomic profiles of human subjects remains limited. Our objective was to obtain metabolite profiles in different matrices (plasma and serum), subject backgrounds (male and female, young and old), and storage conditions (with or without 10 cycles of freezing-thawing). All subjects were Caucasian and overnight fasted to minimize the effect of daily nutrients and ethnic genetic background on metabolite profiles. In total, the levels of 297 endobiotic metabolites were detected by LC/MS and GC/MS, and more than 75% of them were highly represented (present in over 80% of all samples) in all sample groups. OPLS-DA analysis showed that of the variables tested in this study - matrix, gender, age, and freeze-thaw cycles -, and matrix type gave the clearest separation. The levels of more than 100 metabolites, including metabolites related to blood coagulation and several amino acids/dipeptides, were significantly different between plasma and serum in all subject backgrounds. The differences in subject backgrounds were consistent between plasma and serum. Ageassociated differences were more predominant in female than male. Of the metabolites showing different levels between ages, amino acid produced by gut microflora (phenylacetylglutamine and cresol) were common, while progesterone metabolites and fatty acids were only observed in females. Gender-associated difference were more prevalent in younger than older subjects. The levels of androgen metabolites were significantly different in both young and old populations, while those of progesterone metabolites were significantly different only in young population. In addition, the relative standard deviation of the detected levels of each metabolite within same subject background scored from 0.1 to 2.5. Moreover, changes of the metabolite levels caused by freeze-thaw cycles were limited and the effect was much stronger in plasma than serum. Taken together, these results demonstrated the impact of matrix, age or gender, and multiple freeze-thaw cycles on the metabolomic profiles of human subjects. Plasma and serum equally represented the difference in metabolite profiles among subject backgrounds, and thus both of them would be suitable matrices to measure the levels of biomarkers, although serum is more stable against freeze-thaw cycles. In addition, all samples should have a uniform matrix type since the relative metabolite levels detected differed between plasma and serum. Moreover, the detectability, and the inter- and intra-background variations of the metabolite levels in subjects should be considered when designing metabolomic studies to identify biomarkers. This study provides useful fundamental information to evaluate biomarkers for clinical application.

P28. THE ROLE OF ALPHA 1-ACID GLYCOPROTEIN (AAG) AS A BIOMARKER FOR ARRY-520 EXPOSURE Karin D. Brown1, Brian J. Tunquist2, Kevin S. Litlwiler3, Lance A. Wollenberg3, Duncan H. Walker2, Ronald B. Franklin1 and Gary P. Hingorani1 1 Dmpk, Array BioPharma Inc., Boulder, CO, 2Translational Medicine, Array BioPharma Inc., Boulder, CO, 3Clinical Pharmacology, Array BioPharma Inc., Boulder, CO ARRY-520 is a kinesin spindle protein (KSP) inhibitor that has demonstrated clinical activity in patients with relapsed and refractory multiple myeloma (MM). The degree of binding of certain drugs to serum proteins can alter their free fraction (fu) and PK, with a possible impact on clinical activity. Alpha 1-acid glycoprotein (AAG) is an acute-phase reactant protein whose concentrations can vary due to physiological, pathological and genetic factors, and is elevated in the blood of some patients with cancer, including MM. We investigated the significance of the interaction of ARRY-520 with AAG, to determine if AAG levels could affect the pharmacokinetics and pharmacological activity of ARRY-520. ARRY-520 exhibits about a 390-fold higher affinity for AAG than human serum albumin (HSA). In in vitro assays, increasing [AAG] across a clinically relevant range (0.6–3.0 g/L) in the presence of HSA (40 g/L) decreased the unbound concentration of ARRY-520 five-fold. Similar decreases (3- to 5-fold) were demonstrated for the fu of imatinib, imipramine, nelfinavir and docetaxel; drugs for which [AAG] has been correlated with [plasma drug] in vivo. Increasing [AAG] resulted in increased IC50 values (410-fold) for ARRY-520 in MM cell

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line viability assays consistent with a hypothesis that elevated [AAG] decreases free drug leading to a loss of potency. Pharmacokinetic modeling indicated the [AAG] was a significant covariate to explain the disposition of ARRY-520. Increasing [AAG] indicated a decrease in both clearance and volume of distribution of ARRY-520. Moreover, increased [AAG] correlated well with unbound fraction of ARRY-520, decreasing the predicted exposure of unbound ARRY-520. In pre-dose blood samples (n ¼ 159) from 3 clinical studies for a range of oncology indications, [AAG] ranged from 0.2 to 3.9 g/L. Elevated [AAG] in patients was associated with lack of response to ARRY-520 and decreased time on treatment; the majority of patients with ‘‘high’’ baseline [AAG] came off study in less than five months. In patients with relapsed or refractory MM (n ¼ 91), all patients achieving a clinical benefit (4 or ¼ minor response) had ‘‘low’’ [AAG] at screening. These preliminary data suggest that AAG levels may provide a reliable marker for the free fraction of ARRY-520 in plasma over a clinically relevant range.

P29. ATP METABOLISM IN RBC AS IN VIVO BIOMARKER FOR CARDIOVASCULAR TOXICITY


Pollen K. F. Yeung1, Dena Seeto2, Chad Purcell2, Patrick Tillman2 and Hannah Mark2 1 College of Pharmacy and Department of Medicine, Dalhousie University, Halifax, NS, Canada, 2College of Pharmacy, Dalhousie University, Halifax, NS, Canada Although clinical drug development has made significant stride along with pharmaceutical sciences over the last 3 decades, from the application of pharmacokinetics in the 1970’s, controlled clinical studies for efficacy in 1980’s, pharmacodynamics and pharmacogenetics in the 1990s, to a focus on drug safety in the past decade, the success rate to introduce new effective and safe therapeutic agents has not kept up with expectations from the financial investment and those of patients. In another word, there is inadequate improvement in drug therapy or financial reward. Identification and application of biomarkers for lead selection and optimization has been heralded as one of the most likely scientific approach to increase the success of drug development. It is widely conceived that biomarker is a scientific basis and an effective tool for disease management and personalized medicine. We have shown that in a rat model that cardiovascular injury induced by an isoprotereneol injection (30 mg/kg) caused significant mortality and increased break down of ATP to AMP in red blood cell (RBC) in vivo. Those did not survive from the injury had significantly higher RBC concentrations of AMP. We have also shown that exercise increased RBC concentrations of ATP which may be a key factor for post-exercise hypotension. Exercise also reduced mortality and RBC concentrations of AMP in the acute myocardial infraction model induced by isoproterenol. Further, the calcium channel antagonist diltiazem which lowers blood pressure and heart rate was shown to have an attenuating effect on the cardiovascular injury induced by isoproterenol. The presentation will focus on the potential of using ATP metabolism as biomarker target for toxicities and regulation of cardiovascular homeostasis. It will also discuss the opportunities, challenges and obstacles of exploiting ATP metabolism in the RBC as targets for drug development and personalized medicine. The research was supported in part by Canadian Institute of Health Research (CIHR), Nova Scotia Health Research Foundation (NSHRF) and Dalhousie Pharmacy Foundation.

P30. ISONIAZID-INDUCED LIVER INJURY IN HUMANS IS IMMUNE-MEDIATED AND NOT ‘‘METABOLIC IDIOSYNCRASY’’ Imir G. Metushi1 and Jack P. Uetrecht2 1 Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada, 2Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada Background: Idiosyncratic drug reactions remain a significant health problem. An example is isoniazid (INH)-induced liver injury. Previous studies indicated that this toxicity is due to bioactivation of acetylhydrazine (AcHz),1 a metabolite of INH. However, this conclusion was based on an acute model of toxicity in rats. Partially because of a failure to detect anti-INH antibodies in human serum,2 the mechanism of INH-induced liver injury was called ‘‘metabolic idiosyncrasy’’ suggesting that it is not immune-mediated.1 In humans, liver injury has a delayed onset, and mild injury resolves despite continued treatment with drug;3 therefore, the mechanism in the animal model is likely to be different from the mechanism in humans. Recently, we found that INH is directly bioactivated and covalently binds to liver proteins in mice and human hepatic microsomes. Covalent binding of INH in rats is much less than in mice; thus, mice appear to be a better model for humans than rats.4 We have argued that the mechanism of this drug-induced liver injury involves an immune response.5 Hypotheses: Covalent binding of INH to human liver can result in an immune response that causes liver injury and mild cases of hepatotoxicity that resolve despite continued treatment represent immune tolerance. Methods and Results: Covalent binding of INH was identified in a human liver biopsy from a patient with INH-induced liver failure by using the same anti-INH antibody that we used to determine covalent binding to rodent livers. We found anti-INH and/or anti-P450 antibodies in 15 out of 19 patients with INH-induced liver failure, which also strongly suggests that INH-induced liver injury is immune-mediated. In contrast, no anti-drug or anti-P450 antibodies were found in the serum of patients who received INH but did not experience severe liver injury. In five patients who developed mild INH-induced liver injury, we found an increase in Th17 cells and IL-10-producing cells; no change in these cell types was observed in patients taking INH without liver injury. IL-10 is produced by cells that are involved in the induction of immune

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tolerance; therefore, these findings are consistent with INH-induced immune tolerance that limits more severe liver injury. Conclusions: These studies demonstrate that acetylhydrazine is not the only INH metabolite responsible for covalent binding the accepted hypothesis for INH-induced hepatotoxicity for over 40 years. In humans, we identified anti-INH and autoantibodies,6which suggests that the immune response is directed against INH-modified proteins. Mild injury is associated with a Th17 immune response, which appears to be down-regulated by the anti-inflammatory effects of IL10. These results strongly support the hypothesis that INH-induced liver injury is an immune-mediated reaction against INH-modified proteins and not ‘‘metabolic idiosyncrasy’’.

References


1. 2. 3. 4. 5. 6.

Mitchell, J. R. et al. Ann. Intern. Med. 84, 181–192, (1976). Mitchell, J. R.et al. Chest 68, 181–190, (1975). Maddrey, W. C. & Boitnott, J. K. Ann. Intern. Med. 79, 1–12, (1973). Metushi, I. G.et al. Chem. Res. Toxicol. 25, 2567–2576, (2012). Metushi, I. G.et al. Clin. Pharmacol. Ther. 89, 911–914, (2011). Metushi, I. & Uetrecht, J. Hepatology. Accepted, (2013).

P31. COMPARATIVE STUDY OF EXPRESSION OF SMAD3 IN ORAL LICHEN PLANUS AND NORMAL ORAL MUCOSA Shimae Nafarzadeh1, Pouyan amini Shakib2, Amrollah mostafa Zadeh3 and Samad Ejtehadi4 1 Oral and Maxillofacial pathology, Assistant professor,Oral Pahology department,Babol medical school, Babol, Iran, 2Oral Pathology, Assistant professor,Oral PAthology department,Babol medical school,Iran, Babol, Iran,3Immunology, Assistant professor,Oral PAthology department,Babol medical school,Iran, Babol, Iran, 4Dentist, Assistant professor,Oral PAthology department,Babol medical school,Iran, Babol, Iran Background: Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa which the World Health Organization (WHO) considers a premalignant condition. One step in malignant development is so called epithelial mesenchymal transition (EMT), a process whereby epithelial cells acquire mesenchymal characteristics. A factor known to induce EMT is transforming growth factor-b (TGF-b), which uses the Smad proteins as mediators for its signaling. The aim of this study was comparison of expression of Smad3 in Oral Lichen Planus and normal oral mucosa. Methods and Materials: This descriptive analytic study was performed on 30 patients with OLP (21 women and 9 men with mean of age 45.23 2.44) and 20 normal oral mucosa (14 women and 6 men with main of age 46.95 2.21). The samples were supplied from archive of our oral pathology department. 4 microns slices prepared from paraffin blocks, were stained immunohistochemically using standard methods. Data was analyzed with paired T-test and Wilcoxon by SPSS software. Results: Expression of Smad3 in OLP samples was 64.5 30.17 and 11.25 6.85 in normal oral mucosa. There was significant difference between their expression (p50.001). Conclusion: It seems that expression of Smad3 is higher in oral lichen planus compared to normal oral mucosa which might help to discuss its higher potential for malignant transition. Key message: It seems that OLP is a premalignant lesion, so the treatment modalities should be performed based on this consideration to prevent probable malignant transformation.

P32. DNA METHYLATION PROFILES OF MATERNAL PERIPHERAL BLOOD AND INTRAUTERINE GROWTH RESTRICTION Lian Liu, Jie Ping Sr. and Hui Wang Pharmacology, Medical School of Wuhan University, Wuhan, China Intrauterine growth restriction (IUGR) remains a leading contributor to perinatal mortality and morbidity of metabolic syndrome in later life. Nowadays, its early diagnosis is still difficult, and the effect of clinical management is poor. The majority of IUGR are thought to result from the interaction between multiple genetic, epigenetic, environmental and lifestyle factors. Epigenetic mechanisms are attractive targets in the study of complex diseases because they may be altered by environmental factors and dietary interventions. This study was designed to search the possible epigenetic biomarkers of IUGR from maternal peripheral blood. At first, we established a commonly accepted animal model of IUGR. Pregnant Wistar rats were subcutaneously treated with 2.0 mg/kg nicotine from gestational days 8–17. The methylated DNA ImmunoPrecipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify genome-wide maternal DNA methylation in peripheral blood. Some differentially methylated DNA regions were confirmed by bisulfite-sequencing PCR (BSP). The results show that the promoter methylation state of 1798 genes were changed in IUGR group; of these genes, 1302 were hypermethylated and 496 genes were hypomethylated compared with the control. The majority of differentially methylated CpG sites located within CpG islands in promoter region. Gene Set Enrichment Analysis (GSEA) revealed that the genes of interest were enriched in multiple biological processes involved in maternal endocrine and fetal development. For example,

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Wnt signaling pathway, TGF-beta signaling pathway, biopolymer metabolic process, nucleic acid metabolism and organismal development. We present preliminary evidence that alterations in maternal DNA methylation may be associated with IUGR. These differentially methylated genes may be the potential biomarkers to advance IUGR diagnosis. Our results suggest that further studies involving maternal epigenetic patterns and IUGR are warranted. Multiple candidate processes and pathways for future study have been identified. Supported by the National Natural Science Foundation of China (No. 81273107, 81173138, 30800931) and Outstanding Youth Science Fund of Hubei Province (No. 2012FFA017); *Correspondence author: [email protected]

P33. QUANTITATIVE TARGETED ABSOLUTE PROTEOMICS OF THE MALIGNANT BRAIN TUMORS TOWARDS MOLECULAR-TARGETED CHEMOTHERAPY


Tetsuya Terasaki1, Wataru Obuchi1, Sumio Ohtsuki2, Mitsutoshi Nakada3, Yasuo Uchida1, Masanori Tachikawa1 and Jun-ichiro Hamada3 1 Tohoku University, Sendai, Japan, 2Kumamoto University, Kumamoto, Japan, 3Kanazawa University, Kanazawa, Japan Malignant brain tumors are aggressive and incurable, and the molecular-targeted (MT) drugs-based chemotherapy has been expected as a treatment strategy of the malignant brain tumors. Since the MT drugs interact with the membrane receptor proteins for providing their anti-tumor effects, the protein levels of target molecules could be the most potent biomarkers for choosing the MT drug and predicting their efficacy. The purpose of this study was to investigate the individual differences in the expression levels of the membrane proteins such as the MT drugs target receptors and the transporters which affect the MT drug delivery to the tumors by means of LC-MS/MS-based quantitative targeted absolute proteomics (QTAP). Plasma membrane fractions were prepared from the surgical specimen of malignant brain tumors. These protein fractions were denatured and digested by trypsin. The QTAP analysis of the surgical specimens was performed for the membrane proteins of target receptors and transporters. This study was approved by the ethical committees in the authors’ institutions. The wide range of differences in the protein expression levels was detected among the surgical specimens of malignant brain tumors. The expression levels of epidermal growth factor receptor (EGFR) exhibited over 1900-fold individual difference. In contrast, the expression of plateletderived growth factor receptor (PDGFR) a, PDGFRb, and tyrosine-protein kinase c-kit was detected in the limited numbers of the malignant brain tumor patients. As for transporter proteins, the expression levels of several ATP-binding cassette (ABC) transporters, e.g., ABCB1, ABCC1, ABCC4, and ABCG2, were up-regulated in the malignant brain tumors compared to those in the normal brain. In the patient who was diagnosed as anaplastic meningioma, PDGFRb exhibited the highest expression level among the receptor proteins examined. The expression level of PDGFRb in this patient was greater than the average expression levels obtained from other malignant brain tumors. These results of QTAP analysis using the surgical specimen of malignant brain tumors suggest that the efficacy of MT drug-based chemotherapy varies among the patients due to the individual differences in the membrane receptor and transporter protein expressions. Therefore, QTAP-based MT chemotherapy would be a promising strategy to select appropriate MT drugs towards the personalized treatment of the malignant brain tumors.

P34. PLASMA DIHYDROURACIL/URACIL RATIO AS A PREDICTIVE BIOMARKER FOR THE EFFECT AND PHARMACOKINETICS OF 5-FLUOROURACIL IN COLORECTAL TUMOR GROWTH Yukako Ito, Shinji Kobuchi and Kanji Takada Pharmacokinetics, Kyoto Pharmaceutical University, Kyoto, Japan 5-Fluorouracil (5-FU) is an anticancer agent widely used in the treatment of colorectal cancer (CRC). For a personalized medicine approach, current research is focusing on identifying predictive biomarkers of 5-FU efficacy and its incidence of severe toxicity-related side effects. Considering that in a rate-limiting step, uracil (Ura) is metabolized by dihydropyrimidine dehydrogenase (DPD), the enzyme also responsible for the degradation of 5-FU, plasma measurement of Ura and its breakdown product dihydrouracil (UH2) may be useful in predicting 5-FU degradation, as well as DPD activity. Given the relationship between plasma concentrations of 5-FU and its treatment effectiveness, the plasma UH2/Ura ratio (measured prior to 5-FU treatment) may be predictive of 5-FU treatment efficacy. To investigate this hypothesis, 5-FU (20 mg/kg) was administered intravenously to rats with 1,2-dimethylhydrazine-induced CRC (CRC rats) at different times of the day, for 7 days, to determine circadian plasma UH2/Ura ratios and 5-FU pharmacokinetic (PK) outcomes. After 5-FU administration, linear correlations were observed between plasma UH2/Ura ratios and both drug clearance (CL; r2 ¼ 0.905) and the area under the plasma concentration– time curve (AUC0–1; r2 ¼ 0.843), suggesting that the plasma UH2/Ura ratio is a useful predictive biomarker of 5-FU CL and AUC0–1. The 5-FU metabolism showed a circadian variation with the hepatic DPD activity. Furthermore, we conducted pharmacodynamic (PD) studies in CRC rats by using 5-FU for 7 days to investigate a possible correlation between the plasma UH2/Ura ratio determined before 5-FU treatment and the effects of 5-FU on the tumor growth. Interestingly, significant differences in tumor volume (TV) were observed between the treatment groups on Day 10; TV on Day 10 was the highest in rats with high DPD activity (1.56 0.40 mm3) and the lowest in rats with low DPD activity (0.57 0.21 mm3). A linear correlation

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(r2 ¼ 0.988) was obtained when plotting the TV on Day 10 after 5-FU treatments against the plasma UH2/Ura ratio prior to treatment. The fluctuations in 5-FU concentrations caused by a variation in the DPD activity affected TV. These results suggest that plasma UH2/Ura ratio measurement prior to 5-FU treatment may predict DPD activity, 5-FU PK parameters, and treatment efficacy. Predicting the efficacy of 5-FU by using the plasma UH2/Ura ratio as shown here would enable the selection of an optimal therapeutic strategy for each CRC patient.

P35. EVALUATION OF 4BETA-HYDROXYCHOLESTEROL AS AN ENDOGENOUS BIOMARKER OF CYP3A IN CYNOMOLGUS MONKEYS


Ke Li1, Lingling Zhang2, Xiaoyun Wu1, Pan Shu2, Yang Wang1, Hao Feng2, Zheming Gu2, Helen Han Hsu1 and Sylvia Zhao1 1 Drug Safety & Sciences, Asia Pacific, Janssen Research & Development, Shanghai, China, 2XenoBiotic Laboratories (China), Inc, Nanjing, China Cytochrome P450 3A (CYP3A) is well known as the most abundant P450 enzyme involved in the elimination of a wide variety of chemicals including xenobiotics and endogenous compounds of distinct structures. It has been proposed that in human 4b-hydroxycholesterol is formed by CYP3A catalyzed metabolism of cholesterol and thus may serve as an endogenous marker of CYP3A activity. Cynomolgus and rhesus monkeys are widely used as one of the non-rodent preclinical safety species in drug research and development throughout the pharmaceutical industry, partly due to the similar metabolic capabilities of monkey to those of human. In the current study the potential application of 4b-hydroxycholesterol as an endogenous biomarker of CYP3A in response to drug treatment was evaluated in cynomolgus monkeys. Following multiple oral administration of rifampicin at 15 mg/kg/day in cynomolgus monkeys, the mean serum 4b-hydroxycholesterol levels increased by 3-fold from the base line of 55.3 21.7 to 221 53.4 ng/mL. The mean concentration ratios of 4b-hydroxycholesterol/cholesterol also increased by 4-fold. The data suggest that 4b-hydroxycholesterol generation from cholesterol metabolism was induced by rifampicin treatment in the monkeys. This observation correlated with midazolam metabolism, which is often used as exogenous biomarker of CYP3A activity, monitored in the same study. The serum exposure (AUC) of midazolam was markedly decreased by ca. 95%, indicating induction of CYP3A catalytic activity byrifampicin treatment in the monkeys. The involvement of CYP3A8 in 4b-hydroxycholesterol generation from cholesterol is then phenotyped in vitro using monkey liver microsomes and recombinant CYP isozymes. The enzyme kinetics of 4b-hydroxycholesterol generation from cholesterol is also determined. In conclusion, results from the current study suggest that 4b-hydroxycholesterol may be monitored as an endogenous biomarker of CYP3A activity and may find its application in assessing the effect of drug treatment on the enzyme activity in cynomolgus monkeys. In particular it may be used to identify potential strong CYP3A inducers at preclinical stage, which may provide helpful information to predict drug-drug interaction potential in humans.

P36. AD HOC CAFFEINE PHENOTYPING FOR CYP1A2, CYP2A6, XO AND NAT2 ACTIVITY IN ADULTS AND CHILDREN Yvonne S. Lin1, Brian Phillips1, Zachariah Guerrette2, Melinda Vredevoogd2, Eric Vigoren2 and Elaine M. Faustman2 1 Pharmaceutics, University of Washington, Seattle, WA, 2Environmental and Occupational Health Sciences, University of Washington, Seattle, WA Caffeine is a commonly used probe substrate for cytochrome P450 1A2 (CYP1A2), 2A6 (CYP2A6), xanthine oxidase (XO) and N-acetyltransferase 2 (NAT2) phenotyping. Urinary ratios of caffeine metabolites have been used to demonstrate the effect of genetic, environmental and ethnic differences on CYP1A2, CYP2A6, XO and NAT2 activity. The conduct of caffeine phenotyping studies is hampered by the ubiquitous nature of endogenous caffeine metabolites, even after subjects are placed on restricted diets for several days. In this study, we exploit the high background levels of caffeine and its metabolites to determine whether reliable phenotyping ratios can be ascertained from spot urine collections. Participants were part of the University of Washington Center for Child Environmental Health Risks Research (CHC) study. We analyzed urine collected from twelve families (adult-child pairs) on two sample collection days during three agricultural seasons. A specific and sensitive LC-MS/MS method was developed to quantitate the methyl xanthine metabolites of caffeine (1-methyl xanthine, 3-methyl xanthine, 7-methyl xanthine, 3,7-dimethyl xanthine, 1,3-dimethyl xanthine, 1,7-dimethyl xanthine and 1,3,7-trimethyl xanthine (caffeine)) and methyl uric acid metabolites (1-methyl uric acid, 3-methyl uric acid, 7-methyl uric acid, 3,7-dimethyl uric acid, 1,3-dimethyl uric acid, 1,7-dimethyl uric acid and 1,3,7-trimethyl uric acid, and 5-acetylamino-6-formulamino-3-methyluracil (AAMU)). We calculated six metabolite ratios for CYP1A2 activity, three metabolite ratios for NAT2 activity, two metabolite ratios for XO activity, and two metabolite ratios for CYP2A6 activity. For each subject, we determined the interday coefficient of variation for each metabolite ratio. In addition, we tested whether the caffeine metabolite ratios were associated with genetic variants of CYP1A2, CYP2A6, XO and NAT2. Caffeine was detectable in 75% and 43% of urine samples from adults and children, respectively. We found that the metabolite ratios with the lowest mean interday variation from spot urine collections were the same metabolite ratios that are typically used for caffeine phenotyping. Our ad hoc metabolite ratios were similar to ratios reported in the literature following caffeine phenotyping. Moreover, the NAT2 metabolite ratio determined from these spot urine

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samples differed in individuals with slow and fast acetylator genotypes. Though further work will need to be done to validate these findings, ad hoc caffeine phenotyping may be used to predict CYP1A2, CYP2A6, XO and NAT2 activity in adults and children.

P37. LIPIDOMIC PROFILES IN BLOOD FROM FASTED HEALTHY ADULTS VARY BETWEEN PLASMA AND SERUM AND BY SUBJECT’S GENDERS AND AGES


Masaki Ishikawa1, Keiko Maekawa1, Yuya Senoo1, Yoko Tajima1, Kosuke Saito1, Masayo Urata1, Mayumi Murayama1, Yuji Kumagai2 and Yoshiro Saito1 1 Division of Medicinal Safety Science, National Institute of Health Sciences, Tokyo, Japan, 2Clinical Trial Center, Kitasato University East Hospital, Kanagawa, Japan Background and purpose: Blood is the frequently utilized biofluid for biomarker exploration because it contains a wealth of molecules and is collected minimally invasive. Lipid metabolites in the blood have biological functions and thus would be candidate biomarkers to reflect disease states and responses of therapeutic drugs. However, potential confounding factors in biomarker exploration studies which affect lipidomic profiles in the blood have not well evaluated. This study aimed to elucidate the effects of sample collection (difference in matrices and subject’s ages and genders) and storage conditions (with or without repeated freeze-thaw cycle) on lipid metabolites levels in the blood of healthy adults. Methods: Serum and plasma of 60 Caucasians were purchased. According to manufacturer’s data sheets, they were prepared from overnight fasting blood of young (25–34 years old) or elderly (55–64 years old) healthy subjects of both genders. After 2 or 10 freeze-thaw cycle, their lipid metabolites were extracted and measured using liquid chromatography-MS(/MS). Results and discussion: A total of 254 lipid metabolites were identified consisting of 92 glycerophospholipids, 37 sphingolipids, 13 sterols, 1 prenol, 86 simple glycerolipids, and 25 fatty acids and their derivatives. Many lysophosphatidylcholines anddiacylglycerols were present at significantly higher levels in serum than in plasma. In addition, levels of many eicosanoids such as 12-hydroxy eicosatetraenoic acid and thromboxane B2 were significantly different between plasma and serum, suggesting that coagulation process influences levels of many lipid species in serum. Levels of many sphingomyelins were significantly higher in females than in males irrespective of differences in matrices (plasma and serum) and ages of subjects. Age-associated differences were more predominant in females than in males, and many triacylglycerols showed significantly higher levels in elderly females than in young females. Most lipid metabolites were decreased in repeated freeze-thawed plasma and serum. Conclusion: Plasma might be more suitable matrix for measuring lipid metabolites related to coagulation. The metabolites with different levels among gender- or age-biased samples should be paid attention in biomarker exploration and diagnosis using them. Our fundamental information on sample collection and handling issues for measuring blood lipid metabolites is useful for ensuring the quality of biomarker discovery and qualification process.

References Ishikawa M, Tajima Y, Murayama M, Senoo Y, Maekawa K, Saito Y., Plasma and serum from nonfasting men and women differ in their lipidomic profiles. Biol Pharm Bull. 2013;36(4):682–5.

P38. URINARY METABOLIC PATTERN OF VINCLOZOLIN IN ADULT MALE RAT M. Cruz-Hurtado, M.L. Lo´pez-Gonza´lez, D.C. Escobar-Wilches and A. Sierra-Santoyo Toxicologıá, Cinvestav-IPN, Me´xico D.F., Mexico The dicarboximide fungicide vinclozolin (V) is used for agricultural settings (1). V has been classified as an endocrine disruptor by interacting with the androgen receptor (2). In rat serum it is non-enzymatically hydrolyzed to M1 and M2 metabolites and biotransformed to seven more metabolites by cytochrome P450 (3). M4 and M5 glucuronides have been proposed as the main elimination products of V in urine and feces (4). M3 (3,5-dichloroaniline) metabolite has been used as a biomarker of V exposure, however, it is also a metabolic product of other dicarboximides. Thus, this metabolite is not specific for V exposure. The objective of this study was to determine the metabolic pattern of V in rat urine. Adult male Wistar rats were administered an oral dose of V (100 mg/kg) suspended in corn oil. Urine was collected at different times after dosing. V metabolites were extracted from urine using C-18 cartridges, after that they were enzymatically hydrolyzed whit b-glucuronidase/sulfatase of H. pomatia and analyzed by HPLC/DAD. Urinary pharmacokinetic parameters were calculated using adjusted concentrations by creatinine. V and M5, M1, M3 metabolites were detected. M5 remained the main metabolite, it represented about 80% of total excreted metabolites and was easily detected until 168 h after dosing (t1/2 elim 22.6 h). M1 was the second most important metabolite; it was detected until 144 h at trace levels. V was detected only before 48 h at very low levels and M2 was detected until 8 h. These results indicate that M5 may be used as a V biomarker for pharmacokinetic modeling. In addition, they may clarify the relationship between toxicity and tissue dose of V and its metabolites. Funded by Conacyt No. 168384.

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References [1] Szeto, S.Y., Burlinson, N. E., y Oloffs, P. C. Kinetics of hydrolysis of the dicarboximide fungicide vinclozolin. J Agric Food Chem 37, 523–529 (1989). [2] Otto, S., Bentel, O., Elzner, K., y Ohnsorg. Metabolism of 14C-vinclozolin in rats. Unpublished report from BASF AG, Kdwigshafen, German. Submitted to WHO by BASF AG, Ludwigshafen, Germany (1977). [3] Sierra-Santoyo, A., Castan˜eda-Hernańdez, G., y Harrison, R. Pharmacokinetics and dosimetry of the antiandrogen vinclozolin after oral administration in the rat. Toxicol. Sciences 1096:55–63 (2008). [4] Bursztyka, J., Debrauwer, L., Perdu, E., Jouanin, E., Jaeg, J., y Cravedi, J. Biotransformation of vinclozolin in rat precisioncut liver slices: comparason with in vivo metabolic pattern. J. Agric. Food Chem. 56:4832–4839 (2008).


P39. HBMEC/CIB, NEWLY-DEVELOPED CONDITIONALLY IMMORTALIZED HUMAN BRAIN MICROVASCULAR ENDOTHELIAL CELLS, EXPRESS VARIOUS RECEPTORS INVOLVED IN TRANSCYTOSIS AT THE BLOOD-BRAIN BARRIER Tomomi Furihata, Shinya Kawamatsu, Ryo Ito, Atsuko Kamiichi, Satoshi Kishida and Kan Chiba Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University., Chiba-Shi, Japan Biopharmaceutical agents generally cannot cross the blood-brain barrier (BBB), which is formed by brain microvascular endothelial cells (BMEC), together with astrocytes and pericytes. One of the ways to deliver the agents to the brain parenchyma is utilization of transcytotic pathways, which are mediated by the plasma membrane receptors existing in BMEC, such as low density lipoprotein receptor (LDLR)-related protein 1 (LRP1). Therefore, it is expected that BMEC functionally expressing various transcytotic receptor genes can provide useful in vitro BBB models for screening of transcytosis-based central nervous system drugs. Recently, we have established a new immortalized human BMEC line (HBMEC/cib), the immortalized state of which can be reversed by culturing at higher temperature to induce cell differentiation. While the cells show favorable junctional and drug efflux transporter functions, it remains undetermined whether HBMEC/cib express functional transcytotic receptors. Therefore, in the present study, we aimed to clarify expression profile of transcytotic receptors in HBMEC/cib. The results of reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis showed that HBMEC/cib expressed various transcytotic receptor mRNAs at the levels comparable to those in primary human BMEC, along with LRP1, LDLR, transmembrane protein 30A and transferrin receptor protein expression. Then, we examined effects of higher culture temperature on the transcytotic receptor expression levels in the cells by real-time PCR and Western blotting analysis. The results showed that, among the receptors, LRP1 mRNA level increased more than 4-fold in the differentiated culture condition compared with that in the normal condition. Consistently, the LRP1 protein level also significantly increased in the differentiated condition. In conclusion, we have demonstrated that HBMEC/cib expresses various transcytotic receptor genes. Therefore, it is highly expected that HBMEC/cib provides a valuable BBB model for development of biopharmaceutical agents that are efficiently delivered into the brain via the receptor-mediated transcytosis. To the best of our knowledge, this is the first study to identify the immortalized BMEC clearly expressing the LRP1 protein. Accordingly, further studies aimed at clarification of the functional property of LRP1 in HBMEC/cib are currently ongoing.

P40. EVALUATION OF CASSETTE DOSING TO SCREEN CNS COMPOUNDS IN DRUG DISCOVERY Gauri Deshmukh, Jacob Chen, Bilin Chou and Bianca Liederer Dmpk, Genentech Inc., South San Francisco, CA For CNS programs, the efficiency of compounds to cross the blood brain barrier (BBB) is assessed using the ratio of unbound brain to unbound plasma concentration, Kp,uu. Traditional experiments are low throughput, since Kp is determined at steady state after IV infusion or from AUC (area under the curve) of brain and plasma concentrations after single discrete dose. Cassette dosing has been extensively used in the industry as a quick, reliable, and high throughput screening tool to access pharmacokinetics of large number of compounds. The objective of this study was to evaluate brain to plasma and CSF to plasma AUC ratios, Kp of discovery compounds using cassette dosing. This method was validated previously using known P-pg and Bcrp substrates and inhibitors (Liu et al., 2012) and we wanted to establish if it could be implemented to our internal programs. Free plasma and brain binding, fu was determined for 10 discovery compounds by equilibrium dialysis at 37 C for 6 hours (h) using an HTdialysis 96 well equilibrium dialysis block. In vivo pharmacokinetics were evaluated in Sprague-Dawley rats after single bolus subcutaneous administration (n ¼ 1 per time point) as a cassette at 0.6–1 mg/kg/compound or discrete intravenous administration (n ¼ 3 per group for plasma and n ¼ 1 per time point for brain and CSF) at 0.5–1 mg/kg. Plasma, brain, and CSF samples from cassette dosing were analyzed using the mass spectrometer response, namely peak area ratio between compounds and the internal standards in plasma, brain, and CSF mixed matrix, while these samples from discrete dosing were quantitated using standard curves for actual concentrations. AUC values were calculated using mass spectrometer response for plasma (AUCp,R), brain (AUCb,R), and CSF (AUCc,R) in unit of response.hour (R.h) from cassette dosing. AUC values for discrete

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dosing were calculated for the actual brain, CSF and plasma concentrations. Our data demonstrates that the Kp values obtained from discrete and cassette dosing are within 2-fold and showed a good correlation. Samples analysis using mass spectrometer response is an efficient technique to reduce bioanalytical work required to quantitate actual concentration from matrices.

Reference Xingrong Liu, Xiao Ding, Gauri Deshmukh, Bianca M. Liederer, and Cornelis E.C.A. Hop., Use of Cassette-Dosing Approach to Assess Brain Penetration in Drug Discovery. Drug Metab Dispos 40:963–969, 2012.

P41. EVALUATION OF CASSETTE DOSING TO ENHANCE THE THROUGHPUT FOR RAT BRAIN MICRODIALYSIS STUDIES IN DRUG DISCOVERY


Gauri Deshmukh1, Kefeng Sun2, Xiao Ding1, Nicole Valle3, Bianca Liederer1 and Xingrong Liu1 1 Dmpk, Genentech Inc., South San Francisco, CA, 2University of Michigan, Ann Arbor, MI, 3IVS, Genentech Inc., South San Francisco, CA Rat brain microdialysis is a well-established sampling method that allows continous sampling of interstitial fluid (ISF) to determine the unbound drug concentrations in the brain. However, low throughput and high labor intensive procedure limits its application in drug discovery. Recently, we have demonstrated that cassette dosing (N-in-1) can be used to increase the throughput to assess brain penetration in rodents at low dose levels (1–3 mg/kg) with minimal drug-drug interaction risk at the blood-brain barrier (Liu et al., 2012). The objective of this study was to assess whether cassette dosing can be used to increase the throughput of rat brain microdialysis. Jugular and femoral-cannulated male Sprague-Dawley rats with surgically implanted microdialysis guide cannula were used. Eight compounds (Carbamazepine, Citalopram, Desmethylclozapine, Diphenhydramine, Gabapentin, Metoclopramide, Naltrexone, Stavudine) were examined in discrete dosing and cassette dosing in rat brain microdialysis at 0.5–3.3 mg/kg Intravenous (IV) bolus dose followed by IV infusion via femoral cannula at 0.5–3.3 mg/hr/kg for 6 hours. In vitro recovery for these 8 compounds was determined by gain method in discrete and cassette. The in vitro recovery in cassette was 125 40% (mean SD) of that in discrete. For 6 out of the 8 compounds, the ratio at 6 hours for CECF (concentration of extravascular fluid, which was the dialysate concentration corrected with in vitro recovery) to Cup (unbound plasma concentration), CECF to Cub (unbound brain concentration), and CECF to CSF were 0.946 0.415, 1.35 1.25, and 1.39 0.41, respectively. The available data suggest that cassette dosing has potentials in enhancing the throughput of brain microdialysis studies.

Reference Xingrong Liu, Xiao Ding, Gauri Deshmukh, Bianca M. Liederer, and Cornelis E.G.A. Hop., Use of Cassette Dosing Approach to Assess Brain Penetration in Drug Discovery. Drug Metab Dispos 40:963–969, 2012.

P42. ROLE OF PANNEXIN AND CONNEXIN HEMICHANNELS IN THE PATHOLOGICAL HUMAN BLOOD-BRAIN BARRIER TRANSPORT Masanori Tachikawa1, Yosuke Kaneko1, Yasuo Uchida1, Sumio Ohtsuki2 and Tetsuya Terasaki1 1 Tohoku University, Sendai, Japan, 2Kumamoto University, Kumamoto, Japan The blood–brain barrier (BBB) possesses a variety of transporters, receptors, and channels which are involved in the highly regulated transport between the circulating blood and brain. It has long been postulated that the BBB transport dysfunction under the pathological conditions such as stroke, triggers the neuronal damage. Here, we hypothesized that the pannexin and connexin hemichannels would be an initiating factor involved in the BBB transport alteration. Although the hemichannels have been recognized as key components of gap junctions which play a role in electrical and metabolic communication between cells, there has been emerging evidence that they are also functional in single membranes. The purpose of this study was to investigate the hemichanel(s)-mediated transport at the human BBB and to identify drugs which can protect the BBB transport system under pathological conditions. We have established LC-MS/MS-based quantitative targeted proteomics for human and mouse hemichannels using a series of internal standards of stable isotope labeled peptides which sequences were selected by in silico peptide selection criteria. Membrane protein fraction was prepared from the human brain capillary endothelial cell line (hCMEC/ D3), a novel in vitro human BBB model, and digested by trypsin after denaturing. The hemichannel-mediated alteration of the BBB transport under pathological conditions was examined using hCMEC/D3 cells. The LC-MS/MS-based targeted proteomics revealed that two subtypes of pannexin and connexin hemichannels could simultaneously be quantified in hCMEC/D3 cells. The uptake of cationic and anionic fluorescent substrates for hemichannels by hCMEC/D3 cells was significantly increased under the extracellular calcium-free conditions. The uptake was significantly inhibited by carbenoxolone, a potent hemichannel

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inhibitor, various endogenous compounds, the subtype-specific siRNAs and/or a mimetic blocking peptide. The fluorescent substrates uptake by hCMEC/D3 cells exhibited concentration dependence with the approximate Km values of 20 mM. Among the drugs which have been reported to exhibit the brain protection from ischemic damage, several drugs were found as the BBB hemichannel inhibitors. These lines of evidence suggest that the specific subtypes of hemichannels could alter the BBB transport system under pathological conditions, presumably causing exaggeration of the brain damage. The BBB hemichannels inhibitors identified may be potential drugs which can protect from the brain damage.

P43. EXPRESSIONAL AND FUNCTIONAL CHARACTERIZATION OF A RAT PRIMARY CO-CULTURED BLOOD-BRAIN BARRIER MODEL


Houfu Liu, Yang Li, Sijie Lu, Yiwen Wu and Jasminder Sahi Dmpk, GSK R&D China, Shanghai, China Designing potent molecules that traverse the blood-brain barrier (BBB) is a challenge for neuroscience drug discovery. Cell lines of intestinal (e.g. Caco-2) or renal (e.g. MDCK) origin, are typically utilized for early screening of potential central nervous system (CNS) uptake. In contrast, relatively limited studies are conducted with primary endothelial cells isolated from capillaries at the BBB. Since the expression and activity of hepatocyte transporters changes over time in primary culture, our first goal was to assess if transporters in co-cultures of brain microvessel endothelial cells and astrocytes (BMEC) also exhibit temporal expression. For this, we characterized mRNA expression of the major CNS transporters and receptors, and the transporter regulators Pxr, Ahr and Car in rat BMEC over six days of primary culture. This helped define appropriate culture conditions and time post-plating to conduct permeability assessments. Finally, we compared uptake across the rat primary co-cultures with stably transfected MDCKII-MDR1 cells and compared our data to rat brain in situ perfusion data from literature. We demonstrate differential changes of expression of individual transporters and receptors over the culture period. Expression of efflux (ABC) transporters Mdr1a, Bcrp-1, Mdr1b, Mrp1, and Mrp4 was retained better than that of the uptake (SLC) transporters Oatp1a4, Oat1, Oat3, and Glut-1. ABC transporters, with the exception of Mdr1b presented mRNA expression that was 28–101% of vivo levels, while the SLC transporters lost expression rapidly and permanently over the six days in culture. Mdr1b was the only gene upregulated over this time period. The insulin receptor maintained expression similar to in vivo level, while transferrin receptor and low-density lipoprotein receptor-related protein-1 lost expression rapidly in culture. AhR mRNA levels were higher in the rat brain capillaries than Pxr and Car, and expression increased on the last day of cell culture. P-gp, Bcrp-1 and MRP4 were not inducible by agonists of Pxr and AhR in the in vitro system. In line with their high mRNA expression, P-gp and Bcrp-1 were functional, as indicated by directional transport of amprenavir and dantrolene across rBMEC monolayers. Permeability measurements with 17 CNS market drugs indicated that both in vitro models correlate significantly and similarly with rat in situ permeability, corrected for brain unbound fraction. Our data provides a detailed analysis of the potential utilities of primary BMEC cells and facilitates the rational use of this in vitro rat BBB model.

P44. COMBINED CONTRIBUTIONS OF EPILEPTIC SEIZURE AND ANTI-EPILEPTIC DRUG INDUCTION TO OVEREXPRESSION OF P-GLYCOPROTEIN AT BRAIN OF PHARMACORESISTANT EPILEPSY Xiao-dong Liu Sr. College of Pharmacy, China Pharmaceutical University, Nanjing, China Epilepsy is one of the most frequent neurological disorders. Accumulating evidence has shown that 20–30% of patients suffering from epilepsy are pharmacoresistant. Clinic trials and animal experiments indicate that overexpression of P-glycoprotein (P-GP) at the blood–brain barrier (BBB) is considered to be a reason of pharmacoresistant epilepsy. BBB can protect the brain from exogenous toxins, which becomes a main reason that brain is refractory to many drugs. P-GP, widely expressed in the luminal membrane of the microvessel endothelium and in the apical membrane of the choroids plexus epithelium, plays an important role in the integrity of BBB. Here, we reviewed our previous findings about the up-regulation of P-GP function and expression in BBB of chemically kindled-epileptic rats. Kindled-epileptic rats were developed by injecting subconvulsive dose of pentylenetetrazole (PTZ) for 28 days. Typical anti-epileptic drug(ADE) phenobarbital(PB), whose transport across BBB was first identified to be mediated by P-GP (1), served as a model drug. The results showed that PTZ-kindling may significantly increase expression and function of P-GP in brain, resulting in lower accumulation of PB in brain. P-GP inhibitor cyclosporine A may increase brain PB levels and potentiate the anticonvulsive effects of PB (2). It is generally accepted that epileptogenesis is often accompanied by repetitively temporal hypoxia/ischemia. Primary cultured rat brain microvessel endothelial cells were used as in vitro model. The cells were exposure to 15-min hypoxia every day up to 8 days via covering sterile paraffin oil. Function and levels of P-GP in the cells were measured using rhodamine 123 uptake and western blot, respectively. It was found that 8temporal hypoxic exposures induced 1.6-fold increase of P-gp level in cells, accompanied by lower cellular accumulation of rhodamine 123 as well as lower cellular accumulation of PB, inferring that repetitive/temporal hypoxia may be one of the factors resulting in P-gp overexpression in refractory epilepsy (3). Inducing effects of ADEs including PB, phenytoin, carbamazepine and valproic acid on function and expression of P-GP at brain were also identified in vivo and in vitro, indicating roles of ADEs


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in function and expression of P-GP (4,5). All these results suggested that the overexpression of P-glycoprotein in the brains of subjects with pharmacoresistant epilepsy results from both drug inducing effects and seizure activity. Further experiment was documented to verify the deduction. The PTZ-kindled epileptic rats were treated with oral dose of PB for 40 days and anticonvulsive effects of PB were observed during treatment. In accord with behavioral observations, P-GP activity and expression in brain regions were assessed using brain-to-plasma concentration ratios of rhodamine 123, RT-PCR and Western blot analysis, respectively. The distribution of phenobarbital in the brain was also assessed. It was consistent with our expectation that the kindling significantly increased P-glycoprotein activity and expression. Good associations were found among P-GP activity, expression and lower PB concentration in the hippocampus. Short-term treatment with PB showed good anti-epileptic effect; the maximum effect occurred on day 14 when overexpression of P-GP was reversed. However, continuous treatment with PB had gradually reduced anti-epileptic effect. And on day 40, phenobarbital exhibited no anti-epileptic effect, which was accompanied by both a re-enhancement of P-GP and the lower phenobarbital concentration in the hippocampus. It was also found that PB treatment increased P-GP function and expression in brain of age-matched normal rats. All these results gave a conclusion that overexpression of P-GP in the brain of subjects with pharmacoresistant epilepsy is due to a combination of drug inducing effects and epileptic seizures (6). The overexpression of P-GP in the brain is induced by the epileptic seizures during the early stages and by the combined effects of chronic drug treatment and epilepsy at the later stages. In addition, the expression and function of MRP2, another efflux transporter expressed in the BBB were also reported to be up-regulated in brain of PTZkindled epileptic rats (7). Key words: P-glycoprotein, pharmacoresistant epilepsy, blood brain barrier. phenobarbital, drug induction.

References 1. 2. 3. 4. 5. 6. 7.

Yang ZH, Liu XD. Epilepsy Res. 2008;78:40–9. Liu XD, Yang ZH, Yang JS, Yang HW. Neuropharmacology. 2007;53:657–63. Liu XD, Yang ZH, Yang HW. Neurosci Lett. 2008; 432:184–7. Yang HW, Liu HY, Liu X, Zhang DM, Liu YC, Liu XD et al. Neurosci. Lett. 2008;434:299–303. Wen T, Liu YC, Yang HW, Liu HY, Liu XD et al. J Neurol Sci. 2008;270:99–106. Jing XY, Liu X, Wen T, Xie SS, Yao D, Liu XD et al. Brit J of Pharmacol. 2010;159:1511–22. Yao D ,Liu L, Jin S, Li J, Liu XD. Neuroscience. 2012, 227:283–292.

P45. POTENTIAL EFFECT OF ANTI-INFLAMMATORY COMPOUNDS ON HIV-1 GP120 -MEDIATED BRAIN INFLAMMATION Tamima Ashraf, Wenlei Jiang, Tozammel Hoque, Chiping Wu and Reina Bendayan Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada Neuroinflammation is a common immune response associated with brain HIV-1 infection. One obstacle to effective treatment is poor brain penetration of antiretroviral drugs due to functional expression of efflux transporters [P-glycoprotein (P-gp); Multidrug-resistance associated proteins (MRPs); Breast cancer resistance associated protein (BCRP)]. Identifying therapeutic compounds that are known to have better brain permeability and can target signaling pathways (i.e., JNKs, ERK1/2, P38Ks) involved in inflammation may benefit treatment of HIV-associated neurological complications. Studies have demonstrated antiinflammatory and neuroprotective effects of several compounds (i.e., minocycline) in various models of neurological disorders. In this study, by intracerebroventricular administration of the HIV-1 viral coat protein, gp120 in rats, we implemented an in vivo model of brain inflammation and examined anti-inflammatory properties of minocycline, chloroquine and simvastatin. We also investigated regulation of transporters and key signaling pathways involved in HIV-associated brain inflammation in vivo. Male Wistar rats were administered a single dose of gp120ADA (500 ng) daily for 7 consecutive days intracerebroventricularly with or without prior intraperitoneal administration of minocycline or chloroquine or simvastatin. Maraviroc, a CCR5 (chemokine receptor) antagonist, was also administered intracerebroventricularly prior to gp120 administration for 7 days as a control group. Real-time qPCR was used to determine gene expression of inflammatory markers in frontal cortex, hippocampus and striatum. Cytokine secretion in cerebrospinal fluid (CSF) was measured using ELISA. Protein expression of signaling kinases (ERK1/2 and JNKs) and transporters (P-gp, Mrp1, Bcrp) was detected using immunoblot analysis. In gp120ADA-injected rats, transcripts of TNF-a, IL-1b and iNOS were significantly elevated in frontal cortex, whereas, elevated IL-1b and iNOS mRNA were observed in striatum and hippocampus. In CSF, a significant increase in TNF-a and IL-1b was detected. Gp120mediated upregulation of inflammatory markers was attenuated by maraviroc suggesting that interaction of gp120 to CCR5 chemokine receptor is critical for induction of an inflammatory response. Consistent with our previous in vitro findings, gp120 administration resulted in a decrease in P-gp expression and an increase in Mrp1 and Bcrp expression in different brain regions. Minocycline or chloroquine completely attenuated the upregulation of IL-1b and iNOS transcripts in all three regions and prevented IL-1b secretion in CSF. Minocycline was also successful in suppressing TNF-a transcripts in brain tissues and prevented TNF-a secretion in CSF. Simvastatin attenuated both IL-1b and iNOS transcripts in hippocampus and striatum, but only suppressed iNOS in the frontal cortex. Gp120 also resulted in activation of ERK1/2 and JNKs in the hippocampus and

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administration of these anti-inflammatory compounds decreased the phosphorylation of ERK1/2 in this region. Our data suggest that administration of HIV-1 gp120ADA in rodents generates an inflammatory response, activates signaling pathways and alters expression of efflux transporters in several regions of the brain. Furthermore, minocycline, chloroquine and simvastatin can reverse gp120-associated brain inflammation suggesting that these agents could potentially be considered in the prevention/ treatment of HIV-associated cognitive disorders. Supported by an operating grant from the Ontario HIV Treatment Network (OHTN)

P46. IN VITRO P-GP EFFLUX RATIO CAN PREDICT THE IN VIVO BRAIN PENETRATION REGARDLESS OF BDDCS CLASS


J. Cory Kalvass, Ryota Kikuchi and Sonia M. de Morais Drug Metabolism, Pharmacokinetics and Bioanalysis, AbbVie Inc., North Chicago, IL P-glycoprotein (P-gp) is expressed at the blood-brain barrier (BBB) and restricts the CNS penetrations of its substrates. In vitro substrate assessment for P-gp is frequently used in pharmaceutical companies to predict the in vivo relevance of P-gp-mediated efflux at the BBB. Recently, Broccatelli and coworkers have proposed a new framework to predict the brain disposition of drugs using in silico permeability, in vitro P-gp efflux ratio (ER), and the class in the Biopharmaceutics Drug Disposition Classification System (BDDCS)1. They noted that 98% of BDDCS class 1 drugs can penetrate the brain even when those compounds are shown as P-gp substrates in vitro. However, our data analysis focusing on the in vitro-in vivo correlation of P-gp ER suggested that in vitro ER can predict the in vivo brain penetration regardless of the class in BDDCS. It was of note that the in vitro ER in MDCK-MDR1 cell line from NIH was found to be a better predictor of in vivo ER compared with that from NKI (Dr. Borst’s group) with r2 values of 0.813 and 0.531, respectively. Considering that very few marketed CNS drugs are in vivo substrates for P-gp, the in vitro substrate assessment of P-gp should be used in the early stages of drug discovery to select compounds which most likely penetrate the CNS to exert their pharmacological action. Moreover, the bidirectoinal transport assay using P-gp-overexpressing cell lines continues to be the most reliable in vitro tool to assess P-gp liability such as brain penetration.

Reference 1. Broccatelli F, Larregieu CA, Cruciani G, Oprea TI and Benet LZ (2012) Improving the prediction of the brain disposition for orally administered drugs using BDDCS. Adv Drug Deliv Rev 64:95–109.

P47. ROLE OF CYP2ABFGS IN NNK BIOACTIVATION AND LUNG TUMORIGENESIS: INSIGHTS FROM A NOVEL CYP2ABFGS-NULL MOUSE Lei Li, Vandana Megaraj, Yuan Wei and Xinxin Ding Wadsworth Center, New York State Department of Health, Albany, NY CYP2ABFGS enzymes encoded by the mouse Cyp2abfgs gene cluster are preferentially expressed in the respiratory tract, and a number of these are known to be active toward xenobiotic compounds, including many drugs and a tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Previous studies have demonstrated that pulmonary cytochrome P450 (P450)-mediated metabolic activation is the key step in NNK-induced lung tumorigenesis in mice [1]. A recent study on a Cyp2a5-null mouse [2] showed that CYP2A5 mediates a significant fraction, but not all, of NNK bioactivation in the lung [3]. We propose that, besides CYP2A5, other P450s encoded by the Cyp2abfgs gene cluster also play significant roles in mediating NNK bioactivation and NNK-induced lung tumorigenesis. The present study was aimed to test this hypothesis, with use of a novel Cyp2abfgs-null mouse, in which all Cyp2a, 2b, 2g, 2f, and 2s genes are deleted. The mouse model was generated through crossbreeding, first between Cyp2f2-null-loxP and Cyp2s1-null-loxP (yielding a cis-targeted, ‘‘floxed’’ Cyp2abfgs allele), then between theCyp2abfgs-floxed mouse and a Cre-transgenic mouse. Deletion of the constitutive genes in the cluster was confirmed by PCR analysis of the genes and the mRNAs in tissues known to express each gene. The Cyp2abfgs-null mouse was viable, fertile and without any obvious developmental deficits, and they appeared to have no compensatory increase in the expression of other P450s. NNK bioactivation in vitro and NNK-induced DNA adduction and lung tumorigenesis in vivo were studied in wild-type, Cyp2a5-null, and Cyp2abfgs-null mice. The Cyp2abfgs-null mice exhibited significantly lower rates of NNK bioactivation in lung and liver microsomes, compared to either wild-type mice orCyp2a5-null mice. Consistent with the in vitro data, the levels of lung O6-methyl guanine (O6-mG, the DNA adduct highly correlated with lung tumorigenesis) were substantially reduced in the Cyp2abfgs-null mice, compared to either wild-type mice or Cyp2a5-null mice. Moreover, the Cyp2abfgs-null mice were largely resistant to NNK-induced lung tumorigenesis at a moderate NNK dose (50 mg/kg), in contrast to the wild-type mice or Cyp2a5-null mice. These results indicate that, collectively, the CYP2ABFGS enzymes play essential roles in mediating NNK-induced lung tumorigenesis. The Cyp2abfgs-null mouse model will also be valuable for preparation of humanized mice that express human CYP2A6, CYP2A13, CYP2B6, CYP2F1, and/or CYP2S1.

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References 1. Weng, Y., Fang, C., Turesky, R. J., Behr, B., Kaminsky, L. S., and Ding, X.: Determination of the role of target tissue metabolism in lung carcinogenesis using conditional cytochrome P450 reductase-null mice. Cancer Res. 67, 7825–7832, 2007. 2. Zhou, X., Zhuo, X., Xie, F., Kluetzman, K., Shu, Y.-Z., Humphreys, W. G., and Ding, X.: Role of CYP2A5 in the clearance of nicotine and cotinine: insights from studies on a Cyp2a5-null mouse model. J. Pharmacol. Expt. Ther. 332, 578–587, 2010. 3. Zhou, X., D’Agostino, J., Xie, F., and Ding, X.: Role of CYP2A5 in the bioactivation of the lung carcinogen 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mice. J. Pharmacol. Expt. Ther. 341:233–241, 2012.

P48. INFLUENCE OF SEX AND STRAIN ON HEPATOTOXIC AND INFLAMMATORY RESPONSES TO LIVER CARCINOGENS IN THE MOUSE


Daniel Hanna, Debbie Brenneman, Kim S. Sugamori and Denis M. Grant Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada Liver cancer is the third most common cause of cancer-related death worldwide. Many of the risk factors associated with the development of liver cancer are associated with chronic inflammation. Liver cancer also shows a sex bias, with men having a 3-5-fold higher incidence than women even after correcting for differences in risk factor exposure. This sex difference can be modeled in mice using exposure to chemical carcinogens such as the aromatic amine 4-aminobiphenyl (ABP) and the nitrosamine diethylnitrosamine (DEN). One model of ABP carcinogenesis suggests its bioactivation by cytochrome P450dependent N-oxidation followed by arylamine N-acetyltransferase (NAT) mediated esterification and subsequent decomposition to a DNA-binding electrophile. We previously observed that postnatal exposure of mice to ABP produced liver tumors at one year of age only in male wild-type (C57BL/6) mice, while NAT-deficient (Nat1/2/) males, and females of both strains, were protected. However, measures of ABP-induced short-term DNA damage and mutation did not differ between sexes or strains. Others have also observed a sex difference in liver tumors following postnatal exposure of mice to DEN. Reasons for these sex and strain differences are not clear, but acute liver cytotoxicity and inflammation have been hypothesized to play a role, since adult mice show estrogen-dependent protection from DEN-induced acute hepatotoxicity and inflammation. However, it is not known whether similar effects occur in sexually immature mice exposed to carcinogens using postnatal exposure protocols. The goal of the current study was to determine whether postnatal exposure of mice to tumor-inducing doses of ABP or DEN produces sex and/or strain differences in acute cytotoxicity or inflammation. We quantified liver damage using serum alanine aminotransferase (ALT) activity and inflammation using interleukin-6 (IL-6) expression in postnatally exposed male and female wild-type or Nat1/2/ mice. Neither ABP nor DEN increased serum ALT activity levels, and only DEN caused a significant increase in serum IL-6 expression but with no sex difference. These results suggest that at tumor-inducing doses ABP and DEN are poor hepatotoxins, and that the increase in IL-6 expression in DEN-exposed mice may be due to chemical-specific factors. However, we do not know whether these responses occur or persist well after carcinogen exposure, particularly after sexual maturation, and whether sex hormones modulate these responses to influence the proliferation of carcinogen-initiated cells. Future experiments will measure levels of inflammatory cytokines and their downstream targets, and assess their effects on cellular proliferation and survival in ABP- and DEN-exposed mice at subacute and chronic time points after carcinogen exposure, up to the 1 year point when these mice are normally sacrificed and assessed for tumor growth.

P49. CLASSIFICATION MODELS TO PREDICT CARCINOGENICITY USING ANT COLONY OPTIMIZATION AND DECISION TREES Ramamurthi Narayanan1, Vijay Singh1 and Sitarama Gunturi2 1 Innovation Labs, Life Siences R&D Division, Tata Consultancy Services Limited, Hyderabad, India, 2Innovation Labs, Life Sciences R & D Division, Tata Consultancy Services Limited, Hyderabad, India Cancer is one of the leading causes of human death and exposure to physical (radiation and ultraviolet rays), chemicals (carcinogens), and biological (bacteria, virus, and inheritance) factors is one of the major reasons for the onset of the disease. Carcinogens is the most dominant among these three factors [1, 2] and thus it is desirable to carefully assess the carcinogenicity of all the chemical substances; however, animal testing is often very expensive and hence, there is an urgent need for alternative and inexpensive approaches for the assessment of carcinogenic potential of chemicals. Further, regulatory authorities such as REACH, EPA are increasingly recommending the utilization of Quantitative Structure Activity Relationship (QSAR) models following OECD guidelines [3] as reliable alternative animal-free tools for predicting carcinogenicity. Different researchers have reported classification models for predicting carcigenicity using approaches, such as, Machine Learning techniques, Discriminant Analysis etc and most of these models employ more than twenty physico-chemical properties as parameters in their models. [4, 5]

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In this study, we present for the first time the application of Ant Colony Optimization (ACO) algorithm [6] for feature selection and Decision Trees (DT) for building classification models. The classification models are based on i) carcinogenic data set of 1099 chemicals (544 active and 555 inactive) [7, 8] and ii) 445 two dimensional structural descriptors iii) 99 compounds of randomly selected external dataset and iv) five distinct sets of 800 training set of compounds and 200 test sets of compounds. The resultant models were subjected to rigorous internal and external validations and the best models are selected based on the correct classification rate (CCR475%), sensitivity (SE470%) and specificity (SP470%) on the training, test and validation sets. Importantly, the models reported herein are based on only 12 descriptors, which is significantly smaller in number as compared to the published reports, without compromising on the model performance.


References 1. Doll R, Peto R (1981), The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 66:1192–1309 2. Harvard Center for Cancer Prevention (1996) Harvard report on cancer prevention. Volume 1: Causes of human cancer. Cancer Causes Control 7:S3–S59. 3. OECD (2002) Guidance notes for analysis and evaluation of chronic toxicity and carcinogenicity studies. OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 35, Paris, France 4. Prediction of carcinogenicity for diverse chemicals based on substructure grouping and SVM modeling, Kazutoshi Tanabe, Bono Lucic Dragan Amic, Takio Kurita, Mikio Kaihara, Natsuo Onodera Takahiro Suzuki, Mol Divers (2010) 14:789–802 5. QSAR Modeling of Carcinogenic Risk Using Discriminant Analysis and Topological Molecular Descriptors, Joseph F. Contrera, Philip MacLaughlin, Lowell H. Hall and Lemont B. Kier Current Drug Discovery Technologies, 2005, 2, 55–67 6. In silico ADME modelling 2: Computational models to predict human serum albumin binding affinity using ant colony systems, Sitarama B. Gunturi, Ramamurthi Narayanan and Akash Khandelwal, Bioorganic & Medicinal Chemistry 14 (2006) 4118–4129. 7. CPDBAS: Carcinogenic Potency Database Summary Tables, http://www.epa.gov/ncct/dsstox/sdf_cpdbas.html. 8. Carcinogenicity Prediction of Noncongeneric Chemicals by a Support Vector Machine, Min Zhong, Xianglei Nie, Aixia Yan, and Qipeng Yuan, Chem. Res. Toxicol. 2013, 26, 741–749.

P50. PIPERINE DOWNREGULATES HER2 EXPRESSION, LEADING TO INHIBITION OF HER2-MEDIATED PROLIFERATION, MIGRATION AND APOPTOSIS POTENTIALS IN HER2-OVEREXPRESSING BREAST CANCER CELLS Hyung Gyun Kim, Minh Truong Do and Hye Gwang Jeong College of Pharmacy, Chungnam National University, Daejeon, South Korea Piperine is a major component of black pepper, Piper nigrum Linn, used widely in traditional medicine and commonly used for daily consumption. Previous studies demonstrated that piperine exhibits various beneficial bioactivities including antioxidant, anti-inflammation and antitumor properties. In this study, we investigated the molecular mechanisms by which piperine exerts antitumor effects in HER2-overexpressing breast cancer cells. Our results revealed that piperine markedly suppressed proliferation and induced apoptosis through caspase-3 activation and PARP cleavage. Apoptosis in HER2 overexpression cells induced by piperine was related to DNA damage. Furthermore, piperine inhibited HER2 gene expression at the transcriptional level. Blockade of ERK1/2 signaling by piperine significantly reduced SREBP-1 and FAS expression. Piperine significantly repressed EGF-induced MMP-9 expression through suppression of AP-1 and NF-kB activation by interfering with ERK1/2, p38 MAPK, and Akt signaling pathways resulting in a reduction in migration. Finally, piperine pretreatment enhanced sensitization to paclitaxel killing in HER2-overexpessing breast cancer cells. Our findings suggest that piperine may be a potential agent for the prevention and treatment of human breast cancer with HER2 overexpression.

P51. EVALUATION OF ENDOGENOUS METABOLIC MARKERS OF CYP3A ACTIVITY USING METABOLIC PROFILING AND MIDAZOLAM CLEARANCE Kwang-Hee Shin1, Man Ho Choi2 and Joo-Youn Cho1 1 Clinical Pharmacology & Therapeutics, Seoul National University, Seoul, South Korea, 2Future Convergence Research Division, Korean Institute of Science and Technology, Seoul, South Korea Cytochrome P450(CYP) 3A family is a major enzyme involved in drug metabolism and many of the pharmacokinetic interactions between drugs are due to CYP3A enzyme being affected by co-administration. This study was aimed to evaluate endogenous metabolic markers of CYP3A activity in healthy subjects using a metabolomics approach. Twenty-four subjects received the following medication during the following three study periods: 1 mg of iv midazolam alone (control phase), 1 mg of iv midazolam after four days of pretreatment with 400 mg of ketoconazole once daily (CYP3A-inhibited phase), and 2.5 mg of iv

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midazolam after ten days of pretreatment with 600 mg of rifampicin once daily (CYP3A-induced phase). During each study period, 24 h before and after the administration of midazolam, urine samples were collected at 12-h intervals for metabolomic analyses. We derived an equation to predict midazolam clearance based on several of these markers such as 6betahydroxycortisone and 7beta-dehydroepiandrosteron. We demonstrated that a combination of the concentrations and ratios of several endogenous metabolites and the CYP3A5*3 genotype is a reliable predictive marker of CYP3A activity as assessed by administration of midazolam.


P52. PREDICTABILITY OF PLASMA CONCENTRATION-TIME PROFILES IN HUMANS USING CHIMERIC MICE WITH HUMANIZED LIVER Seigo Sanoh1, Mami Fujimoto2, Yoichi Naritomi3, Koya Sato4, Akio Kawamura4, Aya Horiguchi1, Kazumi Sugihara5, Shigeyuki Kitamura6, Toru Horie7, Chise Tateno8 and Shigeru Ohta1 1 Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan, 2Faculty of Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japan, 3Analysis and Pharmacokinetics Research Laboratories, Astellas Pharma. Inc., Tsukuba-shi, Japan, 4Drug Metabolism Research Laboratories, Astellas Pharma Inc., Osaka, Japan, 5Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Japan, 6Nihon Pharmaceutical University, Saitama, Japan, 7 De Three Reseach Laboratories, Ibaraki, Japan, 8R&D Department, PhoenixBio Co., Ltd., Higashihiroshima, Japan Various approaches have been made to predict pharmacokinetics (PK) using human liver microsomes and hepatocytes to decrease the attrition rate during drug development. We used chimeric mice (PXB miceÕ , PhoenixBio, Co., Ltd, Hiroshima, Japan) with humanized liver which were generated from urokinase-type plasminogen activator (uPA)/severe combined immunodeficiency (SCID) mice injected with human hepatocytes to evaluate predictability of the plasma concentration-time profile in humans. The replacement rates of mouse hepatocytes with human hepatocytes were approximately 80% in the liver of PXB mice in this study. Total clearance (CLt) and distribution volume at steady state (Vdss) of several model compounds metabolized by cytochrome P450 (CYP) and non-CYP such as UDP-glucuronosyltransferase, aldehyde oxidase were determined after intravenous administration in PXB mice. CLt and Vdsspredictions were estimated by the single species allometric scaling equations (eq.1 and eq.2). CLt human ¼ CLt PXB mice (B.W. human/B.W. PXB mice)a (eq.1), Vdss human ¼ Vdss PXB mice (B.W. b human/B.W. PXB mice) (eq.2). Both exponential values of allometric equations were calculated from CL and Vdss of each model compound in PXB mice and humans, respectively. Then the plasma concentration-time curves of the model compounds in humans predicted by a complex Dedrick plot (eq.3 and eq.4) using the average exponent values of each model compound were compared with obtained clinical data. Predicted time ¼ Real time PXB mice [(B.W. human)ba/(B.W. PXB mice)ba] (eq.3), Predicted plasma concentrations (Cp) ¼ Cp PXB mice [(B.W. human)1b/(B.W. PXB mice)1b] (eq.4). The plasma concentrationtime curves predicted from PXB mice were similar to observed human data. The single species allometric scaling using PXB mice, which showed accurate prediction for the plasma concentration-time curves proves to be useful. However, the predictability of diazepam which is a CYP substrate was poor because predicted CLt was higher than that observed human. In vitro intrinsic clearance in liver microsomes with low replacement of human hepatocyte of PXB mice was significantly higher than that in high replacement microsomes. The residual mouse hepatocyte in the liver of PXB mice must be concerned to prediction of human PK. It is necessary to evaluate PK in consideration with remaining mouse hepatocytes in PXB mice.

P53. TRANSLATIONAL RESEARCH TOOLS TO IMPROVE DRUG THERAPY IN PEDIATRICS Souzan B. Yanni DMPK Consultants, Inc, Chapel Hill, NC The pharmacokinetic parameters that are influenced by absorption, distribution, metabolism and excretion of drugs were found to be altered as function of age from birth onwards. Consequently, drug disposition in pediatric populations is different compared to adults due to the way they handle (pharmacokinetics) and respond (pharmacodynamics) to drugs. Several in vitro and clinical studies of prototype substrates for drug metabolizing enzymes and few studies of drugs that depend on drug transporters in their dispositions in pediatric populations revealed that the ontogeny is one major factor that implicated in significant differences in the disposition, response, and safety of drugs. The maturation of drug metabolizing enzymes, the uptake and efflux drug transporters, plasma protein binding, liver and kidney sizes relative to body weight , and organ blood flow were found to be some of key factors that influence the changes drug disposition and plasma clearance in pediatric populations compared to adults. To optimize drug safety and its therapeutic benefit in pediatric patients, alternative translational approaches to predict drug dose based on actual drug exposure and clearance in young patients were tested. Case studies based on retrospective clinical investigations of antifungal agents and oncology drugs were used to setup a road map to guide future prospective dose selection in pediatric patients. Here, some criteria were strategically assigned to successfully design the predictive investigation of a specific drug such as: the use of biological samples from specific population of interest, understand the mechanism of drug disposition using adult data, and base in vitro investigation with biological samples from specific organ that play critical role

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in the disposition and clearance of that drug. By applying these steps, the accuracy in predicting drug clearance and dose selection have been improved, thus the efficacy and safety of drugs in pediatric patients have been ensured.

P54. CROSS-VALIDATION OF PRECLINICAL TOOLS FOR HEPATIC DRUG METABOLISM: FROM SUBCELLULAR STUDIES TO IN VIVO


Johan Nicolaı¨1, Paul Van Veldhoven2, Patrick Augustijns1 and Pieter Annaert1 1 Department of Pharmaceutical and Pharmacological sciences, KU Leuven, Leuven, Belgium, 2Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Introduction: Successful quantitative prediction of hepatic drug metabolism often requires integrated interpretation of data obtained in multiple preclinical tools (e.g. microsomes, hepatocytes). In addition, model system-specific scaling approaches are required to extrapolate in vitro data to the in vivo situation. In order to obtain this information, thorough cross-validation of preclinical tools as well as comparison with relevant in vivo data is required. Aim/goal: To cross-validate an array of preclinical tools for hepatic drug clearance prediction with increasing biorelevance going from subcellular preparations all the way up to in vivo, based on clearance measurements for a metabolically cleared compound, namely verapamil. Methods: The suspended hepatocyte assay was first optimized and then applied to determine the hepatic intrinsic clearance (Clhep,int) for verapamil metabolism by freshly isolated as well as cryopreserved rat hepatocytes. These data were compared to values obtained with rat liver microsomes. Finally the isolated perfused rat liver system (IPRL) was used to confirm the obtained Clhep, int values which were ultimately compared to the actual in vivo clearance data. Subsequently SimcypÕ rat was used to compare extrapolated in vitro data to observed blood concentrations. Results: All model systems confirmed that verapamil is a drug with a highextraction ratio (ER40.7), i.e. verapamil’s clearance is blood-flow limited. The obtained Clhep, int values for freshly isolated hepatocytes, cryopreserved hepatocytes, rat liver microsomes, IPRL and in vivo (rat) were comparable. SimcypÕ rat was supplied with the obtained in vitro data and the predicted blood concentrations (full PBPK) only slightly under- predicted the observed data yet supported a comparable elimination rate constant (afe ¼ 1.13). Conclusion: Extensive quantitative exploration of metabolic clearance of selected model drugs in different preclinical tools of the liver, supports reliable inter-system scaling and in vitro-in vivo extrapolation. Subsequent use of these cross-validated model systems for evaluating hepatic disposition of novel compounds may reveal mechanistic information on hepatic drug disposition of these compounds.

P55. ASCORBYLATION AND GLUTATHIONYLATION OF GLUTAREDOXIN-1 BY DEHYDROASCORBATE Ghada Eloraby1, François-Olivier McDuff1, Richard Wagner2 and Klaus Klarskov1 1 Pharmacologie, Universite´ de Sherbrooke, Sherbrooke, QC, Canada, 2De´partement Me´decine Nucleáire et Radiobiologie, Universite´ de Sherbrooke, Sherbrooke, QC, Canada Vitamin C (ascorbic acid; ascorbate ion at physiological pH) protects the organism against reactive oxygen and nitrogen species. In this process, ascorbate is oxidized by the loss of one electron to give initially an ascorbyl free radical (SDA), which subsequently undergoes oxidation to dehydroascorbate (DHA). Under physiological conditions, both SDA and DHA are recycled back to ascorbate through the action of several enzymes including NADH-cytochrome b5 reductase, thioredoxin reductase, protein disulfide isomerase, glutaredoxin-1 (thioltransferase), and certain glutathione-S-transferases. However, in aqueous solution DHA readily degrades to among other a thiol reactive carbonyl, named DHA*, which has been shown to covalently modify glutathione1. Hypothesis: A reactive carbonyl derivative of DHA is capable of modifying glutaredoxin-1 in vitro and DHA induces protein glutathionylation. Human glutaredoxin-1 (also known as thioltransferase) was expressed in E. coli with an additional C-terminal located streptagII (SAWSHPQFEK), TEV protease (ENLYFQS), anti-Myc (EQKLISEEDL) and (His6)-tag. The expressed protein was verified by ESIMS to have a mass of 17219 0.32, which is 129 Da lower than the calculated mass (17348). Further tandem MS analysis of the trypsin digested carbamidomethylated protein indicated that the N-terminal Met residue was absent. Incubation of glutaredoxin-1 with DHA (preincubated for 6 hours to allow decomposition of DHA) suggests that a degradation product of DHA interacts with reduced thiol groups of cysteines. By incubating glutaredoxin with DHA at neutral pH in a Hepes buffer, we observed adducts of 112 Da, 224 and 336 Da by ESIMS. Similarly, incubation with GSH in the presence of DHA induced protein glutathionylation. Following digestion with pepsin at pH 2.0, the entire peptide mixture was analyzed by ESIMS (direct infusion) and LCMSMS. Two peptides exhibited loss of 112 during the ESIMS/MS analysis, confirming the presence of DHA*. The peptides were assigned to the cysteine containing peptides Val5-Val14 and Ileu74-Leu85 and the sites of modification were assigned to cysteine at positions 7, 77 and 81 (glutaredoxin-1 without the N-terminal methionine residue). Identification of the sites of glutathionylation was determined in a similar manner. In conclusion, our results demonstrate that a reactive carbonyl degradation product of DHA and glutathione are capable of modifying cysteine thiols in glutaredoxin, one of the main proteins involved in the recycling of ascorbate and in protein deglutathionylation. Future proteomics studies with cells overexpressing or

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silencing glutaredoxin-1 should reveal if these modifications occur in vivo and could play a role in deregulation of cellular homeostasis following oxidative stress.

Reference (1) Regulus, P.; Desilets, J. F.; Klarskov, K.; Wagner, J. R. Characterization and Detection in Cells of a Novel Adduct Derived from the Conjugation of Glutathione and Dehydroascorbate. Free Radic. Biol. Med. 2010, 49, 984–991.

P56. UNDERSTANDING THE ROLE OF UDP-GLUCURONOSYLTRANSFERASE UGT2B28 IN PROSTATE CANCER


Isabelle Gilbert1, Joannie Roberge2, He´le`ne Hovington3, Anaı¨s Belledant3, Bernard Tetu3, Louis Lacombe3, E´ric Levesque3 and Chantal Guillemette2 1 Faculty of pharmacy, Pharmacogenomics Laboratory, (Que´bec), QC, Canada, 2Pharmacie, Pharmacogenomics Laboratory, (Que´bec), QC, Canada, 3Medecine, Pharmacogenomics Laboratory, (Que´bec), QC, Canada UGT2B28 is a member of membrane-bound proteins of the endoplasmic reticulum. We recently demonstrated that polymorphic UGT2B28 and UGT2B17 deletions are novel prognostic markers associated with an increasing risk of prostate cancer recurrence after radical prostatectomy (Eur Urol. 2011 Dec;60(6):1226–34). Although the function of UGT2B17 in the inactivation of androgens is well documented, UGT2B28 seems to present a distinct enzymatic profile with some activity on estrogens. To investigate further UGT2B28 function, we firstly analyzed its tissue distribution using a specific quantitative PCR strategy revealing a preferentially expression in hormone-dependent tissues, including the prostate and the androgen-dependent prostate tumor cell line LNCaP. In this cell model, we observed a differential regulation of UGT2B17 (-3.3X, p50.001) and UGT2B8 (28X, p50.001) by androgen (R1881 1 nM) mediated through the androgen receptor since these effects were reversed by a combined treatment with the anti-androgen casodex (1uM). We then developed a specific antibody directed against UGT2B28 that does not recognize close family members, namely UGT2B17 and UGT2B15. Using this antibody, we confirmed the strong induction of UGT2B28 expression by androgen at the protein level. Lastly, immunohistochemistry analysis of healthy and tumoral human prostatic tissues exposes an exclusive nuclear staining for UGT2B28 in basal and secretory cells whereas a variable intensity staining in thecytoplasm and the nuclei of tumor cells was observed. This localization is unique to UGT2B28 compared to other UGT2B proteins, suggesting for the first time a potential nuclear-cytoplasmic exchange for UGT2B28 in human prostate tumors.

P57. INTERSYSTEM EXTRAPOLATION FACTORS (ISEF) FOR HUMAN UDPGLUCURONOSYLTRANSFERASE (UGT) ACTIVITY: UTILITY TO ESTIMATE UGT1A1-MEDIATED FRACTIONAL CLEARANCE Jian Lin1, Kimberly Lapham1, Mark Niosi1, Christine C. Orozco1, John K. Fallon2, Hendrik Neubert3, Philip C. Smith2, Ravi Rahavendran4, Louis Leung1 and Theunis C. Goosen1 1 Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, CT, 2Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 3Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Andover, MA, 4Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, La Jolla, CA An increasing number of drug candidates are primarily cleared through metabolism mediated by UDP-glucuronosyltransferases (UGTs). Quantitative prediction of human systemic clearance (CL) therefore requires development of robust in vitro models to predict UGT-mediated intrinsic clearance (CLint). In addition, since UGT1A1 is subject to genetic polymorphism, the importance of determining UGT1A1-mediated fractional metabolism (fm) is highlighted in recent regulatory guidance.1 Quantitative prediction of UGT-isoform specific CL could be estimated if relative substrate activity factors are established with recombinantly expressed UGTs (rUGTs) and human liver microsomes (HLM). In this study, hepatic relative activity factors (RAFs) and intersystem extrapolation factors (ISEFs) were generated for rUGTs 1A1, 1A4, 1A6, 1A9 and 2B7. Unbound CLint was determined with rUGTs and HLM utilizing b-estradiol (UGT1A1), trifluoperazine (UGT1A4), 5-hydroxytryptophol or deferiprone (UGT1A6), propofol (UGT1A9), and zidovudine (UGT2B7) as selective substrates.2 Hepatic RAF values were 0.54, 0.30, 13 or 4.5, 0.84 and 3.3, respectively when determined from UGT activities expressed as mL/min/mg protein. Absolute UGT abundance in rUGTs and HLM were determined from quantitative targeted absolute proteomic (QTAP) analysis to express enzymatic activity as mL/min/pmol protein and determine ISEF values. UGTs 1A1, 1A4, 1A6, 1A9 and 2B7 CLint ISEF values were 24, 1.9, 730 or 260, 17, and 54, respectively. Irinotecan active metabolite (SN-38) glucuronidation, a model substrate of UGT1A1, was studied in rUGTs and CLint values of 12.6, 0.14, and 1.2 mL/min/mg were determined for UGTs 1A1, 1A6, and 1A9, respectively. Based on these estimates, the fm values for SN-38 glucuronidation is estimated at 0.81, 0.072, and 0.12, respectively. These findings confirmed a predominant role of UGT1A1 in the glucuronidation of SN-38 and provide a

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quantitative estimate of fm, which is required for prospective drug-drug interaction predictions. The availability of UGT ISEF values should prove useful in the determination of UGT fm values and in the development of physiologically based pharmacokinetic models where interindividual variation of UGT abundance in hepatic or extrahepatic drug metabolizing organs is incorporated.

References 1. Guidance for Industry: Drug Interaction Studies – Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations, FDA, Draft Guidance, Feb 2012. 2. Walsky RL, Bauman JN, Bourcier K, Giddens G, Lapham K, Negahban A, Ryder TF, Obach RS, Hyland R, and Goosen TC (2012) Optimized assays for human UDP-glucuronosyltransferase (UGT) activities: altered alamethicin concentration and utility to screen for UGT inhibitors. Drug Metab Dispos 40(5):1051–1065.


P58. NOVEL ACTIVITIES OF UDP GLYCOSYLTRANSFERASE (UGT) 8 Peter I. Mackenzie, Anne Rogers, Aravind Shivasami and Robyn Meech Department of Clinical Pharmacol, Flinders University, Adelaide, Australia The UDP glycosyltransferases (UGTs) consist of 4 families of enzymes that add hexose sugars to lipophilic chemicals. The action of UGTs is essential for reducing the toxic and/or biological effects of these chemicals and preparing them for rapid excretion. The addition of the hexose, glucuronic, acid to a variety of drugs and endogenous signalling molecules and metabolites is mediated by the 19 enzymes of the UGT1 and 2 families. Although some members of these two families also have an enhanced capacity to glucosidate specific compounds, their capacity for glucosidation is, in general, lower than their capacity for glucuronidation. The addition of the hexoses, N-acetylglucosamine, glucose and xylose to chemicals is mediated by the two members of the UGT3 family. Hence members of the UGT1, 2 and 3 families are considered to have an essential role in controlling the distribution and biological effects of lipophilic drugs, hormones, ligands of transcription factors, products of metabolism and other endogenous and exogenous lipophilic chemicals. In contrast, the single member of the 4th family, UGT8, adds the hexose, galactose to ceramide and diglycerides, an essential step in the synthesis of glycolipids. Although it is highly expressed in the kidney and intestine, UGT8’s major site of action is the nervous system, where it contributes to myelination of nerves. Hence, UGT8 is usually regarded as a biosynthetic enzyme, rather than a regulator of the distribution and activities of lipophilic chemicals. Given the broad aglycone specificities of members of the UGT1, 2 and 3 families, we speculated that UGT8 may also have a similar capacity to galactosidate a range of compounds. To test this hypothesis, human UGT8 cDNA was cloned and expressed in HEK293 cells and UGT8 activity was assayed with a variety of substrates. In addition to its activity towards ceramides, UGT8 was also active in the galactosidation of bile acids such as cholic and deoxycholic acid and bioflavones, including chrysin. It had little activity towards these substrates when UDP-glucuronic acid and UDP-glucose were used as sugar donors. These novel activities suggest that UGT8 may also have a role in controlling the distribution and activities of lipophilic compounds, in addition to its role in the synthesis of glycolipids and myelination of nerves.

P59. GENERATION OF HERITABLE MUTATIONS IN THE ZEBRAFISH SULT4A1 GENE USING TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES (TALENS) Frank Crittenden, Holly Thomas, John Parant and Charles N Falany Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL Cytosolic sulfotransferase (SULT) 4A1 (SULT4A1) has presented an enigma to the field of drug metabolism for over a decade. Identified in 2000 by Falany et al, this neuronally expressed orphan enzyme has not been shown to have any activity or function. The most conserved of the cytosolic SULTs, the SULT4A1 gene has been identified in every vertebrate investigated to date. Human SULT4A1 and zebrafish SULT4A1 exhibit 88.4% sequence identity and 95.4% similarity, making zebrafish an excellent model organism for the investigation of this protein. This high level of sequence and structural conservation suggests a conserved function. The SULTs are a superfamily of enzymes responsible for a metabolic reaction in which a sulfonate group is transferred from the obligate donor, 50 -phosphoadenosine 30 -phosphosulfate (PAPS), to the substrate. This Phase II conjugation reaction generally renders the substrate more water soluble and easier to excrete. Sequence and structural similarities with the other SULTs suggest that SULT4A1 is a member of the SULT superfamily; however, no substrate has yet been identified. In this study, we use transcription activator-like effector nuclease (TALEN) technology to generate heritable mutations within the SULT4A1 gene in zebrafish. TALENs were designed to target the second exon of the SULT4A1 gene in zebrafish and plasmids containing the sequence were generated using a Golden Gate TALEN and TAL Effector Kit (addgene). TALEN mRNA was then generated and injected into zebrafish embryos at the one or two cell stage of development. Each TALEN consists of a transcription activator-like (TAL) effector of Xanthomonas spp fused to one half of a Fok1 heterodimer endonuclease. Targeted adjacent binding with the genome results in the two halves of the Fok1 joining together and digesting the double stranded DNA between

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the two target sequences. DNA melt curves were then used to identify chimeric fish which were bred with wild type fish to create stable heterozygous lines. Using this method, we have generated and sequenced 3 heritable mutations in the SULT4A1 gene that render the protein incapable of folding correctly. All three mutations resulted in the deletion of two or more amino acids (aa 89–94) that help make up the putative binding pocket of the SULT4A1 protein, and dynamic modeling of these mutants predicts dramatic structural changes within the protein. One mutation resulted in the internalization of the active site histidine, and all three showed disruption of secondary structures adjacent to the mutations. (Supported by NIH grant R21MH095946)


P60. USING TARGETED QUANTITATIVE PROTEOMICS TO BETTER UNDERSTAND VARIABILITY OF THE GLUCURONIDATION PATHWAY Guillaume Margaillan1, Kathrin Klein2, Vincent Meńard3, Lyne Villeneuve3, John K. Fallon4, Patrick Caron5, Ve´ronique Turcotte6, Philip C. Smith7, Ulrich M. Zanger2 and Chantal Guillemette8 1 Universite´ Laval, Que´bec, QC, Canada, 2Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany, 3 Pharmacogenomics Laboratory, CHUQ Research Center and Faculty of Pharmacy, Laval University, Que´bec, QC, Canada, 4 Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 5CHUL Research Center, Quebec, QC, Canada, 62705 boulevard Laurier, CHU de Que´bec Research Center, Quebec, QC, Canada, 7Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 8Pharmacie, Pharmacogenomics Laboratory, (Que´bec), QC, Canada There have been significant recent advances in quantifying membrane-bound enzymes such as UDP-glucuronosyltransferases (UGT) in human tissues using mass spectrometry (MS). Here, we applied a multiplexed method using nanoLC-MS/MS and multiple reactions monitoring (MRM) to provide an absolute quantification of 14 UGT isoforms. This approach was applied to 40 human liver microsomes along with quantification of mRNA transcripts, genotyping of common polymorphisms and assessment of rates of conjugation for selective UGT probe substrates. Ten UGTs could be detected in livers with UGT2B7 as the most abundant (77 pmol/mg protein) followed by UGT2B4, UGT1A1, UGT1A4, UGT2B15 and UGT1A9 (between 24–36 pmol/ mg protein). Co-regulation of their expression was noted with the strongest correlations (r 0.70; p50.0001) between UGT1A3 and UGT1A4, UGT1A6 and UGT1A9, UGT1A4 and UGT2B4, and, UGT1A9 and UGT2B15. A strong correlation was also observed between UGT proteins and rates of glucuronidation for probe substrates (p50.0001) such as UGT2B7-zidovudine, r ¼ 0.82; UGT1A1-bilirubin, r ¼ 0.88; UGT1A9-propofol, r ¼ 0.72; UGT1A4-tacrolimus, r ¼ 0.87 and UGT1A6-deferiprone, r ¼ 0.91. However, poor correlations of protein to mRNA expression levels (qPCR strategies detecting exon 1, exons 2–3 or exons 5–6) were observed for UGT2B7 whereas UGT1A1 protein levels were significantly correlated with mRNA expression (r ¼ 0.74; p ¼ 0.0001) and affected by the presence of the *28 allele (42% vs. *1/*1; p ¼ 0.03). Consistent with the frequent gene deletion reported for UGT2B17, only 30% of livers had significant levels of UGT2B17 protein (45 pmol/mg protein). This extensive comparative investigation demonstrates that several human UGTs can be quantified simultaneously with high reliability and robustness by MS/MS and provides key information about absolute expression levels and for establishing relationships with genetic polymorphisms, mRNA expression patterns and enzyme activity. This approach is thus highly suitable for future work aimed at studying variability of the glucuronidation pathway and to elucidate differences in expression profiles in human tissues. This work was supported by CIHR and The Canada Research Chair program.

P61. IN VITRO SULFATION OF THREE ACTIVE CONSTITUENTS OF CHANSU BY HUMAN LIVER CYTOSOL: IMPLICATIONS ON THE MAJOR METABOLIC PATHWAYS OF BUFODIENOLIDES Jing Ning1, Guang-Bo Ge1, Zhen-Wen Yu1, Jie Hou2, Ling Yang1 and Xiao-Chi Ma2 1 Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Dalian Medical University, Dalian, China Bufadienolides, an important class of natural products with C-24 steroid skeleton and a unique a-pyrone ring at the C-17 site, are recognized as the major bioactive constituents of Chansu (Toad Venom). Three major bufadienolides including resibufagenin (RB), cinobufagin (CB) and bufalin (BF) display excellent biological activities, such as cardiotonic, local anestheticand and anticancer activities. The previous metabolic studies have disclosed that thesebufadienolides can be metabolized by cytochrome P450 enzymes (CYPs) to form a series of hydroxylated metabolites in the phase I metabolic systems, while human UGTs catalyzed these bufodienolides hardly. In the present study, the sulfation pathways of three naturally bufodienolide in Chansu were investigated firstly, by using pooled human liver cytosol (HLC) and recombinant human sulfotransferase (hSULT). The corresponding 3-O sulfates of three bufodienolides were detected in HLC in the presence of 30 -Phosphoadenosine-50 phosphosulfate, by using liquid chromatography-mass spectrometry. hSULT2A1 was identified as the main isoform involved in these biotransformations in HLC on the basis of hSULT isoform phenotyping assays and the chemical inhibition assays using selective inhibitor of hSULT2A1. The formation of 3-O sulfates of RB, CB and BF in both HLC and hSULT2A1 followed the substrate inhibition kinetics. These bufodienolides displayed good affinity on hSULT2A1, the apparent Km values for the formation of 3-O-sulaftes of RB, CB and BF in hSULT2A1 were 0.52 mM, 0.74 mM, and 2.09 mM respectively.

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Meanwhile, similar Km values were also obtained in HLC, the apparent Km values for the formation of 3-O-sulaftes of RB, CB and BF in HLC were 0.46 mM, 1.33 mM, and 2.67 mM respectively. Furthermore, the relative importance of hydroxylations and sulfations in overall metabolism of RB, CB and BF were investigated by in vitro extrapolation. Notably, the hepatic intrinsic clearance for the formation of RB 3-O-sulafte was much larger than that for the formation of hydroxylated metabolite. In contrast, hydroxylation pathway(s) was the major hepatic metabolic pathways of CB and BF. In summary, the sulfation pathways of three major bufodienolides (RB, CB and BF) in human liver were well characterized, while the hepatic intrinsic clearances of major metabolic pathways of these major bufodienolides in Chansu were also determined and well compared. These findings provide vital information to better understand the metabolic behaviors of bufadienolide in human.

P62. UDP-GLUCURONOSYLTRANSFERASE 1A1 IS THE PRINCIPAL ENZYME RESPONSIBLE FOR BAVACHININ GLUCURONIDATION IN HUMAN LIVER MICROSOMES


Xia Lv1, Guang-Bo Ge1, Jing Ning1, Jie Hou2, Ling Yang1 and Xiao-Chi Ma2 1 Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Dalian Medical University, Dalian, China Psoralea corylifolia, commonly known as babchi, is a popular herb, which has since long been used in traditional Ayurvedic and Chinese medicine for its magical effects to cure various skin diseases. Bavachinin (BCI) is one of the most essential bioactive components in Psoralea corylifolia, possessing a marked anti-inflammatory, antipyretic, and mild analgesic properties. Although its pharmacological activities are recognized, the metabolic pathways of BCI in human tissues have not been reported. In the present study, the glucuronidation pathway(s) of BCI was investigated by using pooled human liver microsome (HLM) and recombinant human UDP glucosyltransferase (UGT). One mono-glucuronide was detected in HLM in the presence of uridine-50 diphosphoglucuronic acid using liquid chromatography-mass spectrometry. The conjugate was further confirmed by b-glucuronidase-mediated hydrolysis reactions. Furthermore, the glucuronide was biosynthesized using liver microsomes from human and rat, and was identified as bavacinin-40 -O -glucuronidation (BCIG) by using nuclear magnetic resonance. The enzymes participating in BCI glucuronidation were UGT1A1, UGT1A3 and UGT1A8. Among these isoenzymes, UGT1A1 displayed the best catalytic ability, while UGT1A3 and UGT1A8 display limited activities. The kinetic studies on BCI glucuronidation in HLM, UGT1A1 and UGT1A3 were characterized, with the Km values of 14.3 mM, 9.7 mM, and 9.4 mM, respectively. The similar Km values for BCI glucuronidation in UGT1A1 and HLM implied that UGT1A1 played important role in BCI glucuronidation in human liver. 20(S)-Protopanaxatriol, a potent and selective inhibitor of UGT1A1, displayed similar inhibitory effects against the formation of this conjugate mediated by UGT1A1 and HLM. Furthermore, a strong correlation was observed between the formation rate of BG and the formation rate of b-estradiol-3-glucuronide, using a panel of nine individual HLMs, with the coefficient parameter of 0.97. In summary, UGT1A1 is the major isoform responsible for glucuronic acid conjugated reaction of BCI in human liver. The findings not only disclosed the metabolic behaviors of BCI in human, but also push us to believe that BCI can be used as a good probe substrate of UGT1A1 for measuring the activity of this enzyme in vitro and in vivo.

P63. UGT1A9 PLAYS PREDOMINANT ROLE IN GLUCURONIDATION OF SHIKONIN Guiyuan He1, Guang-Bo Ge1, Liang-Liang Zhu2, Yang-Liu Xia1, Xiao-Yu Sun1 and Ling Yang1 1 Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2School of Life Science, Anqing Normal University, Anqing, China Shikonin is one of the major ingredients isolated from medicine herb Zicao (Lithospermum erythrorhizon). Shikonin drew many attentions recent years for its anticancer, anti-inflammatory and anti-virus activities. Although its pharmacological activities are recognized, the metabolic pathways of shikonin in human have not been reported. In this work, the metabolic pathways of shikonin in human tissues were characterized. Shikonin can be metabolized to several trace metabolites by CYPs in human liver microsome (HLM), in the presence of NADPH. Shikonin also can be easily catalyzed by human UGTs and form a monoglucuronide (SG); however it can not be catalyzed by SULTs. The glucuronidation site which was identified by LC-MS/MS is not at the two phenolic hydroxyl groups but at the alcohol hydroxyl group on the side chain of shikonin. The enzymes participating in shikonin glucuronidation were UGT1A9, UGT1A8 and UGT2B7. Among these isoenzymes, UGT1A9 displays the best catalytic ability, while UGT2B7 and UGT1A8 only display limited activities. The kinetic studies on shikonin glucuronidation in UGT1A9, UGT1A8 and UGT2B7, as well as in HLM, human intestine microsome (HIM) and human kidney microsome (HKM) were characterized. In recombinant enzymes, UGT1A9 shows the highest affinity to shikonin with Km 4.95 mM, the Km of UGT2B7 and UGT1A8 are 27.10 mM and 81.06 mM respectively. The intrinsic clearance (Vmax/Km) of UGT1A9, UGT2B7, and UGT1A8 are 264.87 pmol/min/mg, 13.35 pmol/min/mg, 0.49 pmol/min/mg respectively. In tissue microsomes, HKM shows the highest affinity to shikonin with Km 3.52 mM, followed by HLM 7.71 mM and HIM 64.88 mM. The intrinsic clearance in HKM, HLM and HIM are 281.03 pmol/min/mg, 283.49 pmol/min/mg and 0.62 pmol/min/mg respectively. Except UGT1A8 followed Michaelis-Menten kinetic model; UGT1A9, UGT2B7, HLM, HIM and HKM all displayed substrate

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inhibition models. The similar Km and intrinsic clearance for shikonin glucuronidation in UGT1A9, HLM and HKM implied that UGT1A9 plays important role in human liver and kidney. Furthermore, a strong correlation was observed between the formation rate of SG and the formation rate of propofol glucuronide, using a panel of ten individual HLMs, with the coefficient parameter of 0.89. To sum up, glucuronidation is the major metabolism pathway of shikonin in human. UGT1A9 is the major isoform responsible for shikonin glucuronidation. Shikonin glucuronidation mainly happened in liver and kidney; GI tract will not be a significant barrier for shikonin oral administration.

P64. GLUTATHIONE TRANSFERASE P1 (GSTP1) IS STRONGLY ASSOCIATED WITH CELLULAR MEMBRANES AND MODIFIED BY PALMITATE


Vanessa Marensi1, Megan Yap2, Sohail S. Qazi1, Luc G. Berthiaume2 and Elaine M. Leslie1 1 Physiology, University of Alberta, Edmonton, AB, Canada, 2Cell biology, University of Alberta, Edmonton, AB, Canada Glutathione transferase P1 (GSTP1) is involved in the detoxification of reactive compounds, through conjugation with the tripeptide glutathione (g-Glu-Cys-Gly) and also has important roles in cellular signaling. GSTP1 is described in the literature as a cytosolic enzyme; however, we have previously reported the strong association of GSTP1 with the plasma membrane. Thus, GSTP1 is not removed from the plasma membrane under harsh stripping conditions [e.g., treatment of plasma membrane sheets with potassium iodide (KI), KI-EDTA or alkaline carbonate], known to remove peripherally associated membrane proteins. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins through a thioester bond and promotes the anchoring of proteins to membranes. In order to test the possibility that GSTP1 could be palmitoylated on a cysteine residue, we subjected membranes to alkali treatment (0.1 N NaOH) known to cleave thioester bonds. Exposure to NaOH caused GSTP1 to be released from cellular membranes. These results suggested that GSTP1 could be palmitoylated via an alkali sensitive thioester bond. To confirm the possibility that GSTP1 is a palmitoylated protein, we labelled cells using an alkynylpalmitate analog and monitored the incorporation of that label using azido-biotin after click chemistry. We show data that demonstrates that GSTP1 is palmitoylated via a thioester bond. Therefore, palmitoylation of GSTP1 is likely the mechanism by which it strongly associates with the plasma membrane. The close association of GSTP1 with the plasma membrane could be important for numerous cell functions. For example, GSTP1 might be in close proximity to the plasma membrane for efficient conjugation and efflux of either labile conjugates (e.g., arsenic triglutathione) or hydrophobic toxicants (e.g.,4-hydroxynonenolglutathione).

P65. RECOMBINANT ARYLAMINE N-ACETYLTRANSFERASES OF NON-HUMAN PRIMATES AS MODELS FOR IN VITRO EVALUATION OF PHARMACEUTICAL COMPOUNDS Sotiria Boukouvala1, Theodora Tsirka1, Audrey Sabbagh2, Edith Sim3, Brigitte Crouau-Roy4 and Giannoulis Fakis1 1 Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece, 2Institut de Recherche pour le De´veloppement (IRD), Universite´ Paris Descartes, Paris, France, 3Faculty of Science, Engineering and Computing, Kingston University, Kingston-upon-Thames, United Kingdom, 4ENFA, UMR5174EDB (Laboratoire E´volution & Diversite´ Biologique), CNRS, Universite´ Paul Sabatier, Toulouse, France Human arylamine N-acetyltransferases, NAT1 and NAT2, are drug metabolizing enzymes that catalyze the acetyl-coenzyme A dependent N-acetylation of arylamines and arylhydrazines. The human NAT1 isoenzyme shows wide developmental/tissuespecific distribution and is considered to have an endogenous role, possibly in folate catabolism. Human NAT1 is also investigated as a putative cancer therapeutic target, and specific inhibitors have been developed. The human NAT2 isoenzyme is liver-specific and has a well-validated role in phase II metabolism of many drugs, including the anti-tubercular drug isoniazid. Human NAT proteins are difficult to express in E. coli to high levels of yield and purity, complicating the discovery and evaluation of novel specific compounds. The NAT proteins of laboratory rodents have been used as alternatives for in vitro screening, but their genetic homology and functional similarity to human NATs is relatively low. To overcome these problems, we have undertaken cloning and recombinant expression of the NAT1 and NAT2 genes of 11 primate species [1], including human, followed by comparative enzymatic analysis of the protein products. Recombinant human NATs were consistently expressed at significantly lower yields and appeared to be substantially less pure after affinity chromatography, compared with the majority of other primate NATs. The recombinant NAT proteins of human and non-human primates were then assayed against a panel of established NAT1- and NAT2-specific substrates, to assess enzymatic activity. The NAT1 proteins of the rhesus monkeyMacaca mulatta and the barbary ape Macaca sylvanus (which are identical in primary sequence) were the most active (20-fold higher activity measured with p-aminobenzoate, relative to human NAT1), while the most active NAT2 protein was that of the red guenon Erythrocebus patas (15-fold higher activity measured with sulfamethazine, relative to human NAT2). The recombinant NATs of available primate species were further studied by differential scanning fluorimetry, providing highest melting temperatures for the NAT1 protein of the red-cheeked gibbon Nomascus gabriellae and the NAT2 protein of M. sylvanus. Our comparative assessment of recombinant protein yield, purity, stability and enzyme activity, suggest that the conserved NAT proteins of non-human primates, particularly the gibbon N. gabriellae which is phylogenetically closest to

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human in our panel, could serve as good working models for use in chemical library screens for pharmaceutical purposes. Functional characterization is also in progress for various NAT1 and NAT2 polymorphic alleles, found in a population of rhesus monkeys, with results indicating specific effects on enzymatic activity.

Reference [1] Sabbagh, A., Marin, J., Veyssie`re, C., Lecompte, E., Boukouvala, S., Poloni, E.S., Darlu, P., and Crouau-Roy, B. (2013). Rapid birth-and-death evolution of the xenobiotic metabolizing NAT gene family in vertebrates with evidence of adaptive selection. BMC Evol. Biol. 13:62.

P66. A NOVEL HUMANIZED UGT2 MOUSE MODEL CONTAINING THE HUMAN UGT2 CLUSTER FOR ASSESSING DRUG GLUCURONIDATION


Tsuneo Deguchi1, Yasuhiro Kazuki2, Atsushi Kurihara1, Shoko Takehara3, Mitsuo Oshimura2 and Takashi Izumi1 1 Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo, Japan, 2Graduate School of Medical Science, Tottori University, Yonago, Japan, 3chromocenter Inc., Yonago, Japan The prediction of human pharmacokinetics (PK) from preclinical data is an important goal in the drug discovery and the developmental processes. The purpose of the present study was to generate a novel humanized UDP-glucuronosyl transferase 2 (UGT2) mouse model to evaluate the UGT2-dependent glucuronidation. The human UGT2 locus spans nearly 1.5 Mb on chromosome 4 and encodes nine UGT2 proteins that play prominent roles in drug and xenobiotic metabolism. However, transgenic mouse lines carrying the entire human UGT2 cluster have not been produced yet because of limitations in conventional cloning techniques. Recently, to introduce regions of large human genes into animals, we have developed a mouse artificial chromosome (MAC) vector. To generate ‘humanized’ trans-chromosomic mice with the entire human UGT2 cluster, the 1.5 Mb region of the UGT2 cluster was cloned into the MAC vector by chromosome engineering technology. The UGT2MAC was introduced into mouse embryonic stem (ES) cells using microcell-mediated chromosome transfer. Then, healthy chimeric mice containing the UGT2-MAC were generated from the ES cells, and the UGT2-MAC was transmitted through the germline. The transferred UGT2-MAC was stably maintained in vitro and in vivo. The UGT2 genes were expressed mainly in the liver, kidney and small intestine, consistent with the expression profile in humans. Plasma concentration profiles and excretion into urine and bile were investigated using the humanized UGT2 mice and wild-type mice (WT). Zidovudine (AZT), a UGT2B7 substrate, and SN-38, an exclusive UGT1A1 substrate were used as test drugs. Plasma concentration of AZT glucuronide showed the greatest increase in the humanized UGT2 mice after iv or po administration of AZT compared with WT, while that of SN38 glucuronide after iv or po administration of SN-38 was not altered in the humanized UGT2 mice. Urinary and biliary excretion of AZT glucuronide after iv administration of AZT were much higher in the humanized UGT2 mice compared with WT. However, those of SN38 glucuronide after iv administration of SN-38 were not altered in humanized UGT2 mice. In vitro enzyme activities were determined using liver microsomes prepared from the humanized UGT2 mice or WT, and results showed patterns consistent with the in vivo PK data for AZT and SN-38. Novel humanized UGT2 mice exhibited an efficient capacity of drug glucuronidation similar to that in humans, suggesting that they could be useful animal models in the drug development. The next step is to develop humanized UGT2 mice in which the endogenous Ugt2 gene cluster is disrupted.

P67. REFINING THE SULT1B1 MECHANISM: IMPORTANCE OF THE HIGHLY CONSERVED DIMERIZATION DOMAIN Zachary E. Tibbs1 and Charles N. Falany2 1 Pharmacology & Toxicology, University of Alabama at Birmingham, Birmingham, AL, 2Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL In order to predict physiological drug actions using in silico models, the basic biological properties and processes of the enzymes involved must be understood. Specifically, cytosolic sulfotransferase (SULT) mediated sulfonate conjugation represents one avenue by which drugs, environmental chemicals, and xenobiotics are subjected to metabolic processing in the body. While extensively studied, the complex mechanism by which these enzymes perform their function is not fully understood. Comparing the primary sequences and tertiary structures of all human SULT isoforms allowed for the generation of a hSULT conservation map, allowing identification of highly conserved residues and secondary/tertiary structures which likely contribute heavily to the underlying SULT mechanism. One highly conserved region, the SULT dimerization domain, is oriented as an antiparallel interface spanning residues 266-275 (hSULT1B1) with amino acid sequence KXXXTVXXXE. This domain is located proximal to the cofactor binding site, leading us to the hypothesis that the domain plays a vital role in maintaining SULT kinetic properties through regulation of PAPS binding. As determined by gel filtration, a single K266L point mutation rendered the model SULT (hSULT1B1) monomeric, allowing for in vitro characterization of monomeric and dimeric forms of SULT1B1. Thermal stability tests, molecular dynamic (MD) simulations, kinetic analyses, binding studies, and investigation of existing crystal structure data

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were used to address our hypothesis. When subjected to 42 C incubation, monomeric SULT1B1 exhibited a significant decrease in stability compared to the dimeric enzyme. Following a 20 ns MD simulation, per residue root mean square fluctuation (RMSF) analysis of a-C atoms supports this conclusion with the largest stability divergence presented in the dimerization domain and the adjacent Loop 3. The dynamics of Loop 3 are known to alter substrate specificity in a manner dependent on cofactor binding, suggesting a relationship between dimerization and substrate specificity. Monomeric and dimeric forms of the enzyme both exhibit relatively high, but similar Km’s for PAPS around 4 mM as determined by 2-naphthol sulfation. Likewise, both the monomer and dimer SULT1B1 exhibit a similar Kd for PAPS around 100 mM as shown by intrinsic fluorescence binding studies. However, time-dependent (burst) sulfation of a well-known hSULT1B1 substrate, triclosan, by each enzyme presents altered kinetic profiles when compared to one another, leading to the conclusion that SULT1B1 dimerization contributes to kinetic properties of the enzyme, at least with specific substrates, via a dimer cross-talk mechanism. Further support for this cross-chain communication is provided in uninvestigated crystal structure data, in which nucleotide free, PAPS bound, and PAP bound dimer chains demonstrate altered orientation with respect to one another. Our data suggests that the dimerization domain contributes to the underlying mechanism of SULT1B1 through dynamic regulation while providing increased enzymatic stability. Use of dimeric SULT structures in future in silico studies involving SULTs may provide more relevant drug-interaction prediction models than the computationally advantageous monomeric models.

P68. MICE DEFICIENT IN INTESTINAL EPITHELIUM CYTOCHROME P450 REDUCTASE HAVE ELEVATED LEVELS OF SECRETARY IMMUNOGLOBIN A AND ARE PRONE TO ACUTE TOXININDUCED MUCOSAL DAMAGE Fang Xie1, Sarita Ahlawat2, Yi Zhu1, Rebecca D’Hondt2, Xinxin Ding1, Qing-Yu Zhang1 and Nicholas J. Mantis2 1 Division of Environmental Health Sciences, Wadsworth Center, NYSDOH, Albany, NY, 2Division of Infectious Diseases, Wadsworth Center, NYSDOH, Albany, NY Cytochrome P450 (P450) enzymes constitute a ubiquitous superfamily of heme-containing enzymes that mediate the biotransformation/detoxification of numerous drugs and other xenobiotics. P450 enzymes are also involved in the metabolism of various endogenous compounds, including retinoids, glucocorticoids, and eicosanoids, that are postulated to participate in the maintenance and/or development of inflammatory and immune reactions in the intestinal mucosa. To investigate the role of P450 enzymes in intestinal inflammation and immunity, we took advantage of the recently described IE-Cpr-null mice [1], which are deficient specifically in intestinal epithelium of NADPH-cytochrome P450 reductase (CPR), the obligate redox partner of all microsomal P450 enzymes. We report that IE-Cpr-null mice, following an acute ricin toxin challenge, had higher levels of the pro-inflammatory chemokine MCP-1 and increased tissue damage, as compared to wild type mice. While retinoic acid and glucocorticoid levels in serum and intestinal epithelium were not significantly altered in IE-Cpr-null mice as compared to wildtype animals, basal levels of two P450-produced arachidonic acid metabolites (11,12-DiHETE and 14,15-DiHETE) with known anti-inflammatory property were significantly lower. In addition, levels of the endocannabinoid 2-arachidonoylglycerol were significantly higher in the intestinal mucosa of IE-Cpr-null mice than in wild-type mice. In terms of mucosal immunity, IE-Cprnull mice had normal Peyer’s patch numbers and were unaffected in their capacity to elicit a secretory IgA (SIgA) antibody response following intragastric immunization with cholera toxin. SIgA baseline levels in IE-Cpr-null mice were, however, elevated 2-3 fold over wild-type littermate controls, further underscoring a role for intestinal P450 enzymes in mucosal homeostasis and immunity.

Reference [1] Zhang, Q.-Y., Fang, C., Dunbar, D., Zhang, J., Kaminsky, L.S., and Ding, X. An intestinal epithelium-specific cytochrome P450 reductase-knockout mouse model: direct evidence for a role of intestinal cytochromes P450 in first-pass clearance of oral nifedipine. Drug Metab. Dispos. 37, 651–657, 2009. PMC2645477

P69. TRANSCRIPTOMIC AND METABOLOMIC EFFECTS OF INDIVIDUAL CYP EXPRESSION IN HEPG2 CELLS Aurelie J. Krol1, Anne Bolopion1, Mary Dutheil1, Ce´line Lepeigneux1, Ahmad Al Ali1, Monique Diry1, Gildas Bertho2, Philippe Beaune3 and Isabelle de Waziers1 1 INSERM U775, Paris, France, 2Umr 8601, Uninersite´ Paris Descartes, Paris CX 06, France, 3U775, INSERM, Paris, France The cytochrome P450 (CYP) family is a multigene family of around 50 heme-containing enzymes, out of which 23 are involved in the metabolism of xenobiotics. If their roles in xenobiotics metabolism is well recognized and described, the effects of those enzymes on endobiotics is not well understood. In this work we investigated the effects of the expression of some of the main xenobiotics-metabolizing CYP on HepG2 cells. The HepG2 hepatic cell line does not express CYPs. We infected HepG2 cells with recombinant lentiviruses expressing either


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GFP alone or GFP and one CYP under the control of the hepatic promoter Phospho Glycerate Kinase (PGK). It allowed a stable expression of the transgene in the HepG2 cells. We checked that the expressed P450 protein had a catalytic activity. We established stable HepG2 cell lines expressing GFP (control), CYP2C9, CYP2D6 and CYP3A4. We harvested cells from the different cell lines, and performed analysis of the transcriptome, using pangenomic affymetrix microarray GeneChip Human Gene 1.0 ST. Preliminary analysis of the results showed that the effects of each CYP was different, both in terms of the number of differently expressed genes and of the nature of those genes. CYP3A4 expression influenced genes linked to carbohydrate and lipid metabolism, CYP2D6 influenced genes linked to xenobiotic metabolism, as well as cellular adhesion, and carbohydrate binding, whereas CYP2C9 influenced genes linked to cell adhesion and motility, as well as glycoprotein metabolic process, and coagulation factors. Further experiments are currently under way to validate those results (qPCR). We also analyzed by RMN the metabolites present in the culture medium and planned to analyze the intracellular medium for the GFP, CYP2C9, CYP2D6 and CYP3A4 cell lines. Culture medium composition from GFP and CYP3A4 cell lines were not distinguishable by RMN, whereas CYP2D6 lines had an increase in glutamine concentration, and a decrease in lactate concentration, compared to the GFP and CYP3A4 cell lines. The culture medium from CYP2C9 cell line is now under investigation. These results could lead to the idea that the xenobiotics metabolizing CYP also metabolizes CYP specific endogenous substrates.

P70. TOWARDS THE ABSOLUTE QUANTIFICATION OF CYP450 ISOFORMS: A PROTEOMICS STUDY Jingkai Gu collage of life science, Research Centre for Drug Metabolism, Jilin University, Changchun, China ‘‘Drug metabolism’’ or ‘‘biotransformation’’ is the process by which the drug is chemically converted in the body to a metabolite. Biotransformation is usually an enzymatic process.The most important enzymes responsible for oxidation and reduction of drugs are cytochrome P450 enzyme group, which consists of closely related isozymes that differ somewhat in amino acid sequence and drug specificity. It involves in the biotransformation of many endogenous and exogenous compounds including activate many drugs, pre-carcinogens, pre-toxins and mutagens. In recent years, the genetic polymorphism of cytochrome P450 and its relationship with drug metablism is gradually paid more and more attention. The purpose of this study was to determine the absolute protein expression levels of cytochrome P450 enzyme using liquid chromatography-tandem mass spectromery(LC-MS/MS). The absolute level of cytochrome P450 isozymes was measured by the determination of isoform specific peptides released by enzymatic digestion of the rat liver microsomes, using multiple reaction monitoring(MRM). The method uses synthetic stable isotope-labeled peptide as internal standards which has tryptic cleavage sites, attempting to create an internal standard that has cleavage characteristic more similar to the protein being quantified. Results shown that the method could be used for the determination of cytochrome P450 isoforms and improved the accuracy and precision comparing with reported studies.

P71. EXPRESSION OF CLINICALLY RELEVANT METABOLIZING ENZYMES AND NUCLEAR RECEPTORS ALONG THE ENTIRE HUMAN INTESTINE Stefan Oswald1, Jette Penski2, Christian Groër1, Susanne Bru¨ck1, Marek Drozdzik3 and Werner Siegmund4 1 Clinical Pharmacology, University of Greifswald, Greifswald, Germany, 2Clinical Pharmacology, University of Greifswald, Greifswald, 3Clinical Pharmacology, Pomeranian Medical University, Szczecin, Poland, 4Department of Clinical Pharmacology, University of Greifswald, Greifswald, Germany Introduction: The oral bioavailability of many drugs is influenced by intestinal metabolizing enzymes such as cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs). The expression of these enzymes is regulated by nuclear receptors (NR) including pregnane X receptor (PXR), farsenoid X receptor (FXR) and constitutive androstane receptor (CAR) the activation of which was shown to cause substantial drug-drug interactions. In order to estimate or even predict their impact on oral drug absorption, data on their intestinal expression levels are an indispensable prerequisite. Therefore, it was the aim of this study to comprehensively analyse the expression of clinically relevant phase I and II enzymes as well as nuclear receptors along the entire human intestine. Materials and Methods: Intestinal tissue was collected from 8 organ donors (age: 24–56, cause of death: suicide or intracranial bleeding). In each case, 10 samples from duodenum, jejunum, ileum and colon were taken and mRNA expression of CYP2B6, 2C8, 2C9, 2C19, 2D6, 3A4 and 3A5, SULT1A family, UGT1A family, UGT2B7 and UGT2B15 as well as PXR, FXR, CAR, LXRalpha/beta was measured by real-time RT-PCR (TaqManÕ Low Density Arrays). In parallel, the absolute protein expression the aforementioned enzymes and nuclear receptors will be measured using LC-MS/MS-based targeted proteomics. Results: With exception of CYP2C8, all investigated CYP enzymes could be detected in nearly all intestinal segments. Expression (mRNA) of CYP3A4, CYP3A5, CYP2B6, CYP2C9, CYP2C19 and CYP2D6 was found to be the highest in duodenum and jejunum, and significantly decreased from the upper intestine to colon. In duodenum and jejunum, the relative

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CYP expression was as follows: CYP3A44CYP2C94CYP2C194CYP2B64CYP3A5 and CYP2D6. In the entire colon only very small amounts of all previously mentioned enzymes have been observed. Intestinal SULT1A and UGT1A enzymes as well as UGT2B7 demonstrated similar expression pattern along the human GI tract. The highest quantity of these enzymes was found in jejunum, followed by a significant decline in ileum and colon. SULT1A- and UGT1A- enzymes could also be determined in colonic tissue. In contrast to this, UGT2B15 could not be detected in any of the intestinal samples. PXR was the most abundant NR in all gut segments, with highest expression in jejunal tissue. The relative NR expression in the intestine was PXR44LXRalpha4LXRbeta4FXR4CAR (only traces). In addition, absolute protein expression data will be presented. Conclusion: The observed expression pattern for phase I and II enzymes as well as NR may be an explanation for site-dependent oral drug absorption and different contribution of each enzyme to intestinal drug-drug interactions.

P72. INDUCTION OF PROCARCINOGEN-ACTIVATING ENZYMES, CYP1A1, CYP1A2, AND CYP1B1 BY TRIMETHYL ARSINE OXIDE (TMA) IN C57BL/6 MICE


Osama H. Elshenawy and Ayman O.S. El-Kadi Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada Arsenic is the most common cause of acute heavy metal poisoning in adults and was found to be associated with skin and several types of internal cancers. The Agency for Toxic Substances and Diseases Registry (ATSDR) and the Canadian Environmental Protection Act Registry (CEPA) have ranked arsenic as the most hazardous, toxic and carcinogenic substance in the environment. Arsenic biomethylation is widespread in nature and has been observed in bacteria, archaea, fungi, algae, plants, animals and humans. Although, methylation of arsenic was considered to be a detoxification mechanism, recent reports revealed that methylation could activate the toxic and carcinogenic potential of arsenic. We hypothesized that the pentavalent organic metabolite, trimethyl arsine oxide (TMA), activates procarcinogen-activating enzymes cyp1a1, cyp1a2 and cyp1b1. For this purpose, C57BL/6 mice were received single intraperitoneal injected of TMA (13 mg/kg) with or without the prototypical aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzodioxin (TCDD; 15 mg/kg) for 6 (for gene expression determination) and 24 h (for protein expression and catalytic activity determination). The livers were harvested for total tissue RNA isolation (6 h treatment), and microsomal protein preparation (24 h treatment). The gene expression of different cyp enzymes as well as oxidative markers such as NADH quinone oxidoreductase (NQO1), glutathione S-transferase A1 (Gsta1) and heme oxygenase-1 (Ho-1) were determined by reverse transcription-polymerase chain reaction (RT-PCR) followed by real-time PCR. Protein expression levels of cyp enzymes were determined using Western blot analysis, whereas catalytic activities were measured using 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-deethylase (MROD) assays. Gene expression results showed that TMA alone increased cyp1a1, cyp1b1, Gsta1 and Nqo1 mRNA levels. Upon co-exposure to TMA and TCDD, TMA significantly potentiated the TCDD-mediated induction of the cyp1a1 and Nqo1. Ho-1 mRNA was found to be increased significantly in the presence of both TMA and TCDD. Western blotting revealed that, TMA alone significantly increased cyp1a1 and cyp1a2 protein levels. Furthermore, TMA significantly potentiated the TCDD-mediated induction of cyp1a1 and cyp1b1 protein levels. In addition, TMA alone significantly increased EROD and MROD activities and significantly potentiated the TCDD-mediated induction of EROD. In conclusion, our results demonstrate for the first time that the TMA, induce the procarcinogen activating enzymes at gene expression, protein, and activity levels. This represents a novel mechanism by which arsenic causes carcinogenicity and would help designing strategies to treat arsenic-induced carcinogenicity and toxicity. Support: This work was supported by NSERC Discovery Grant RGPIN 250139 to A.O.S. O.H.E. is the recipient of University of Alberta Doctoral Recruitment Scholarship.

P73. INDUCTION OF PROCARCINOGEN-ACTIVATING ENZYMES, CYP1A1, CYP1A2, AND CYP1B1 BY TRIMETHYL ARSINE OXIDE IN C57BL/6 MICE Osama H. Elshenawy and Ayman O.S. El-Kadi Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada Arsenic is the most common cause of acute heavy metal poisoning in adults and was found to be associated with skin and several types of internal cancers. The Agency for Toxic Substances and Diseases Registry (ATSDR) and the Canadian Environmental Protection Act Registry (CEPA) have ranked arsenic as the most hazardous, toxic and carcinogenic substance in the environment. Arsenic biomethylation is widespread in nature and has been observed in bacteria, archaea, fungi, algae, plants, animals and humans. Although, methylation of arsenic was considered to be a detoxification mechanism, recent reports revealed that methylation could activate the toxic and carcinogenic potential of arsenic. We hypothesized that the pentavalent organic metabolite, trimethyl arsine oxide (TMA), activates procarcinogen-activating enzymes cyp1a1, cyp1a2 and cyp1b1. For this purpose, C57BL/6 mice were received single intraperitoneal injected of TMA (13 mg/kg) with or without the prototypical aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzodioxin (TCDD; 15 ¼ g/kg) for 6 (for gene expression determination) and 24 h (for protein expression and catalytic activity determination). The livers were harvested for total tissue RNA isolation (6 h treatment), and microsomal protein preparation (24 h treatment). The gene expression of different cyp

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enzymes as well as oxidative markers such as NADH quinone oxidoreductase (NQO1), glutathione S-transferase A1 (Gsta1) and heme oxygenase-1 (Ho-1) were determined by reverse transcriptionpolymerase chain reaction (RTPCR) followed by real time PCR. Protein expression levels of cyp enzymes were determined using Western blot analysis, whereas catalytic activities were measured using 7-ethoxyresoruuˆn O-deethylase (EROD) and 7-methoxyresoruuˆn O-deethylase (MROD) assays. Gene expression results showed that TMA alone increased cyp1a1, cyp1b1, Gsta1 and Nqo1 mRNA levels. Upon co-exposure to TMA and TCDD, TMA significantly potentiated the TCDD-mediated induction of the cyp1a1 and Nqo1. Ho-1 mRNA was found to be increased significantly in the presence of both TMA and TCDD. Western blotting revealed that, TMA alone significantly increased cyp1a1 and cyp1a2 protein levels. Furthermore, TMA significantly potentiated the TCDD-mediated induction of cyp1a1 and cyp1b1 protein levels. In addition, TMA alone significantly increased EROD and MROD activities and significantly potentiated the TCDD-mediated induction of EROD. In conclusion, our results demonstrate for the first time that the TMA, induce the procarcinogen activating enzymes at gene expression, protein, and activity levels. This represents a novel mechanism by which arsenic causes carcinogenicity and would help designing strategies to treat arsenic-induced carcinogenicity and toxicity. Support: This work was supported by NSERC Discovery Grant RGPIN 250139 to A.O.S. O.H.E. is the recipient of University of Alberta Doctoral Recruitment Scholarship.

P74. ROLE OF HEPATIC AND INTESTINAL P450 ENZYMES IN THE METABOLIC ACTIVATION OF THE COLON CARCINOGEN AZOXYMETHANE IN MICE Vandana Megaraj, Cheng Fang, Nataliia Kovalchuk, Yi Zhu, Qing-Yu Zhang and Xinxin Ding Molecular Toxicology, Wadsworth Center, New York State Department of Health, Albany, NY Azoxymethane (AOM) is a potent colon-specific carcinogen in rodents. It undergoes sequential metabolic activation by P450 enzymes, forming the reactive methyldiazonium ion. The latter alkylates DNA at guanine, forming adducts, of which O6methylguanine (O6-mG) is the most mutagenic and contributes to colon tumorigenesis. The mechanistic details of the in vivo metabolic activation of AOM remain unclear. The aim of this study was to determine whether P450 enzymes of the intestine, the target organ, contribute to AOM bioactivation in vivo. We compared tissue levels of O6-mG and N7-mG adducts between wildtype (WT) B6 mice and intestinal epithelium-specific Cpr-null (IECN) mice, which have little P450 activity in the intestine, following treatment of the mice with AOM. We further assessed the contributions of hepatic P450 enzymes to AOM-induced DNA damage, by comparing between liver-Cpr-null (LCN) and WT mice. We also determined rates of in vitro AOM metabolism, including N7-mG DNA adduct formation, by liver, small intestine and colon microsomes from WT, IECN and LCN mice. We found that, at 6 h following AOM treatment (at 14 mg/kg, s.c.), O6-mG and N7-mG levels were highest in liver, followed by colon, and lowest in the small intestine, in WT mice. In IECN mice, compared to WT mice, the hepatic and colonic DNA adduct levels were unchanged, but the small intestinal adduct levels were significantly decreased (by 40%). In LCN mice, compared to WT mice, the hepatic O6-mG and N7-mG adduct levels were significantly decreased (by 60%), while the colonic O6-mG and N7-mG levels were significantly increased by 1.5- and 2-fold, respectively. The tissue-specific impact of the CPR loss in IECN and LCN mice on microsomal AOM metabolic activity was confirmed. Compared to WT mice, the IECN mice had significant decreases in AOM metabolic activity in both small intestine and colon, but not in liver, while the LCN mice had essentially no microsomal AOM metabolic activity in liver, but had normal activity in small intestine and colon. Taken together, these results indicate that hepatic P450 enzymes have high capacity to bioactivate AOM both in vitro and in vivo, but they are not essential for AOM-induced O6-mG formation in the colon. Furthermore, intestinal-epithelial P450 enzymes are clearly capable of bioactivating AOM, and they likely contribute to AOM-induced O6-mG formation in the colon. Our findings support the hypothesis that both hepatic and target-tissue bioactivation of AOM by P450 enzymes plays an important role in AOM-induced colon carcinogenesis. (Supported in part by NIH grants CA092596 and GM082978)

P75. HUMAN LIVER S9 FRACTIONS STORED AT -80 C MAINTAIN HIGH PHASE I AND PHASE II ENZYMATIC ACTIVITIES OVER MULTIPLE FREEZE/THAW CYCLES AND FOR AT LEAST 10 YEARS Clayton JM Otwell, Zell Woodworth and David B. Buckley XenoTech, LLC, Lenexa, KS The use of S9 fractions in metabolic stability, clearance and phenotyping studies has markedly increased during the past five years with expanded interest in the biotransformation of xenobiotics by phase II enzymes and other cytosolic enzymes (e.g. aldehyde oxidase). To ensure integrity of the in vitro test system, it is important to understand the stability of metabolic enzymes in S9 fractions during long-term storage conditions and throughout multiple freeze/thaw cycles. Therefore, the present study evaluates the effects of long-term storage at 80 C and multiple freeze/thaw cycles on the enzymatic activities in human liver S9 fractions. Various lots of pooled (n ¼ 16 to 200) human liver S9 samples were prepared over a ten year period and stored at 80 C for up to 10 years. These S9 fractions were analyzed for their ability to catalyze reactions for various phase I and phase II enzymes. Additionally, a single lot of pooled (n ¼ 200) human liver S9 samples were subjected to up to 10 freeze/thaw cycles and further evaluated for potential loss of enzymatic activities with increasing freeze/thaw cycles. Enzyme activities were

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evaluated with marker reactions for cytochrome P450 (CYP) UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), sulfotransferase (SULT), aldehyde oxidase (AO), and N-acetyltransferase (NAT). Metabolite formation was quantified by LC-MS/MS analysis. There was little to no effect of long-term storage at 80 C on the enzyme activities tested. Sampleto-sample variation in the activity of CYP, GST, UGT, NAT and AO activities was nominally effected by long-term storage of liver S9 at 80 C. The observed variation in enzyme activities from lots prepared over numerous years was within the typical lot-to-lot variation observed as donors are replaced in pooled subcellular fractions. Similarly, CYP, UGT, GST, SULT, AO, or NAT enzyme activities in liver S9 samples subjected to up to 10 freeze/thaw cycles did not exhibit significant loss (typically less than 10%). In the worst case, GST activity decreased by 13% after 10 freeze/thaw cycles. These results suggest that our methods for preparation and storage of human liver S9 fractions are well suited to preserving enzymatic activities for up to 10 freeze/thaw cycles and for at least 10 years.


P76. EVALUATION ON THE ACTIVITIES OF CYTOCHROME P450 ISOFORMS BY COCKTAIL APPROACH IN STREPTOZOTOCIN INDUCED DIABETIC RATS Qin LI1, Jian-Jie Jiao1, Weizhen GAO2, Lin Meng2, Yanxia LIU3, Jianing Shang4 and Jianshi Lou2 1 Pharmacology, Tianjin Medical University, Tianjin, China, 2Department of Pharmacology, TIANJIN MEDICAL UNIVERSITY, Tianjin, China, 3Pharmacology, TIANJIN MEDICAL UNIVERSITY, Tianjin, China, 4Department of Pharmacology, Tianjin Medical University, Tianjin, China Backgrounds: It is now becoming increasingly evident that the induction and regulation of the cytochrome P450-dependent mixed-function oxidases is not determined solely by exposure to xenobiotics, but may also be triggered by pathophysiological conditions. Objective: To study on the variations of metabolic activities and protein contents of CYP450 isoforms CYP1A2, CYP 2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 in streptozotocin (STZ)-induced diabetic rats by a six-probe cocktail approach composed of caffeine, tolbutamide, omeprazole, metoprolol, chlorzoxazone and midazolam. Methods: Diabetic rats were prepared by STZ induction. Rats were randomly divided into 4 groups: control group, diabetic 1-week group, diabetic 4-week group and diabetic 8-week group. The six-probe cocktail including caffeine (2.5 mg/kg), tolbutamide (2.5 mg/kg), omeprazole (10 mg/kg), metoprolol (10 mg/kg), chlorzoxazone (5 mg/kg) and midazolam (5 mg/kg) was intravenously injected to all the rats. Before and after injection, the blood samples were collected at a series of time-points and the concentrations of six probe drugs in plasma were determined by a HPLC method with UV detection in a single run. The main pharmacokinetic parameters were calculated by the DAS 2.0 software (Drug And Statistics 2.0 edition, provided by Chinese Pharmacological Society). The protein levels of CYP450 isoforms were measured by ELISA. Results: Compared with control group, AUC of caffeine in diabetic 4 and 8 week group decreased by 62.78% and 79.31%, CLz increased by 5.12 and 52.35 times, respectively. There were no significant differences of AUC, t1/2z, CLz of metoprolol in all diabetic groups. AUC of chlorzoxazone in diabetic 8 week group decreased by 60.58%, CLz increased by 1.2 times. AUC of omeprazole in diabetic 4- and 8-week group decreased by 55.60% and 81.42%, CLz increased by 27.50 and 41.25 times, respectively. AUC of tolbutamide in diabetic 8-week group decreased by 45.39% and CLz increased by 83.33%. AUC of midazolam in diabetic 1- and 8-week group decreased by 43.23% and 53.87%, CLz increased by 1.33 and 2.04 times, respectively. Compared with control group, the protein level of CYP1A2 in diabetic 1- and 4-week group increased by 24.60% and 26.41%, the protein level of CYP3A4 in diabetic 1-week group decreased by 24.62%. The protein levels of CYP2D6, CYP2E1, CYP2C19 and CYP2C9 in all groups were not different. Conclusion: The metabolic activities of CYP1A2, CYP2E1, CYP2C19, CYP2C9 and CYP3A4 were induced, but CYP2D6 was not changed in STZ-induced diabetic rats. This work was supported by a grant from Tianjin Natural Science Foundation key project of China, No. 09JCZDJC21500. *corresponding author: [email protected]

P77. THE ROLE OF HEPATIC CYTOCHROME P450 2A5 DURING ENDOPLASMIC RETICULUM STRESS Larry J. Morgan, Monica Antenos and Gordon M. Kirby Biomedical Sciences, University of Guelph, Guelph, ON, Canada Cytochrome P450 2A5 is a murine orthologue of the human variant, CYP2A6, and is predominantly found within the endoplasmic reticulum (ER) of the liver. CYP2A5 differs from its P450 counterparts in that it is induced during liver injury caused by hepatitis (microbial), liver cancer, and hepatotoxicity [1]. All of these events induce ER stress and protein malfolding, eventually resulting in apoptosis if not rectified [2]. ER stressors Thapsigargin (Tg) and Tunicamycin (Tu) induce Nuclear factorE2 p45-related factor 2 (Nrf2) translocation [3]. Moreover, Nrf2 transcriptionally regulates Cyp2a5 [4]. Furthermore, the oxidized form of DTT (DTTox) induces CYP2A5 in primary mouse hepatocytes while Tg and Tu do not, suggesting a reductive ER environment may be required for induction [5]. The purpose of this study is to investigate the mechanism of Cyp2a5 gene regulation by DTTox and to assess the cytoprotective role of CYP2A5 during reductive ER stress. Treatment of mouse liver hepatoma (Hepa 1-6) cells with DTTox induced nuclear translocation of Nrf2 within 1 h. In reporter assays, DTTox treatment of primary hepatocytes transfected with a Cyp2a5 promoter construct showed a 50% increase in Cyp2a5 reporter activity.

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Treatment of hepatocytes with the reducing agent 2-mercaptoethanol increased hepatic CYP2A5 levels by 3-fold, further indicating that CYP2A5 levels increase in response to reductive stress in the ER. Furthermore, DTTox induces the ER stress biomarkers glucose-related protein 78 (GRP78) mRNA, and spliced x-box binding protein-1s (XBP-1s) mRNA, ER stress biomarkers involved in activation of the Unfolded Protein Response (UPR). Transient overexpression of CYP2A5 in Hepa 1–6 cells blocked XBP-1s mRNA splicing and caspase-3 cleavage by approximately 40% and 25% respectively while GRP78 levels were unaffected. These results indicate that Cyp2a5 is transcriptionally up-regulated in response to reductive stress and performs a cytoprotective role in the ER of hepatocytes. Future studies will investigate the mechanisms underlying the cytoprotective role of CYP2A5 during ER stress and to determine if Nrf2 is required for the transactivation of Cyp2a5 by DTTox.


References 1. Kirby, G.M., K.D. Nichols, and M. Antenos, CYP2A5 induction and hepatocellular stress: an adaptive response to perturbations of heme homeostasis. Curr Drug Metab, 2011. 12(2): p. 186–97. 2. Malhi, H. and R.J. Kaufman, Endoplasmic reticulum stress in liver disease. Journal of hepatology, 2011. 54(4): p. 795–809. 3. Cullinan, S.B., et al., Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival. Molecular and cellular biology, 2003. 23(20): p. 7198–7209. 4. Abu-Bakar, A., et al., Regulation of CYP2A5 gene by the transcription factor nuclear factor (erythroid-derived 2)-like 2. Drug Metab Dispos, 2007. 35(5): p. 787–94. 5. Gilmore, W.J. and G.M. Kirby, Endoplasmic reticulum stress due to altered cellular redox status positively regulates murine hepatic CYP2A5 expression. J Pharmacol Exp Ther, 2004. 308(2): p. 600–8.

P78. REGULATION OF CYTOCHROME P450 2A5 BY 6,7-DIMETHYLESCULETIN IN MURINE HEPATOCYTES Sangsoo Daniel Kim, Elyse J. Hargreaves and Gordon M. Kirby Biomedical Sciences, University of Guelph, Guelph, ON, Canada 6,7-dimethylesculetin (DE) is a compound present in Artemisia capillaris, an herb widely used in Asia to prevent and treat neonatal jaundice. Previous studies have identified the constitutive androstane receptor (CAR) as a key regulator of bilirubin metabolism in the liver and DE activates CAR in mice [1, 2]. Additionally, recent studies have shown that cytochrome P450 2A5 (CYP2A5) is a novel inducible bilirubin oxidase and plays a cytoprotective role against bilirubin toxicity as an alternative to BR conjugation by uridine-diphosphate glucuronyltransferase 1A1 (UGT1A1) [3, 4]. Recently, we have shown that CAR is required for phenobarbital induction of CYP2A5, suggesting that DE may also regulate Cyp2a5 [5]. Thus, we hypothesized that Cyp2a5 is regulated by DE in murine hepatocytes via CAR. We investigated the effect of DE on the expression of Cyp2a5 and further examined the regulatory mechanism of Cyp2a5 by DE. Cultured primary hepatocytes were treated with various doses of DE (0, 10, 25, and 50 mM) at different time points (0, 1, 2, 3, 6, 24 h), and CYP2A5 mRNA and protein levels were determined. DE induced CYP2A5 expression at the mRNA and protein levels in a dose-dependent manner. DE treatment also caused nuclear translocation of CAR at 1 h, but not at 2, 3, 6, 24 h. In reporter assays, DE treatment of hepatocytes transiently transfected with a Cyp2a5promoter-luciferase reporter construct increased reporter activity by 8-fold at 1 hr, but no activities were seen at 2, 3, 6, and 24 h. Furthermore, cotransfection of hepatocytes with a CAR expression vector resulted in an increase inCyp2a5 reporter activity by approximately 2-fold. These results indicate that DE regulates CYP2A5 in transcriptional manner via CAR activation and suggests that DE may be useful in the treatment of jaundice by increasing both UGT1A1- and CYP2A5-mediated bilirubin metabolism.

References 1. Huang, W., et al., Induction of bilirubin clearance by the constitutive androstane receptor (CAR). Proc Natl Acad Sci U S A, 2003. 100(7): p. 4156–61. 2. Huang, W., J. Zhang, and D.D. Moore, A traditional herbal medicine enhances bilirubin clearance by activating the nuclear receptor CAR. J Clin Invest, 2004. 113(1): p. 137–43. 3. Abu-Bakar, A., et al., Inducible bilirubin oxidase: a novel function for the mouse cytochrome P450 2A5. Toxicol Appl Pharmacol, 2011. 257(1): p. 14–22. 4. Kim, S.D., et al., Cytochrome P450 2A5 and bilirubin: Mechanisms of gene regulation and cytoprotection. Toxicology and Applied Pharmacology, 2013. 270(2): p. 129–138. 5. Kirby, G.M., K.D. Nichols, and M. Antenos, CYP2A5 induction and hepatocellular stress: an adaptive response to perturbations of heme homeostasis. Curr Drug Metab, 2011. 12(2): p. 186–97.

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P79. THE ROLE OF CYTOCHROME P450 ENZYMES IN THE FORMATION OF EPOXYEICOSATRIENOIC ACIDS, AND MID-CHAIN, SUBTERMINAL, AND TERMINAL HYDROXYEICOSATETRAENOIC ACIDS


Ahmed A. El-Sherbeni and Ayman O.S. El-Kadi Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada Cytochrome P450 (P450) enzymes oxidize arachidonic acid (AA) to several epoxy-, and hydroxy metabolites with prominent physiological effects. However, these metabolites possess diverse biological functions; whereas, epoxymetabolites, epoxyeicosatrienoic acids (EETs), exhibit potent vasodilatory, anti-platelet, anti-inflammatory, and fibrinolytic properties, most hydroxyeicosatetraenoic acids (HETEs) are harmful. Since P450 levels have been reported to be altered during several diseases, it is imperative to determine the full spectrum of AA metabolism by different P450 enzymes. We investigated the potential role of recombinant rat CYP1A1, CYP1A2, CYP2A1, CYP2B1, CYP2C6, CYPC11, CYP2C13, CYP2D1, CYP2E1, and CYP3A1 and recombinant human CYP1B1, CYP2J2, CYP4A11, and CYP4F2 in the formation of 5,6-, 8,9-, 11,12-, and 14,15-EET, 5-, 8-, 9-, 11-, 12-, and 15-HETE (mid-chain HETEs), 16-, 17-, 18-, and 19-HETE (!-1 HETEs) and 20-HETE. P450 enzymes were incubated with a physiological concentration of AA (75 mM) for 15 to 25 min. The levels of arachidonic acid metabolites were determined by liquid chromatography/mass spectroscopy (LC/MS). A sensitive, specific, and robust LC/MS method was developed that allows simultaneous analysis of the targeted metabolites. The metabolites were separated using a gradient elution of 8% methanol, 8% isopropanol, and 84% acetonitrile with 0.1% formic acid and water with 0.1% formic acid (v/v) at a flow rate of 200 mL/min with a C18 column (250mm 2.1 mm). All P450 were able to oxidize AA to different metabolites but with different extent. Among the P450, rat CYP1A1, CYP1A2, CYP2C6, and CYP2C11 and human CYP4A11, and CYP4F2 possessed the highest activity towards AA. On the other hand, rat CYP2A1, CYP2C13, and CYP2E1 were the lowest. CYP1A1, and CYP1A2 produced mainly !-1 HETEs by 88%, and 61%, respectively. The prominent metabolites for CYP2C11 were EETs (64%), whereas for CYP2C6 were mid-chain HETEs (47%), and EETs (31%). For CYP4A11, and CYP4F2, they produced mainly 20-HETE as 71%, and 81% of the total metabolites, respectively. Also, P450 enzymes were found to exhibit a characteristic regioselective oxidation of AA. For CYP1A1, it preferentially oxidized AA to produce 19-HETE, while CYP1A2 produced mainly 16-HETE. The main epoxy-metabolite for CYP2C11, and CYP2C6 were 11,12-, and 14,15-EET, respectively. In conclusion, the data suggested that CYP2C subfamily demonstrates the major P450-AA epoxygenase activity, while CYP4 family is the main 20-HETE producer. Interestingly, CYP1A subfamily is the predominant !-1 HETEs producer. These results provide important insights into the activity of arachidonic-acid-P450 monooxygenases, and link alterations in P450s levels to physiologic, or pathologic changes in the tissues. Support: This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) [Grant MOP 106665]. AAE is the recipient of Egyptian Government Scholarship.

P80. DIFFERENTIAL ROLE OF CENTRAL AND PERIPHERAL D2 RECEPTORS IN THE REGULATION OF CYP METABOLIC ACTIVITY Michaela Sabova1, Ondrej Zendulka1, Jan Jurica1, Gabriela Dovrtelova1 and Alexandra Sulcova2 1 Department of Pharmacology, CEITEC MU, Masaryk University, Brno, Czech Republic, 2Experimental and Applied Neuropsychopharmacology Research Group, CEITEC MU, Brno, Czech Republic The cytochrome P450 (CYP) enzymes belong to the major biotransformation complex in the metabolism of xenobiotics including drugs and numerous endogenous substrates. Activity of CYP is not rigid but it can be affected by many factors, which can ultimately change the effectiveness of the drug. Recently it was shown that many neuroregulatory mechanisms are involved in the regulation of CYP metabolic activity including the dopaminergic modulations. Dopamine release in the tuberoinfundibular pathways can influence the expression of CYP in the liver via dopaminergic D2 receptor system which might be a cause of interidividual differences in its metabolic activity. Modification of dopaminergic transmission may also affect the activity of CYP through changes in the plasma levels of various hormones (adrenocorticotropic hormone, corticosterone, growth hormone and/or thyroid stimulating hormone). The aim of this study was to determine the impact of the selective D2/D3 receptor antagonist raclopride, on the CYP 1A2, 2C11, 2D2 metabolic activities. Raclopride was administered in various application schemes to determine differences in the role of both central and peripheral D2 receptors. Raclopride influence on the CYP metabolic activity was tested as follows: I. raclopride administered intraperitoneally at the dose of 0.3 mg/kg/day for 7 days –antagonistic effect preferably at peripheral D2 receptors; II. raclopride administered intracerebrally at the dose of 0.3 mg/day for 10 days – antagonism at D2 receptors in the nucleus accumbens; III. liver tissue was directly influenced by raclopride added into the perfusion medium in the final concentration of 3.5 mg/l in the perfusion medium - direct interaction between raclopride and CYP; the last group were controls treated intraperitoneally with proportional volume of saline. The CYP metabolic activity was tested in the model of isolated perfused rat

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liver. Systemic administration of raclopride significantly decreased the CYP1A2 metabolic activity in the 60th and 120th min (p ¼ 0.03 and 0.01. In CYP2C11 we observed that direct interaction of raclopride significantly decreased in 30th and 120th min (p ¼ 0.1 and 0.003), whereas the CYP2C11 metabolic activity was increased in 120th min (p ¼ 0.05) after raclopride intracerebral administration. The metabolic activity of CYP2D2 was not affected by raclopride regardless of the administration scheme used. Supported by the project ‘‘CEITEC - Central European Institute of Technology’’ (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund and by Czech Ministry of Education: MSM0021622404.


P81. IDENTIFICATION OF CYTOCHROME P450S INVOLVED IN THE METABOLISM OF ARACHIDONIC ACID IN HUMAN PLATELETS Yazun B. Jarrar1, Eon-Jeong Shim2, Sun-Ah Cho1, Woo Young Kim3, Kyung-Suk Oh4, Dong-Hyun Kim1, Jae-Gook Shin5 and Su-Jun Lee3 1 Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Busan, South Korea, 2Department of Pharmacology and Pharmacogenomics Research Center, Department of Clinical Pharmacology, Inje University College of Medicine and Busan Paik hospital, Busan, South Korea, 3Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, South Korea,4Dept. of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Busan, South Korea, 5Department of Pharmacology and Pharmacogenomics Research Center, Department of Clinical Pharmacology, Inje University College of Medicine, Busan, South Korea Identification of cytochrome P450s involved in the metabolism of arachidonic acid in human platelets Yazun B. Jarrar1, Eon-Jeong Shim1,2, Sun- Ah. Cho1, Woo-Young Kim1 ,Kyung-Suk Oh1, Dong-Hyun Kim1, Jae-Gook Shin1,2, Su-Jun Lee 1 1Department of Pharmacology and Pharmacogenomics Research Center, 2Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Inje University College of Medicine, Inje University, Gaegum2dong, Busanjin-gu, Busan, South Korea. Abstract Although cytochrome P450s (CYPs) have been identified in most human cells, identification of CYPs in human platelets remains poorly explored. CYP expressions in human platelets were screened by using reverse transcriptase-polymerase chain reaction and Western blot analysis followed by functional assays using arachidonic acid (ARA). CYP1A1, 2U1, 2J2, 4A11, 4F2, and 5A1 were expressed as both proteins and mRNAs in platelets. Ethoxyresorufin-O-deethylase activity was observed in platelets and this activity was significantly decreased after treatment with the general P450 inhibitor SKF-525A and the CYP1A inhibitor, ¥a´-naphthoflavone (40–45%, p50.001). Seventeen ARA metabolites were detected in ARA-treated platelets. Among these, the levels of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids were significantly decreased with the treatment of the P450 ¥ø-hydroxylase inhibitor 17-octadecynoic acid (p50.05–0.001). In summary, multiple ARA-metabolizing P450s were identified in human platelets. These findings may provide an important resource for understanding physiological function of P450s in platelets.

P82. IMPACT OF AGE ON HEPATIC CYTOCHROME P450 OF PIGS Steven Hu Veterinary Medicine Research and Development, Zoetis, Inc, Kalamazoo, MI Cytochrome P450s (CYPs) are an important family of enzymes in the metabolism of many therapeutic agents and endogenous metabolic reactions. Age is one of the factors which has significant impact on quantities and activities of those enzymes in liver of human and animals. Pig is a mammalian species of particular interest in pharmacological and toxicological studies because it can be used as laboratory model for human metabolism without the requirement to induce the enzymes that carry out biotransformation. Pig is also an important animal protein source globally which represents 40% of worldwide meat production. In this study, CYP450 in pig livers at various ages was investigated. Livers of pigs of a domestic breed in pork industry were collected at the ages of day 1, 14, 35 70 and 140 which is close to the typical finishing phase of pig growth for slaughtering in pork industry. Liver microsomes were prepared through a low temperature and multiple step procedure. Total CYP450 was measured by a spectrophotometric method. Activities of CYP1A, 2A, 2C, 2D and 3A were evaluated through metabolism of their specific substrates. No CYP450 was able to be detected using spectrophotometric method and the activities of each CYP isoform were minimum in the liver microsomes of the pigs at day 1. CYP450 content and activities in pig liver microsomes changed with age. The CYP content increased rapidly from day 1 to 14 and showed a plateau from day 14 to 35 before increasing continuously to the highest level at day 140. The hepatic CYP activities to the substrates of CYP1A, 2A, 2C, 2D and 3A also changed with age of pigs. The age patterns of CYP450 activity were different among the CYP isoforms. These results provide valuable information for R&D of drug discovery in both human and swine medicines.

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P83. Abstract Withdrawn

P84. IDENTIFICATION AND CHARACTERIZATION OF A NOVEL CYP3A4 VARIANT RESULTING FROM MISSENSE MUTATION FOUND IN KIDNEY TRANSPLANTED PATIENT WITH DEFECTS IN TACROLIMUS CLEARANCE


Anneke N. Werk1, Stefanie Lefeldt2, Henrike Bruchmueller1, Georg Hemmrich-Stanisak3, Andre Franke3, Marcel Roos2, Klaus Thu¨rmel2, Marcus Baumann2, Dominik Steubel2, Christoph Schmarderer2, Jan H. Bra¨sen4, Uwe Heemann2, Ingolf Cascorbi1 and Lutz Renders2 1 Institute for Clinical and Experimental Pharmacology, Kiel, Germany, 2Department of Nephrology, University Hospital Klininkum rechts der Isar of technical university of Munich, Munich, Germany, 3Institute for Clinical Molecular Biology, University Hospital Schleswig-Holstein, Kiel, Germany, 4Institute of Pathology, University Hospital Eppendorf, Hamburg, Germany A 19-year-old kidney transplanted patient suffering from Alport syndrome, experienced unexpected high tacrolimus blood trough levels during immunosuppressant therapy. After a starting dose of 5 mg/day, high tacrolimus levels (23 ng/ml) were reached. Even dosage reductions (2.5 mg/day) and discontinuation of therapy resulted in increased plasma levels of 41 ng/ml tacrolimus. As liver failure or drug interactions could be excluded we hypothesised a deteriorated metabolism of the drug caused by mutations in the main detoxification enzyme CYP3A4. Exome sequencing revealed a novel SNP (c.802C4T) resulting in a premature stopp codon in exon 5. Analysis of the CYP3A4 in the Alport syndrome patient’s kidney revealed lack of variant protein expression. In-vitro analysis in transient transfected HepG2 cells confirmed no expression of CYP3A4 cDNA carrying the 802C4T variant. Functional analysis of CYP3A4 variant enzyme using a fluorescent substrate showed significantly diminished metabolism compared with the wild type giving strong evidence of variant enzyme being inactive (p 0.05; n ¼ 6). Additionally, the patient harboured the inactive CYP3A5*3 genotype, resulting in loss of function of the entire CYP3A locus contributing to the deteriorated tacrolimus clearance. This is to our knowledge the first example of a complete failure of CYP3A4 enzyme.

P85. OPTIMIZATION OF CYP KNOCK-OUT IN VIVO MODELS – RATS AND MICE Upendra A. Argikar1, Myrtha Damzal2, Adam Amaral2, Panos Hatsis2, Jennifer Bushee2, Danuta Lubicka2 and Suzie Ferreira2 1 Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research Inc., Cambridge, MA, 2Novartis Institutes for Biomedical Research Inc., Cambridge, MA 1-Aminobenzotriazole (1-ABT) is a well-known non-specific mechanism-based inhibitor of both human and non-human CYP450 enzymes. It has been widely used to distinguish CYP- from non-CYP-mediated metabolism because of its ability to inactivate almost all CYPs. Optimization of an in vivo 1-ABT pre-treatment model was performed in rats using buspirone, a compound known to undergo oxidative metabolism by CYPs, as a probe substrate. To complement this, similar study design was applied in mice along with exploiting the HRN (Hepatic CYP450 Redcutase Null) mice as a CYP knockout (KO) model. In vitro metabolite identification studies were carried out with and without 1-ABT to compare inhibition of the rate and extent of metabolism in mouse and rat liver microsomes. For in vivo studies in rats and mice, 1-ABT was administered via IP route, at 50 or 100 mg/kg, either as a 2 or 16 h pre-treatment before dosing test or probe compounds. All probe substrates were administered at 1 mg/kg IV. Buspirone was utilized as an in vivo probe and compared to controls pre-treated with saline). In 1-ABT pre-treated in rats, dose but not time dependency was observed. Clearance of buspirone was observed to be changed from 28 to 16 to 10 (mL/min/kg) after pretreatment with 1-ABT at 0, 50 or 100 mg/ kg, respectively. Similar results were seen for Compound 1, with clearance of Compound 1 changing from 96 to 45 to 15 mL/min/kg after pretreatment with 1-ABT at 0, 50 or 100 mg/kg, respectively. Interestingly, in vivo in mice, clearance of buspirone increased after pre-treatment with 1-ABT in a time dependent, but not dose dependent manner. Buspirone clearance changed from 81 (control) to 155 (2 h pre-treatment) to 500 (24 h pre-treatment) mL/min/kg. Investigations of compound 1 in 1-ABT treated mice are on-going. In vitro metabolite identification studies revealed similar extent of inhibition of oxidative metabolites in 1-ABT treated in rat and mouse liver microsomes when compared with the respective control groups. Compound 1, clozapine and perphenazine were also evaluated in HRN mice and results were compared with wild type (C57BL/6) mice matched with HRN mice for age and gender. For Compound 1, clozapine and perphenazine, clearances of 37, 6 and 22 mL/min/kg were observed in HRN mice in comparison with 89, 19 and 146 mL/min/kg in wild type mice. This investigation presents optimization of 1-ABT in rats and mice and reveals optimal CYP KO models in rodents for investigation of CYP vs. non-CYP mediated clearance.

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P86. APPLICATION OF THE CYP3A4 SELECTIVE MECHANISM-BASED INACTIVATOR CYP3CIDE TO DELINEATE CYP3A4 AND CYP3A5 CONTRIBUTIONS TO THE METABOLISM OF CYP3A CLEARED DRUGS


Elaine Tseng1, Robert L. Walsky2, Rick Luzietti2, Jennifer Harris2, Rachel E. Kosa1, Michael A. Zientek3, Theunis C. Goosen1 and R. Scott Obach1 1 Pfizer Global Research and Development, Groton, CT, 2AstraZeneca, Waltham, MA, 3Pfizer Global Research and Development, La Jolla, CA Delineation of the role of CYP3A5 to the metabolism of drugs is important because this enzyme is subject to a major genetic polymorphism. However, since CYP3A4 and CYP3A5 have overlapping substrate and inhibitor specificities, determining the relative contributions of these two enzymes to the metabolism of a drug is difficult. With the identification of a CYP3A4 selective inactivator, CYP3cide1, a tool compound is now available to delineate the contributions of CYP3A4 and CYP3A5 to the metabolism of drugs in human liver microsomes. In the present study, we determined the relative contribution of CYP3A4 and CYP3A5 to the metabolism of 36 common CYP3A substrates using human liver microsomes (HLM) with CYP3A5 *1/*1 and *3/*3 genotypes in the presence and absence of ketoconazole and CYP3cide. The contribution of CYP3A5 can be estimated by determining the difference in inhibition by ketoconazole versus inhibition by CYP3cide in *1/*1 HLM. Of the 36 substrates tested, CYP3A5 contribution ranges from none (i.e., all CYP3A4) to 80% (predominantly CYP3A5). Drugs with the greatest contribution of CYP3A5 include vincristine, atazanavir, and midazolam. Drugs with minimal, if any, CYP3A5 contribution include erythromycin, tamoxifen, and lidocaine. Furthermore, enzyme kinetic parameters (Km and Vmax) for six probe reactions of CYP3A were measured in the presence and absence of these inhibitors and were used to calculate an inter-system extrapolation factor (ISEF) for recombinant CYP3A5. This ISEF value will be useful for relating metabolism in recombinant CYP3A5 to that which would be expected in vivo. Overall, the approach described in this work offers a method to utilize CYP3cide to predict the potential impact of CYP3A5 to metabolism, and can better inform the need for clinical pharmacogenetic studies for CYP3A metabolized drugs.

Reference 1. Walsky RL, Obach RS, Hyland R, Kang P, Zhou S, West M, Geoghegan KF, Helal CJ, Walker GS, Goosen TC, Zientek MA. (2012) Selective mechanism-based inactivation of CYP3A4 by CYP3cide (PF-04981517) and its utility as an in vitro tool for delineating the relative roles of CYP3A4 versus CYP3A5 in the metabolism of drugs. Drug Metab. Dispos. Sep:40(9):1686–97

P87. IC50 SHIFT ASSAY FOR DETECTING CYTOCHROME P450 TIME-DEPENDENT INHIBITION OF DRUG CANDIDATES IN DRUG DISCOVERY AND DEVELOPMENT HyunJi Jeong, DongJin Ha and MyoungKi Baek Translational Medicine Team, SK Biopharmaceuticals, Daejeon, South Korea New draft US FDA guidance on drug interactions studies (DDI) and European Medicines Agency (EMA) guideline on the investigation of drug interactions (DDI) were released in 2012. Both of these guidance recommend determining whether an NME (New molecular entity) is a reversible and time-dependent inhibitor of major drug-metabolizing cytochrome P450 (CYP) enzymes. According to these guidance, time-dependent inhibition (TDI) should be studied in standard in vitro screening protocols by pre-incubating the drug candidate before the addition of a substrate. In order to obtain more details on drug candidates, we developed IC50 shift assay for detecting TDI rather than single point TDI assay by using human liver microsomes and various reference compounds known as time-dependent inhibitors and specific substrates of major CYP enzymes. Multiple concentrations of time-dependent inhibitors were pre-incubated with human liver microsomes for 30 min in the absence and presence of NADPH. Following pre-incubation, the mixture was incubated with the marker substrate and the reactions were terminated by combining with ice-cold acetonitrile. The supernatant from each sample after centrifugation was analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Time-dependent inhibitors as reference compounds showed from 5 to 51 fold shift in IC50 values for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 using 2 substrates when NADPH was included in the pre-incubation. These inhibitors were obvious time-dependent inhibitors in this IC50 shift assay system and these results demonstrate that our IC50 shift assay can be used to determine TDI of drug candidates in drug discovery and development.

References 1. FDA Draft Guidance, Guidance for industry, Drug interaction studies - study design, data analysis, implications for dosing, and labeling, Recommendations, 2012.

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2. FDA Draft Guidance, Guidance for industry, Drug interaction studies - study design, data analysis, and implications for dosing and labeling, 1–55, 2006. 3. European Medicines Agency Guideline on the investigation of drug interactions, 1–60, 2012. 4. Scott W. Grimm, Heidi J. Einolf, et al., The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: A perspective of the pharmaceutical research and manufacturers of America, 37(7), 1355–1370, 2009.

P88. A NEW APPROACH TO HIGH THROUGHPUT ADME ASSAY SCREENING


Jing Wang1, Gino Segre2 and Sean Xiang Wu3 1 Applications, Labcyte, Inc., Sunnyvale, CA, San Francisco, CA

2

Marketing, Labcyte, Inc., Sunnyvale, CA,

3

Quintara biosciences, So.

Early ADMET assessment is expected to not only improve the overall quality of drug candidates, but also shorten the drug discovery and development process. Time-dependent CYP450 inhibition and metabolic stability assays are critical in identifying drug candidates that may have undesirable drug-drug interactions or sub-optimal pharmacokinetic properties. The cost of profiling these assays during early drug development with large number of compounds, however, can dramatically increase the expense of drug discovery. One objective for pharmaceutical companies is to find less expensive, more reliable and high throughput ADMET methods that can be moved upstream. Here, we present a miniaturization method using the EchoÕ liquid handler to evaluate ADMET characteristics. The Echo liquid handler uses non-contact acoustic energy to transfer low volume compounds and reagents. This new method not only generates high quality and reliable IC50 and intrinsic clearance values, but also significantly reduces reagent costs.

P89. DEVELOPMENT OF A SELECTIVE CYP2B6 AND CELL VIABILITY DUPLEX ASSAY James J. Cali1, Dongping Ma1, Hui Wang2 and Poncho Meisenheimer2 1 R&D, Promega Corporation, Madison, WI, 2R&D, Promega Biosciences, San Luis Obispo, CA An important mechanism of adverse drug interactions involves induction of cytochrome P450 (CYP) activity: one drug induces a CYP that accelerates clearance and reduces efficacy of another co-administered drug. Within the CYP family CYP1A2, -2B6 and -3A4 contribute prominently to induction-based drug interactions in humans. In vitro assays that predict these interactions use cultured hepatocytes exposed to a test compound and a probe CYP substrate. Compounds that accelerate probe conversion are scored as CYP inducers. However, in this single parameter approach, induction may be underestimated or overlooked due to cytotoxic concentrations of test compounds because CYP activity declines as cells die, and comparisons to an untreated control are skewed by reduced cell viability in treated samples. Here we describe the development of a novel selective CYP2B6 assay and its combination with a cell viability measurement to control for cytotoxicity by normalization to cell number. Both assay parameters provide bioluminescent signals in a rapid add-then-read approach. For CYP2B6 we synthesized 6-((4,4-dimethoxybutan-2-yl)oxy)benzo[d]thiazole-2-carbonitrile as a selective probe substrate. This compound is a luciferase pro-substrate that only makes light in a luciferase formulation after oxidation by a CYP. The probe is exposed to a CYP enzyme(s) and then the sample is combined with a luciferase formulation that produces light in direct proportion to CYP activity. We expected this probe and its reaction product to move freely into and out of cells for an effective cell based assay because this was observed with similar probes described previously for CYP1A2 or -3A4. Since hepatocytes express multiple potentially cross reacting CYPs it was important to demonstrate the probe is selective for CYP2B6. An initial screen of 21 recombinant human CYPs showed a strong CYP2B6 preference. CYP2B6 inhibitors against recombinant CYP2B6 or human liver microsome reactions with the probe provided further evidence for selectivity by producing IC50s that correlate well with published values from conventional CYP2B6 assays. With adherent hepatocytes in 96 well plates initial test compound or vehicle treatments were followed by incubation with the probe in balanced salt solution. Then half of the solution was removed and mixed with a luciferase formulation in a separate plate that was read on a luminometer to give CYP values. The hepatocytes remained in the original plate, undisturbed by the CYP measurement and available for a lytic ATP-based cell viability assay performed by adding a luciferase reagent to cells and then reading luminescence. This viability assay couples the [ATP] of the cell lysate to luciferase activity, which in turn reflects viable cell number. Values from CYP measurements were normalized to the respective viability values. Like the biochemical data the cell based data was consistent with CYP2B6 selectivity. Basal CYP activities were enhanced by known CYP2B6 inducers (e.g. phenobarbital, CITGO) and both basal and induced activities were sensitive to a selective CYP2B6 inhibitor (e.g. Clopidogrel). Fold inductions correlated well with the values from a mass spectrometry based CYP2B6 assay reported for specific hepatocyte donors. This approach adds value and biological relevance by deriving double measurements from each sample.

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P90. HPLC-MS DETERMINATION OF FIVE PROBE DRUGS AND EVALUATION OF THP ENANTIOMERS ON THE ACTIVITY OF CYTOCHROME P450 ISOZYMES IN RATS


Zhanying Hong1, Jian Le2, Yinying Zhang3, Jun Wen3 and Yifeng Chai3 1 Department of Pharmaceutical Analysis, Second Military Medical University, Shanghai, China, 2Shanghai Institute for Drug and Food Control, Shanghai, China, 3Second Military Medical University, Shanghai, China Backgrouds: Tetrahydropalmatine (THP), a racemic mixture of d- and l-enantiomer, is a biologically active ingredient isolated from a traditional Chinese medicine Rhizoma Corydalis (yanhusuo) 1,2,3. A number of drug-drug interactions resulting from the induction and inhibition of CYP450 isozymes have highlighted the importance of understanding drug-metabolizing enzymes4,5. The aim of this study was to investigate the effects of THPenantiomers on the activity of cytochrome P450 isozymes in rats. Methods: A liquid chromatography- mass spectrometry method(HPLC-MS) has been developed for simultaneous determination of five major CYP450 probe drugs, metoprolol (CYP2D6), caffeine(CYP1A2), dapsone(CYP3A4), chlorzoxazone(CYP2E1) and tolbutamide(CYP2C9) in rat plasma samples. The method validation was performed and the linearities of five probe substrates were good;the lower limits of quantification of metoprolol,caffeine, dapsone, chlorzoxazone, tolbutamide were 0.025,0.05,0.05,0.05,0.25 mg/mL ,respectively; relative recoveries were between 85.0% and 115%;intra- and inter day precisions were 15% lower (RSD);the matrix effect ranged from 87.5% to 109.2%.Effects of THP enantiomers on the activity of CYP450 isozymes in rats were evaluated using a cocktail probe substrates method. Three groups of the rats(each n ¼ 6) was separately given d-THP, l-THP and rac-THP once daily for 7 days while the control group received orally saline once daily. Seven days later, a mixture of the above five cocktail probe drugs was given to rats by intravenous injection. The concentrations of the five probe drugs were determined by HPLC-MS and pharmacokinetic parameters were calculated. Results: The metabolism of caffeine and metoprolol slowed down after receiving d-THP, with longer t1/2. After receiving l-THP, the metabolism of caffeine slowed down with longer t1/2, but the metabolism of tolbutamide and dapsone speeded up, with shorter t1/2. The metabolism of caffeine slowed down after receiving dl-THP, with longer t1/2, while that of dapsone, chlorzoxazone and tolbutamide speeded up, with shorter t1/ 2. There were significantly different of these t1/2values from that in blank control group (p50.05). Conclusion: We can confer that there were stereoselective differences between the two enantiomers; d-THP had the potential to inhibit the activities of CYP2D6 and CYP1A2 isozymes,however, l-THP inhibited CYP1A2 isozyme and induced CYP3A4 and CYP2C9 isozymes.

References 1. Pharmacopoeia commission of RPC. Chinese Pharmacopoeia (Part I, 2005 edition). Chemical Industry Press, Beijing, 2005,pp.94. 2. Xiang H.,Wang Z.T.,Yu G.D.,Ruan B.F.,Li J. Alkaloids from Rhizoma corydalis. Journal of China Pharmaceutical University 2002;33(6):483–486. 3. Jin G.Z. Advances in pharmacological study of ()-tetrahydropalmatine and its analogs. Acta Pharmaceutica Sinica 1987; 22:472–474. 4. Zhang QH, Hu JP, Wang BL, Li Y. Effects of capsaicin and dihydrocapsaicin on human and rat liver microsomal CYP450 enzyme activities in vitro and in vivo. J Asian Nat Prod Res. 2012;14(4):382–95. 5. Han YL, Yu HL, Li D, Meng XL, Zhou ZY, Yu Q, Zhang XY, Wang FJ, Guo C. In vitro inhibition of Huanglian [Rhizoma coptidis (L.)] and its six active alkaloids on six cytochrome P450 isoforms in human livermicrosomes.Phytother Res. 2011;25(11):1660–5.

P91. PREDICTION OF CYP3A4 INDUCTION POTENTIAL WITH RIS IN HEPARGä CELLS AT MRNA AND ACTIVITY LEVELS Jun Sun, Mohamad Shebley, Yau Yi Lau, Sonia de Morais and Ganesh Rajaraman Department of Drug Metabolism, Abbvie Inc, North Chicago, IL CYP3A4 is a major drug metabolizing enzyme in humans and the induction of this enzyme can cause drug-drug interactions (DDIs). Primary human hepatocytes are the gold standard to study CYP induction but suffer from limitations such as higher cost, donor variability, and availability. HepaRGÔ cells are a human hepatic progenitor cell line that retains many characteristics of primary human hepatocytes. The use of HepaRGÔ cells avoid these limitations without sacrificing critical hepatocyte traits such as drug-metabolizing enzyme expression, functional transport proteins, and expression of key nuclear receptor pathways. HepaRGÔ cells respond to prototypical P450 inducers such as omeprazole (OMP), phenobarbital (PB), and rifampicin (RIF), demonstrating the utility of this cell system in the evaluation of in vitro enzyme induction. In our studies, we determined the maximal fold induction (Emax) and the concentration causing half maximal induction (EC50) by Rifampicin, Phenobarbital, Carbamazepine, Phenytoin, Efavirenz, Pleconaril, Pioglitazone, Rosiglitazone and Troglitazone at both mRNA (determined by qtPCR) and activity levels (measured by P450-GloÔ, a luminogenic IPA probe from Promega) and calculated relative induction score (RIS) in HepaRGÔ cells and cryopreserved primary human hepatocytes. The in vitro-in vivo extrapolation (IVIVE)

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showed good correlation between in vitro RIS values (from both enzyme activity and mRNA levels) and in vivo observed percentage AUC decreases of Midazolam and Ethinyl Estradiol. The results indicate that CYP3A4 induction potential can be assessed with RIS in the both cell types. CYP induction screening using enzyme activity with IPA is an efficient method that increases throughput without sacrificing data quality. HepaRGÔ cells combined luciferin IPA is a novel approach to screen compounds in early discovery to assess potential CYP3A4 induction liability.

P92. METABOLIC BIOACTIVATION OF PIPERAZINE IN BNAPHTHOFLAVONE AND PHENOBARBITAL OR AROCROL 1254 INDUCED RAT LIVER S9 FRACTIONS


Mithat Gunduz, Amin Kamel, Peter Klimko, Kevin Colizza and Shawn Harriman Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research Inc., Cambridge, MA Compound 1 exhibited genotoxicity in mini AMES assay. This work aimed to understand the mechanism leading to genotoxicity in order to alleviate this concern for other compounds in the series. The SD rats induced liver S9 fractions were purchased from Xeno Tech (Lenexa, Kansas), or Molecular Toxicology Inc. (Boone, NC). The SD rats treated with b-Naphthoflavone and Phenobarbital or Aroclor 1254 to induce the CYP1A1, CYP1A2 and CYP2B1 enzymes and the rat liver than processed to obtain the S9 fractions. CYP1A1 and CYP1A2 activate polycyclic aromatic hydrocarbons (PAHs) to carcinogenic species and heterocyclic aromatic amines/amides (HAAs) to procarcinogenic species Salmonella bacteria strain used in the mini AMES assay require exogenous histidine for survival (E-Coli strains require exogenous tryptophan). After 48 h, only mutated bacteria will have formed colonies and the genotoxicity is results of either a base-pair substitution or frame-shift mutation. Most commonly used tester strains:TA97a, TA98, TA100, TA1535 and TA1537. Our data highly suggested that the genotoxicity of Compound 1, was mainly arising from the bioactivation of the piperazine ring. The bioactivation pathway of the piperazine most likely occurred by a ring opening to form imine/aldhyde and/or potential release of a glyoxal. The biotransformation studies on Compound 1 were carried out using b-Naphthoflavone and Phenobarbital or Aroclor 1254 induced rat liver S9 fractions fortified with NADPH in presence of trapping agents such as glutathione, methoxylamine or phenylenediamine. The negative controls lacked either glutathione, methoxylamine, S9 fractions, phenylenediamine or NADPH. Structural elucidation was carried out on a Thermo-Finnigan LTQ-Orbitrap Õ with accurate mass measurement and MSn capabilities. A total of two metabolites have been observed and identified in induced rat liver S9 fractions. Metabolism of Compound 1 involved ring cleavage of piperazine to form M1 and hydroxylation on piperazine moiety to form M2 and In the incubation containing the methoxyl amine, two additional metabolites (M3 and M4) were observed and both of them from the trapped imine/aldehyde intermediate indicating a ring opening. Treating the post incubate of Compound 1 in induced rat S9 fractions with phenylenediamine at 80 C for 30 minutes, yielded the quinoxaline, which highly suggest the presence of glyoxal in incubation. The glutathione conjugate of Compound 1 was not observed. Modification of the piperazine ring in this chemical series lead to synthesis of AMES negative compounds.

P93. POTENTIAL OF THALIDOMIDE FOR INDUCTION OF DRUG METABOLIZING ENZYMES IN HUMAN LIVERS Norie Murayama1, Hiroshi Suemizu2, Kanako Yajima1, Masahiro Utoh1, Masato Nakamura2 and Hiroshi Yamazaki1 1 Showa Pharmaceutical University, Machida, Japan, 2Biomedical Research, Central Institute for Experimental Animals, Kawasaki, Japan Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/5 by the teratogen thalidomide was confirmed in our previous in vitro studies using recombinantly expressed human P450 3A enzymes, human liver microsomes, primary human hepatocytes, and differentiated HepaRG cryopreserved (Biopredic International, Rennes, France) with the model P450 3A substrate midazolam. Recently we demonstrated that transplanted livers of humanized mice mediated an enhancement of midazolam clearance by thalidomide via heterotropic cooperativity in vivo. However, it is not fully elucidated the effects of thalidomide on drug metabolizing enzymes in human livers. The purpose of the present study is to assess the potential of thalidomide for enzymes induction in human livers to understand the thalidomide-mediated drug interactions. In vivo drug interactions of thalidomide were investigated in TK-NOG mice with humanized livers. The clearance of midazolam (administered intravenously, 10 mg kg1) in chimeric mice was enhanced by pre-treatment with orally administered thalidomide (100 mg kg1) daily for 3 days. A higher area-under-the-curve of the minor metabolite 4-hydroxymidazolam (3.5-fold) was seen, presumably because of human P450 3A induction. In the medium of HepaRG cells after 48–72 incubation, thalidomide (500– 1000 mM) enhanced the formation of hydroxymidazolam, hydroxybupropion, and glucuronides of 10 -hydroxymidazolam and hyodeoxycholic acid (2-fold). Immunoblotting analysis for the cell lysates indicated increases of P450 3A protein levels. Peptide microarrays (PamChip arrays, PamGene International, Netherlands) revealed that a high concentration of thalidomide was the potential modulator for PXR and CAR. These results suggest that thalidomide would be potential ligands for PXR and

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CAR and induce P450 2B6 and 3A4/5 and UDP-glucuronosyltransferase 2B7 in human livers associated with enhanced drug metabolizing activities The possibilities of drug interactions during thalidomide therapy in humans should be evaluated further.

P94. REDUCTION OF THYROID HORMONES TRIGGERS DOWN-REGULATION OF HEPATIC CYP2B THROUGH NUCLEAR RECEPTORS CAR AND TR IN A RAT MODEL OF ACUTE STROKE


Jiang Yue1, Guicheng Dong2, Yuntao Bing1, Siying Zhu1 and Jie Li3 1 Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan, China, 2Baotou Teachers’ college of Inner Mongolia University of Science and Technology, Baotou, China, 3Basic Medical School of Wuhan University, Wuhan, China Stroke is one of the most devastating neurological conditions known and may cause changes in hepatic drug-metabolizing enzymes. Hepatic cytochrome P450 2B is involved in the metabolism of a variety of centrally active substances, including clinical drugs (e.g., propofol), drugs of abuse (e.g., cocaine), and neurotoxins (e.g., chlorpyrifos). We investigated the influence of brain ischemia/reperfusion (I/R) injury on hepatic CYP2B1 in a rat model of acute stroke. The brain I/R injury was induced by a middle cerebral artery occlusion (MCAO) for 120 min, and the animals were sacrificed at 0, 2, 12, or 24 h after the reperfusion. After a severe MCAO (120 min), the necrotic area has been previously shown to extend over the striatum to large portions of the adjacent neocortex, amygdala, and hypothalamus. We found FT3 and FT4 were markedly decreased before the appearance of the change in hepatic CYP2B1 mRNA and protein expression in the brain I/R-operation rats, while no change in the plasma levels of adrenocorticotropic hormone (ACTH), IL-1 beta, AST and ALT was observed at that time point. Both constitutive androstane receptor (CAR) and retinoid X receptor alpha (RXR alpha) were significantly dissociated from the phenobarbital-responsive enhancer module (PBREM), the distal promoter of the CYP2B1gene, after a MCAO operation in rats not subjected to reperfusion. The data from chromatin immunoprecipitation assay revealed that thyroid hormone receptors (TRs) share the binding site with CAR in the PBREM region, and that no significant change in the binding of thyroid hormone receptors (TRs) to the PBREM region was observed after reperfusion. Nuclear TRs, CAR and RXR alpha formed the polymer determined bycoimmunoprecipitation, while the levels of the polymer were significantly decreased in rats not subjected to reperfusion. The CAR mRNA expression and nuclear protein levels and the nuclear TRs proteins were respectively decreased after reperfusion, and the nuclear TRs and CAR levels were positively associated with thyroid hormone levels. These results indicate that thyroid hormones can regulate the expression or translocation of these nuclear receptors. The reduction of thyroid hormone levels after a brain I/R injury may be the initial trigger for the down-regulation of hepatic CYP2B1 via induction of the dissociation of CAR from the TRs and from the PBREM region. Our data suggest that patients with acute ischemic stroke may have a decreased CYP2B-mediated metabolism of exogenous and endogenous compounds because of the low level of thyroid hormones. This work was supported by the Program for New Century Excellent Talents in University (NCET-11-0399) and the National Natural Science Foundation of China (No. 81173122).

P95. IDENTIFICATION OF POTENT CYP2D6 INHIBITORS IN NELUMBO NUCIFERA LEAVES Ye Lin hu, Kong Ling ti, Xiao Bing xin, Cao Fang rui and Chang Qi Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Nelumbo nucifera (lotus) is utilized not only as an ornamental plant and a dietary staple, but also as a traditional Chinese medicine in China. The leaves of N. nucifera exhibit several pharmacological effects such as hyperlipidemia reduction, antiobesity, diuretic, antidiarrheal, antipyretic, antioxidant, antimicrobial and hypoglycemic activities. Recently, the leaves are becoming popular in China as an ingredient in health functional foods for losing body weight. But the potential inhibitory effect of lotus leaves on human cytochrome P450 (CYP450), the most important Phase I drug-metabolizing enzyme system involved in metabolism of various clinical drugs has not been systemic investigated to date. The aim of this study is to assess the potential inhibitory effects of the leaf extract and their major fractions, as well as main compounds on five CYP450 isoenzymes including CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 in vitro. Five probe substrates were incubated with human microsome in the presence or absence of the leaf extract, the two major fractions from the extract (alkaloid fraction and flavonoid fraction), or three aporphine alkaloids namely nuciferine (NF), N-nornuciferine (N-NF) and 2-hydroxy-1-methoxyaporphine (HMA). The relative metabolites produced were analyzed after incubation by liquid chromatography/tandem mass spectrometry. The inhibition of each tested substance was characterized with kinetic parameters IC50 and Ki values. The results showed that leaf extract strongly inhibited CYP2D6 catalyzed dextromethorphan o-demethylation formation with IC50 value of 12.05 ƒI`g/ml and weakly inhibition of the other isoform enzymes with IC50 values higher than 60 ƒI`g/ml. The flavonoid fraction weakly inhibited CYP2D6, while, the alkaloid fraction possessed great inhibition against CYP2D6 activity with IC50 value of 0.96 ƒI`g/ml. And the three alkaloidal compounds NF, N-NF and HMA isolated from alkaloid fraction, significantly inhibited CYP2D6 activity with IC50 values of 3.78, 3.76 and 3.15 ƒI`M. The Lineweaver-Burk plots and Dixon plots showed that the NF, N-NF and HMA competitively inhibited CYP2D6 activity with Ki value of 1.88, 2.34 and 1.56 ƒI`M, respectively. The in vitro study reveals that alkaloid compounds existed in Nelumbo nucifera leaves has potent inhibitory activity on CYP2D6 isoenzyme and suggests that

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caution should be paid to the possible drug interactions of the leaves and its preparations. This study was financially supported by the funds (1108 and 2012ZX09301002-001) from Ministry of Science and Technology of China.

P96. STUDIES OF IN VITRO MODULATORY EFFECT OF LABISIA PUMILA ON CYP1A2, CYP2A6 AND CYP3A4


Chin Eng Ong1, Yan Pan2, Badrul Amini Abd-Rashid3, Zakiah Ismail3, Rusli Ismail4 and Joon Wah Mak5 1 School of Pharmacy, Monash University Sunway Campus, Selangor, Malaysia, 2School of Biomedical Sciences, University of Nottingham Malaysia Campus, Semenyih, Malaysia, 3Herbal Medicine Research Unit, Division of Biochemistry, Institute for Medical Research, Kuala Lumpur, Malaysia, 4Level 17, Wisma R&D, Centre of Excellence for Research in AIDS (CERiA), Kuala Lumpur, Malaysia, 5School of Pharmacy and Health Sciences, International Medical University, Kuala Lumpur, Malaysia Labisia pumila (local name Kacip Fatimah), has been used by many generations of Malay women to induce and facilitate childbirth and as a post-partum medicine in Southeast Asia [1]. Cytochrome P450 (CYP) is a superfamily of enzymes involved in the metabolism of a large number of currently prescribed drugs and many environmental chemicals. CYP1A2, CYP2A6 and CYP3A4 are among the most important CYP isoforms responsible for the metabolism of many drugs and environmental chemicals [2]. Concurrent administration of drug substrates of these CYPs with L. pumila may lead to drug-herb interaction with possible consequence of adverse drug reaction or altered drug response in patients. In this study, three enzyme assays (3-cyano7-ethoxycoumarin oxidase, coumarin 7-hydroxylase and testosterone 6b-hydroxylase assays for CYP1A2, CYP2A6 and CYP3A4 respectively) were established to determine inhibitory potentials of various L. pumila extracts (aqueous, dichloromethane, ethanol and hexane) on CYP activities. Results showed that all extracts exhibited negligible inhibition on the three isoforms over the concentration ranges studied (from 1 to 1000 mg/ml) with the exception of three extracts. The ethanol and hexane extracts were found to inhibit CYP3A4 with IC50 values of 252.9 mg/ml and 101.5 mg/ml respectively. Relatively stronger inhibition was observed for the dichloromethane extract which inhibited CYP1A2 with an IC50 of 35.2 mg/ml. Further kinetic study revealed that the dichloromethane extract exhibited mixed type inhibition on CYP1A2 with a Ki of 41.1 mg/ml. These inhibitory effects indicated that some lipophilic phytochemicals may be involved in the observed modulatory effect. The inhibition however was considered moderately weak due to relatively high IC50 and Ki values. As a conclusion, it is unlikely that significant drug-herb interaction may result from the concomitant ingestion of L. pumilaand substrates of CYP1A2, CYP2A6 and CYP3A4.

References [1] Shahrim (2006) The in vitro rodent micronucleus assay of ‘Kacip Fatimah’ (Labisia pumila) extract. Tropical Biomedicine 23: 214–219. [2] Emoto C, Murayama N, Rostami-Hodjegan A, Yamazaki H (2010) Methodologies for investigating drug metabolism at the early drug discovery stage: prediction of hepatic drug clearance and P450 contribution. Current Drug Metabolism 11: 678–85.

P97. DOWN-REGULATION OF TESTICULAR CYTOCHROME P450 AND RELATED ENZYMES BY ESTRADIOL IN ADULT RAT TESTIS Ravindranath Reddy Gilibili1, A. Wayne Vogl2, Thomas K. H. Chang3 and Stelvio Bandiera4 1 Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada, 2Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada, 3Pharmaceutical Sciences, Univ of British Columbia, Vancouver, BC, Canada, 4 Fac of Pharm Sciences, Univ of British Columbia, Vancouver, BC, Canada There is a growing body of evidence that exposure to endocrine disrupting chemicals and to estrogenic compounds in particular, can affect the testis and disrupt sperm production. In the present study, the constitutive expression of steroidogenic and nonsteroidogenic cytochrome P450 (CYP) and related enzymes was investigated in adult rat testis using immunoblot and immunofluorescence analyses, and their regulation by estradiol was determined. Testicular microsomes prepared from untreated adult Sprague-Dawley rats were separated by SDS-PAGE, electrophoretically transferred onto membranes, and probed with different antibodies. Immunoblot results indicated the presence of CYP1B1, CYP2A1, CYP17A1, NADPH-cytochrome P450 reductase (POR) and epoxide hydrolase (mEH) in rat testis. In contrast, CYP1A, CYP2B, CYP2E, CYP2D, CYP2C, CYP3A and CYP4A enzymes were not detected. Immunofluorescence analysis of tissue sections prepared from frozen testis indicated that CYP1B1, CYP2A1 and CYP17A1 were localized in interstitial cells, but not in seminiferous tubules, whereas mEH and POR were localized in both interstitial cells and in seminiferous tubules. Treatment of adult rats with 17beta-estradiol benzoate at dosages of 0.004, 0.04, 0.4 or 4 micromol/kg (n ¼ 4 rats/group), once daily for 14 days, was found to decrease testicular CYP1B1, CYP2A1 and CYP17A1 protein levels in a dosage-dependent manner. POR and mEH protein levels were decreased to a lesser extent and only at the three highest dosages of estradiol. In contrast, expression of 3beta-hydroxysteroid dehydrogenase,

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a key steroidogenic enzyme localized in Leydig cells, was unaffected at the lower dosages. In conclusion, expression of testicular CYP1B1, CYP2A1, CYP17A1, mEH and POR exhibited differential sensitivity to the suppressive effect of estradiol.

P98. TIME COURSE FOR THE MODULATIONS OF CYTOCHROME P450 EPOXYGENASES AFTER ISOPROTERENOL ADMINISTRATION TO INDUCE CARDIAC HYPERTROPHY


Hassan Althurwi, Osama H. Elshenawy and Ayman O.S. El-Kadi Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada We have previously shown that seven days of isoproterenol treatment causes significant changes to several cytochrome P450s (P450s) gene expression. These changes have led to lower formation of the cardioprotective, epoxyeicosatrienoic acids (EETs) metabolites in the hypertrophied hearts. However, the cause and effect relationship of P450s during the development of cardiac hypertrophy are still unknown. Therefore, we hypothesized that P450s epoxygenases play crucial role in the early phases of cardiac hypertrophy development. For this purpose, male Sprague–Dawley rats were treated with isoproterenol (intraperitoneal (IP) injection, 5 mg/kg/day) for 1, 3, 7 or 10 days. Weight-matched control group received saline (IP). Animals were euthanized 24 hours after the last injection. Thereafter, the heart was harvested, the heart weight to body weight ratio was measured, and the total tissue RNA was isolated. The expression of hypertrophic markers, fibrotic markers and different P450s genes were determined by real time-polymerase chain reaction (RT-PCR). Moreover, heart microsome were prepared and incubated with arachidonic acid (AA) to determine P450s epoxygenases activity using liquid chromatography–mass spectrometry (LCMS). Our results show that isoproterenol caused a significant induction of hypertrophic markers, atrial natriuretic peptide (ANP) and b-myosin heavy chain (b-MHC), as well as the heart to body weight ratio after 24 h of treatment and continued thereafter. In addition, isoproterenol induced the gene expression of the fibrotic markers, procollagen I and procollagen III at 3, 7, 10 days. In different manner, P450s epoxygenases enzyme; CYP2B1 and CYP2B2, were induced only after 7 days of isoproterenol treatment whereas CYP2J3 was inhibited at early time points (1 and 3 days) and induced at 10 days compared to control group. On the other hand, no significant changes were detected in CYP2C11 gene expression at all tested time points whereas CYP2C23 mRNA was inhibited at 1 day and back to normal after that. In accordance with gene expression, incubation of heart microsomes with AA reveals that the total epoxygenases activity was decreased at 1, 3, 7, and 10 days compared to control group. All in all, these results suggest that the expression of P450s epoxygenases differentially modulated during the early phases of cardiac hypertrophy development, which has led to significant decrease in epoxygenases activity as early as 1 day. This further confirms the role of P450s epoxygenases in cardiac hypertrophy which could reveal novel points of intervention to be exploited in the development of new therapies for the prevention of cardiac hypertrophy at early stages. Support: This work was supported by a grant from the CIHR to A.O.S.E. H.N.A is the recipient of Salman Bin Abdulaziz University scholarship, Saudi Arabia. O.H.E. is the recipient of University of Alberta Doctoral Recruitment Scholarship.

P99. REGULATION OF THE RETINOIC ACID-METABOLIZING CYP2C22 BY NITRIC OXIDE Edward T. Morgan1, Bang-sub Lee1, Samuel L.M. Arnold2, Leslie J. Dickmann3, Nina Isoherranen2 and Choon-myung Lee1 1 Department of Pharmacology, Emory University, Atlanta, GA, 2Department of Pharmaceutics, University of Washington, Seattle, WA, 3Pharmacokinetics and Drug Metabolism, Amgen Inc., Seattle, WA CYP2C22 is a retinoic acid-inducible cytochrome P450 (P450) enzyme in rat liver that catalyzes the oxidation of retinoic acid to more polar metabolites (Qian L, Zolfaghari R, Ross AC. Liver-specific cytochrome P450 CYP2C22 is a direct target of retinoic acid and a retinoic acid-metabolizing enzyme in rat liver. J Lipid Res 2010;51:1781–92). We used an iTRAQ-based proteomic scan to identify proteins in rat hepatocytes that are rapidly down-regulated by nitric oxide, and CYP2C22 was identified as such a protein. Therefore, we hypothesized that cellular NO generated from nitric oxide synthase-2 (NOS2) stimulates the degradation of CYP2C22 via ubiquitination and proteasomal degradation, a mechanism that contributes to the degradation of CYP2B1 and CYP3A enzymes. Antisera to CYP2C22 were generated by immunizing rabbits with the peptide corresponding to amino acids 366-390 of the protein. The antisera were specific for CYP2C22 in Western blot assays, as the signal was ablated by preincubation with the immunogen, and they failed to recognize rat 2C6, 2C11, 2C12 or any other rat or human P450s tested. Consistent with its mRNA, expression of CYP2C22 protein was limited to the liver. In rat hepatocytes treated with interleukin1b (IL-1), CYP2C22 protein was rapidly down-regulated without any effect on its mRNA expression, and its time course closely followed the induction of NOS2 protein and NO production by the cells. CYP2C22 down-regulation was significant by 8 h of IL-1 treatment, reaching a nadir of 11% of control at 12 h. This suppression was blocked by the NOS inhibitor L-NGNitroarginine methyl ester (100 mM). The long-lived NO donor 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) down-regulated CYP2C22 protein (but not its mRNA) within 6 h of incubation, with an EC50 of approximately 400 mM. The short-lived NO donor (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate (DPTA, 500 mM) downregulated CYP2C22 in the presence of the protein synthesis inhibitor cycloheximide (10 mg/ml), demonstrating that NO promotes CYP2C22 proteolysis. DPTA reduced the half-life of CYP2C22 protein from 8.7 h to 3.4 h. Treatment of hepatocytes with IL-1, NOC18 or DPTA caused a decreased conversion of retinoic acid to 4-hydroxyretinoic acid by S9 fractions from the

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cells, which was correlated with the down-regulation of CYP2C22. In contrast, oxidative stress elicited by hydrogen peroxide did not down-regulate CYP2C22 expression. Under conditions in which proteasome inhibitors blocked the NO-mediated degradation of CYP2B1, they failed to affect the down-regulation of CYP2C22 protein elicited either by IL-1 or DPTA. Inhibitors of calpain, lysosomal function or lysosomal transport also had no effect on CYP2C22 degradation. Together, our results indicate that cellular NO synthesized by NOS2, or NO released from NO donors, stimulate the proteolytic degradation of CYP2C22 via a novel mechanism that does not require proteasomal activity. Supported by grants R01 GM 069971 (ETM) and R01 GM081569 (NI) from the National Institutes of Health.

P100. CONSUMPTION OF A HIGH-FAT DIET DURING PREGNANCY CHANGES THE EXPRESSION OF CYTOCHROME P450 IN THE LIVERS OF INFANT MALE MICE


Masataka Tajima1, Nobutomo Ikarashi1, Takehiro Okaniwa1, Yukari Imahori1, Kanako Saruta1, Takahiro Toda1, Makoto Ishii1, Wataru Ochiai1, Harumi Yamada2 and Kiyoshi Sugiyama1 1 Department of Clinical Pharmacokinetics, Hoshi University, Tokyo, Japan, 2Department of Pharmaceutical Sciences, International University of Health and Welfare, Tochigi, Japan It has recently been reported that the consumption of a high-fat diet during pregnancy exerts various effects on fetuses and newborn mice. The purpose of this study was to determine the effects of a high-fat diet during pregnancy on the expression of cytochrome P450 (CYP) in the livers of offspring. The mechanism responsible for the change in the expression of CYP3A and CYP2C was also examined. Mouse dams were fed a high-fat diet during pregnancy from the time of conception. After their birth, the newborn mice were fed a normal diet until 12 weeks of age. In the livers of the infant male mice whose mothers consumed a high-fat diet, the protein expression of CYP3A and CYP2C was decreased, and the protein expression of CYP1A and CYP2E was increased at 6 and 12 weeks of age. However, almost no changes were observed in the CYP proteins at 6 and 12 weeks of age in the livers of the infant female mice whose mothers consumed a high-fat diet. The nuclear translocation of the nuclear receptors PXR and CAR was examined in the livers of infant male mice at 6 weeks of age. The nuclear translocation of PXR was significantly lower in the high-fat diet group compared to the control group. However, there was no change in nuclear translocation of CAR between the control group and the highfat diet group. We have previously reported that an increase in lithocholic acid (LCA) produced by enteric bacteria contributes to an increase in the expression of CYP3A in the liver. Therefore, we examined the levels of enteric bacteria (Bacteroides fragilis) that produce LCA in the large intestines of infant males born to mothers that consumed a high-fat diet during pregnancy. No differences were observed in the levels of enteric bacteria between the control group and the high-fat diet group. These data suggested that CYP3A and CYP2C might decrease as a result of the decrease in the amount of nuclear PXR in infant male mice whose mothers consumed a high-fat diet. The results of this study suggest that the consumption of a high-fat diet by pregnant mothers may be one explanation for individual differences in pharmacokinetics.

P101. CYP4F2 IS THE MAJOR CYTOCHROME P450 ENZYME INVOLVED IN CMX001 METABOLISM Tim Tippin1, Jenni Chladek2, Laurie Keilholz1, Etsuko Usuki2, Brian Ogilvie2, Kathy Van Sickle1, Herve Mommeja-Marin1, Irma M. Grossi1 and Lawrence Trost1 1 Chimerix, Inc., Durham, NC, 2XenoTech, LLC, Lenexa, KS CMX001 is a novel, broad spectrum, orally administered, lipid acyclic nucleotide phosphonate in development for prevention and/or treatment of diseases caused by double-stranded DNA viruses, including cytomegalovirus, BK virus and adenovirus. Previous in vitro studies identified CYP3A4, 2C8, 2C19 and 2E1 as the cytochrome P450 (CYP) enzymes responsible for CMX001 metabolism. The objective of this study was to quantify the percentage of CMX001 metabolized by each CYP, and to explore potential metabolic pathways for CMX001 via less traditional CYPs known to metabolize endogenous long chain fatty acids. Pooled human liver microsomes, recombinant human CYP enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2J2, 3A4, 4A11, 4F2, 4F3a, 4F3b and 4F12) and specific CYP inhibitors were used for this study. Incubations with recombinant human CYP enzymes suggested that only CYP4F2 metabolizes CMX001. The complete inhibition of CMX001 metabolism in human liver microsomes by N-hydroxy-N’-(4-n-butyl-2-methylphenyl)formamidine (HET0016), a specific CYP4F2/3 inhibitor, and the partial inhibition by ketoconazole, a dual CYP3A and CYP4F2 inhibitor, confirmed the recombinant CYP results. The near complete inhibition of CMX001 metabolism by quercetin (CYP2C8 inhibitor), disulfiram (CYP2E1 inhibitor) and tranylcypromine (CYP2C19 inhibitor) was also observed in human liver microsomes; however, subsequent experiments performed with recombinant CYP4F2 alone indicated that these chemicals also inhibit CYP4F2-mediated metabolism of CMX001. Thus, inhibition of microsomal metabolism of CMX001 by these inhibitors is likely due to inhibition of CYP4F2 rather than inhibition of CYP2C8, CYP2C19, CYP2E1, or CYP3A4. Posaconazole, fluconazole and voriconazole, which, like ketoconazole, are known to inhibit CYP3A, did not inhibit metabolism of CMX001, indicating their CYP inhibitory activity does not extend to CYP4F2.

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In conclusion, these data indicate that CYP-mediated metabolism of CMX001 is primarily by CYP4F2, and that other CYP enzymes previously identified as having a potential role in CMX001 metabolism, namely 3A4, 2C8, 2C19 and 2E1, likely do not contribute significantly to the metabolism of CMX001. The results from this study confirm the dual inhibition of CYP3A and CYP4F2 by ketoconazole, and extend the list of compounds known to inhibit CYP4F2 to include quercetin, tranylcypromine, and disulfiram. By contrast, voriconazole, fluconazole and posaconazole were not observed to inhibit CYP4F2 at concentrations below or up to 2x above the reported peak human plasma concentration for each drug; therefore, no clinically relevant interaction is expected between CMX001 and these azoles.

P102. CROCIN INCREASES METABOLIC ACTIVITY OF CYP2C6 Gabriela Dovrtelova1, Michaela Sabova1, Jan Jurica1, Ondrej Zendulka1 and Alexandra Sulcova2 1 Department of Pharmacology, CEITEC MU, Masaryk University, Brno, Czech Republic, 2Experimental and Applied Neuropsychopharmacology Research Group, CEITEC MU, Brno, Czech Republic Crocin is a natural carotenoid that is one of elemental substances of saffron (Crocus sativus L.) and it is also a component of the curry powder. Saffron extracts proved antidepressant effects in preclinical and pilot clinical studies. Many other effects of saffron were described in series of experiments such as antioxidative, hypolipidemic, hypnosedative, anxiolytic and antiproliferative effect. Several food supplements containing saffron extracts are already available worldwide. It was described that the metabolic activity of cytochrome P450 (CYP), one of the most important enzymatic systems for xenobiotic biotransformation, is influenced by large number of natural carotenoids. The modulation of CYP metabolic activity could lead to clinically relevant changes in plasmatic concentrations and thus effects of concurrently administered drugs. The influence of crocin on CYP metabolic activity was not evaluated yet. Therefore it is not clear whether its high intake in diet or food supplements could interfere with co-administered drugs. The aim of this study was to determine the effect of crocin on the metabolic activity of CYP2C6 in a rodent model. Forty Wistar albino male rats were randomly allocated into four groups. The control group was intraperitoneally treated with saline (1 ml/kg), the other groups were injected i. p. with crocin dissolved in saline for 9 consecutive days in the following doses: 4, 20 and 100 mg/kg/day. The CYP2C6 specific marker diclofenac was administered i. p. on the Day 10 and blood samples were drawn after 20, 60, 100 and 180 min. The samples were analysed using HPLC and levels of diclofenac and its CYP2C6 specific metabolite 4-hydroxydiclofenac were determined and molar ratios of diclofenac to 4-hydroxydiclofenac (metabolic ratios, MR) were calculated. Our results showed that systemic administration of crocin significantly increased the metabolic activity of CYP2C6 independently on the dose used. The most expressive increase was monitored in samples drawn 20 minutes after administration of the marker, when the metabolic activity was elevated in the range 184–235% of metabolic activity in the control group. Induction of CYP2C6 metabolic activity by crocin may lead to clinically significant interactions changing the effects of co-administered drugs metabolised by the same pathway.This work was supported by the project ‘‘CEITEC - Central European Institute of Technology’’ (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.

P103. CYP2E1 AND CYP2U1 PROTEIN EXPRESSION IN HUMAN AMYGDALA AND PREFRONTAL CORTEX: INFLUENCE OF ALCOHOLISM AND SMOKING Francesca Toselli, Simon Worrall, Peter A. Wilce, Peter R. Dodd and Elizabeth M.J. Gillam School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia Considerable interindividual variability is observed in the therapeutic response to, and side effects of, neuroactive drugs. Alterations in metabolism by cytochrome P450 enzymes (CYP, P450s) contribute to this variability. Alcohol and cigarette smoke are addictive substances known to modulate the levels of several human P450s in liver and extrahepatic tissues. Both substances target prefrontal cortex (PFC) and amygdala (AMG), brain areas that mediate the reinforcing effects of drugs of abuse. However, little is known about their effects on P450 protein expression in the AMG and PFC. The aim of this study was to analyze the expression of CYP2 forms in human AMG and PFC from alcoholics and smokers by immunoblotting. Antisera raised against purified recombinant P450s were affinity purified to enhance selectivity, then incubated with whole membranes prepared from PFC and AMG samples from alcoholic smokers, alcoholic non-smokers, non-alcoholic smokers and non-alcoholic non-smokers. Of the P450s studied, only CYP2E1 and CYP2U1 were detected in AMG and PFC; no conclusive evidence was obtained for CYP2A6, CYP2B6, CYP2D6, CYP2J2, CYP2S1 or CYP2W1 protein expression. CYP2E1 and CYP2U1 were expressed in each

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sample analyzed (n ¼ 26 and 22 for CYP2E1 and CYP2U1, respectively), and elevated in alcoholics. CYP2U1 expression was also increased in smokers. In AMG, CYP2E1 expression was increased 2.6 1.0 fold in alcoholics compared to non-alcoholics (p ¼ 0.026, two-tailed, unpaired Student’s t-test), whereas CYP2U1 expression was only increased in PFC (1.3 0.2 times higher in alcoholics compared to non-alcoholics, p ¼ 0.0015; 1.3 0.2 times higher in smokers compared to non-smokers, p ¼ 0.03). Expression of both P450s was increased in AMG compared to PFC of the same individuals by a factor of 2.8 1.7 (p ¼ 0.02) for CYP2E1 and 2.0 0.4 (p ¼ 0.0003) for CYP2U1. No correlation was found for either form between protein and transcript expression previously analyzed in the same samples by quantitative RT-PCR1, suggesting post-translational regulation of protein levels. These results are in general agreement with previous findings on CYP2E1, CYP2A6, CYP2S1 and CYP2W1 expression in the PFC. Although CYP2J2 and CYP2S1 transcript expression has been detected in these samples previously1, the respective proteins have only been detected in white matter in human frontal cortex (CYP2J2) or brain (CYP2S1). Cell specific CYP2B6 and CYP2D6 protein expression in human frontal cortex has been previously reported, and both forms were detected at the mRNA level in the samples used here1 suggesting these proteins may have been below the level of detection in whole membranes. In conclusion, these results show that alcohol and tobacco may affect metabolism of xenobiotics and endogenous compounds in the brain by altering CYP2E1 and CYP2U1 expression. In particular, this is the first report of potential modulation of CYP2U1 expression by alcohol and tobacco smoke.

Reference 1. Toselli F, de Waziers I, Wilce PA, Dodd PR, Loriot MA, Gillam EMJ. Quantification of cytochromes P450, ABC transporters and their principal transcription factors in the amygdala and prefrontal cortex of alcoholics, smokers and drug-free controls by qRT-PCR. In submission.

P104. 1-AMINOBENZOTRIAZOLE INEFFECTIVELY INACTIVATES CYP2C9 DUE TO PREFERENTIAL BINDING TO THE DISTAL WARFARIN BINDING SITE OF CYP2C9 Jasleen Sodhi1, Jason Halladay2, Kevin Ford3, Sophie Mukadam4, Cyrus Khojasteh5 and Susan Wong6 1 Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA, 2Drug Metabolism and Pharmacokinetics, Genentech, Inc., South San Francisco, CA, 3Safety Assessment, Genentech, South San Francisco, CA,4Dmpk, Genentech, Inc., South San Francisco, CA, 5Dmpk, Genentech Inc., South San Francisco, CA, 6Genentech Inc, South San Francisco, CA 1-Aminobenzotriazole (ABT) is considered to be a non-selective, mechanism-based, pan-inactivator of cytochrome P450 (CYP), and is used in drug discovery in in vitro and in vivo studies to discern CYP- from non CYP-mediated metabolism. In in vitro metabolism experiments, a 30 minute pre-incubation with ABT (1 mM) in human liver microsomes is often used and results in various degrees of inactivation of several major human CYP isoforms. At these conditions, ABT effectively inactivated the major CYP isoforms, except for CYP2C9, with only 9.73% and 0.417% inactivation observed when diclofenac and (S)-warfarin were used as the probe substrates, respectively. In silico docking experiments placed ABT at the warfarin binding site, which is distant from the active site (near the heme). This suggests that for CYP2C9, ABT is not bioactivated to the benzyne that is required for covalent modification of the heme and subsequent CYP inactivation. Based on this, in vitro studies were designed to ‘‘displace’’ ABT from the warfarin binding site to the active site to make ABT an efficient inactivator of CYP2C9. In vitro inactivation studies with various combinations of (S)-warfarin, diclofenac, and/or ABT were conducted to displace ABT from the warfarin binding site to the active site. Interestingly, potent inactivation (72.8%) of CYP2C9 was observed following co-pre-incubation of ABT and (S)-warfarin with diclofenac as the probe substrate. Weak inactivation (16.2%) of CYP2C9 was observed following copre-incubation of ABT and diclofenac with (S)-warfarin as the probe substrate. The difference in the effectiveness of ABT to inactivate CYP2C9 is due to the (S)-warfarin-mediated displacement of ABT from the warfarin binding site to the active site, which was not observed when diclofenac was co-pre-incubated with ABT. The in vitro and in silicowork presented here provides the first detailed molecular analysis of (S)-warfarin, diclofenac, and ABT and the differential binding that leads to CYP2C9 probe sensitivity and ABT insensitivity.

P105. COCULTURE OF ANTIGEN-PRESENTING CELLS AND KERATINOCYTES: MODULATION OF COMPOUND-RELATED INCREASE OF CYP1 MRNA IN ANTIGEN-PRESENTING CELLS Jennifer Hennen1, Christopher Cohrs1, Andrea John2, Albrecht Seidel2 and Brunhilde Blo¨meke1 1 Department of Environmental Toxicology, University Trier, Trier, Germany, 2Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer Foundation, Grosshansdorf, Germany Small chemical compounds may induce skin sensitization. A subgroup of chemicals (prohaptens) induces sensitization only following metabolic activation, e.g. by cytochrome P450 (CYP), whereas prehaptens can be activated abiotically. Using a coculture model consisting of THP-1 cells as DC-like cells and HaCaT keratinocytes we demonstrated that cross talk between these two cell types improves activation of DC as wells as CYP1-related xenobiotic metabolism in keratinocytes upon

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stimulation with several prohaptens. In this study we investigated whether coculture also impacts on metabolic potential of THP1 cells. In single cultured THP-1 cells, benzo[a]pyrene (B[a]P) neither induces CYP1 enzymes nor CD86 expression, whereas after coculture with HaCaTs CD86 as well as CYP1A1 and CYP1B1 are clearly augmented. Increase of CYP1A1 depended on the continuous presence of HaCaT cells. For the prohapten eugenol and the prehapten para-toluylenediamine (PTD) we also detected a dose-dependent increase of CYP1A1 mRNA in cocultured THP-1 cells. It is not known whether proximate or ultimate metabolites are transported between these two cell types. Using B[a]P as prohapten, we detected several B[a]P metabolites in both HaCaT and THP-1 supernatants in preliminary experiments, but the suspected ultimate metabolite was only formed in HaCaT cells and in coculture as indicated by the presence of B[a]P tetrol in the supernatants. These results suggest that certain compounds are indeed able to modulate CYP1 level in cocultured THP-1 cells, although it is not yet clear whether this CYP1 increase provides the basis for the observed CD86 upregulation or active transport of metabolites is needed.


P106. SIGNALING PATHWAYS OF IL-6-MEDIATED DOWN-REGULATION OF DRUG DETOXIFICATION CAPACITY IN HUMAN HEPATOCYTES Marcus Klein1, Benjamin A. Kandel1, Maria Thomas1, Ute Hofmann1, Ute Metzger2, Markus Templin2, Georg Damm3 and Ulrich M. Zanger1 1 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany, 2The Natural and Medical Science Institute (NMI) at the University of Tuebingen, Reutlingen, Germany, 3Department of General-, Visceral- and Transplantation surgery, Charite´ University Medicine Berlin, Berlin, Germany Circulating pro-inflammatory cytokines, such as interleukin-6 (IL-6), are responsible for the activation of the acute phase response (APR) but also for dramatic reduction of drug detoxification capacity due to impaired gene expression of numerous genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs. There is evidence that not only the JAK/ STAT- but also the MAPK- and PI3K-pathways are activated by IL-6 (1). The mechanisms of the coordinated down-regulation of ADME gene expression upon IL-6 stimulation remained, however, largely unexplained. Here we aimed to elucidate the importance and contribution of each signaling pathway to the cellular response. To stimulate the inflammatory response, primary human hepatocytes (PHHs) were treated with IL-6 (10 ng/ml). For the inhibition of key molecules in the JAK/STAT-, MAPKand PI3K-pathways we used selective chemical inhibitors. Signaling protein activation was determined by reverse-phase array and Western Blot analysis using phospho-specific antibodies. Gene expression was measured using high-throughput TaqmanÕ qPCR (Fluidigm), including more than 90 gene expression assays to quantify effects on a broad range of ADME and acute phase response genes at different time points. Protein expression of major cytochrome P450 (CYP) enzymes was analyzed by Western Blot. A mass-spectrometric cocktail activity assay comprising highly specific probe substrates was carried out to simultaneously determine the in vitro activities of eight CYPs. IL-6 treatment of PHHs led to substantial transcriptional down-regulation of many CYPs including CYP3A4 as well as other ADME genes such as phase II enzymes, transporters, transcription factors and nuclear receptors, whereas CYP2E1 and major acute phase response genes were strongly up-regulated. For some major CYPs like CYP1A2 and CYP3A4 gene expression changes also translated into altered protein-levels and activities. Phospho-protein analyses by reverse-phase protein-array confirmed activation of the JAK/STAT-, PI3K- and MAPK-pathways upon IL-6 treatment. Inhibition of the JAK/STAT- pathway appeared to have no significant effects on IL-6-mediated ADME gene expression changes, in agreement with an earlier report (2). In contrast, co-inhibition of the MAPK- and PI3K-pathways almost completely abolished the down-regulation of most ADME genes by IL-6. Individual inhibition of these pathways also suggested ‘‘gene-group’’ specific patterns of regulation. Our data indicate a pronounced IL-6-mediated hepatocellular response in a quite strongly coordinated fashion with many important ADME genes significantly down-regulated. PI3K- and MAPK-signaling appear to play major roles in this regulation. With siRNA-mediated knock-down of key signaling molecules we are currently validating our findings. This experimental approach and data should be useful for systems biology modeling in order to better predict the effect of IL-6 and other cytokines on drug metabolism capacity. Supported by the German BMBF (Virtual Liver grants 0315755 and 0315742) and the Robert Bosch Foundation, Stuttgart, Germany.

References (1) Eulenfeld, R. et al. Interleukin-6 signalling: more than Jaks and STATs. Eur. J. Cell Biol. 91, 486–495 (2012). (2) Jover, R., Bort, R., Go´mez-Lechoń, M. J. & Castell, J. V. Down-regulation of human CYP3A4 by the inflammatory signal interleukin-6: molecular mechanism and transcription factors involved. Faseb J. Off. Publ. Fed. Am. Soc. Exp. Biol. 16, 1799–1801 (2002).

P107. CYP2E1 HYDROXYLATION OF ANILINE INVOLVES COOPERATIVITY Jessica H. Hartman1, Katie Knott2, Gunnar Boysen3 and Grover P. Miller1 1 Dept of Biochem & Molec Biol, Univ of Arkansas for Med Sci, Little Rock, AR, 2Dept of Chemistry, University of Central Arkansas, Conway, AR, 3Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR


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CYP2E1 hydroxylates aniline into 4-aminophenol, a toxic metabolite, and thus understanding the reaction mechanism is critical for assessing risks associated with aniline exposure. Some previous human liver microsomal studies reported that CYP2E1 metabolism of aniline displayed traditional Michaelis-Menten kinetics, while others observed substrate inhibition. These conflicting reports result in distinctly different predictions of in vivo metabolic clearance of aniline by CYP2E1. Consequently, we analyzed steady-state aniline metabolism by recombinant CYP2E1 for the first time and then investigated its overall contribution to aniline metabolism relative to other cytochrome P450s present in human liver mcirosomes. The recombinant enzyme exhibited a non-hyperbolic kinetic profile for aniline metabolism, which statistically fit the Hill Equation better than that for the Michaelis-Menten mechnanism. The Hill coefficient was 0.56, suggesting negative cooperativity. We identified a twobinding site cooperative mechanism in which two active forms of the enzyme exist as the most probable one from a set of 6 possibilities. At low concentrations, aniline binds CYP2E1 with high affinity (Ks ¼ 30 mM) and oxidation proceeds at 2.7 nmol/ min/nmol. At higher concentrations, a second aniline molecule binds weakly to CYP2E1 (Kss ¼ 1100 mM) and the oxidation rate increases nearly three-fold (8.8 nmol/min/nmol). Scaled modeling of the in vitro cooperative mechanism for CYP2E1 metabolism of aniline to its in vivo clearance led to significant deviations from the traditional model based on Michaelis-Menten kinetics. Low aniline levels were notably much more toxic than generally assumed. We are currently validating the cooperative mechanism using the CYP2E1-specific inhibitor and drug 4-methylpyrazole, which we have shown binds two CYP2E1 sites. Preliminary experiments performed with human liver microsomes revealed multi-phasic kinetics and were suggestive of the contribution of multiple cytochrome P450 isoforms. Through inhibitor phenotyping experiments, we confirmed the major role of CYP2E1 in aniline metabolism as well as identified contributions from CYP2A6 and CYP2C9 for the first time. Ongoing experiments will inhibit their contributions to confirm the cooperative mechanism for CYP2E1 metabolism of aniline also occurs in microsomal fractions. Taken together, these results expand the array of CYP2E1 substrates reported by our group and others that involve cooperative mechanisms during metabolism and force a re-evaluation of efforts to model and predict the role of CYP2E1 in the metabolic activation or detoxication of drugs and pollutants.

P108. CHANGE IN EXPRESSION OF CYTOCHROME P450 IN ULCERATIVE COLITIS AND ANALYSIS OF THE MECHANISM OF CHANGE Yoshiki Kusunoki, Nobutomo Ikarashi, Yoshitaka Hayakawa, Risako Kon, Makoto Ishii, Wataru Ochiai and Kiyoshi Sugiyama Dept of Clin Pharmacokinet, Hoshi Univ, Shinagawa-ku, Japan It is known that the quality and quantity of the drug-metabolizing enzyme cytochrome P450 (CYP) changes in different pathologies such as diabetes and cirrhosis. In this study, we examined the expression and activity of hepatic CYPs using ulcerative colitis model mice. Moreover, the mechanism of this expression change was analyzed for CYP3A, the most prevalent CYP. Ulcerative colitis model mice were prepared by giving 3.5% dextran sulfate sodium (DSS) solution in water to male ICR mice ad libitum for 9 days. After confirming the ulcerative colitis pathology, the expression of CYP in the liver was analyzed. For CYP3A, the metabolic activity was evaluated using the liver microsome fraction. Moreover, inflammatory cytokines, nuclear factor-kB (NF-kB), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) were analyzed to clarify the mechanism of the change in CYP3A. The mRNA and protein expression levels of CYP3A, CYP1A, CYP2C, CYP2D, and CYP2E were significantly reduced in the liver of ulcerative colitis model mice. In addition, the metabolic activity of CYP3A decreased compared to the control group. Moreover, the expression levels of PXR and CAR in the nucleus decreased significantly. There was also an increase in the expression levels of inflammatory cytokines (IL-1b, IL-6, and TNF-a) and NFkB in the liver. These results suggested that at the onset of ulcerative colitis, LPS that penetrated the inflammatory site in the large intestine triggered the induction of inflammatory cytokines in the liver. This led to the increase in the nuclear translocation of NF-kB and the decrease in the nuclear translocation of PXR and CAR, resulting in the reduction in the CYP3A expression level. The present results show that care should be taken when using substrate drugs of CYPs because the CYPs decrease at the onset of ulcerative colitis.

P109. EFFECT OF GENETIC POLYMORPHISM IN CYTOCHROME P450 2A6 ON THE ENANTIOSELECTIVE METABOLISM OF TEGAFUR TO 5-FLUOROURACIL Ikuo Yamamiya1, Kunihiro Yoshisue1, Yuji Ishii2, Hideyuki Yamada2 and Masato Chiba1 1 Pharmacokinetics Research Laboratory, Tsukuba Research Center, Taiho Pharmaceutical Co. Ltd., Tsukuba, Japan, 2Labratory of Molecular Life Sciences Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan Tegafur [5-fluoro-1-(2-tetrahydrofuryl)-2, 4 (1H, 3H)-pyrimidinedione] (FT), a prodrug of 5-fluorouracil (5-FU), is used in cancer chemotherapy at a wide range of doses as a combination drug, for example, as S-1 and tegafur-uracil. FT is a chiral molecule: a racemate of R- and S-FT. In humans, cytochrome P450 (CYP) 2A6 is mainly responsible for the conversion of both FT enantiomers to 5-FU in hepatic microsomes. Many forms of genetic polymorphisms in theCYP2A6 gene have been reported, which may be responsible for the inter-individual differences in CYP2A6 activity. In the present study, variant CYP2A6s (CYP2A6 *7, *8, *10 and *11) as well as the wild type together with NADPH-CYP reductase were expressed in

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baculovirus-infected Sf9 cells. Furthermore, we performed a kinetic analysis of 5-FU formation from R- and S-FT, mediated by CYP2A6 and its variants. Intrinsic clearance was higher for R-FT than forS-FT, owing to the considerably lower Km. In reference to the effect of the genetic polymorphisms of CYP2A6 on the metabolism of FT, all allelic variants caused a reduction in the activity for 5-FU formation from both R- and S-FT. Enzyme kinetic analysis showed that the Vmax values of CYP2A6*7 (I471T) and CYP2A6*10 (I471T and R485L) for both FT enantiomers were markedly decreased to approximately one-tenth of those of the wild type. Furthermore, CYP2A6*10 yielded higher Km values for the conversion of FT to 5-FU than the wild type gene. On the other hand, Km values for both FT enantiomers were increased for the CYP2A6*8 (R485L) and CYP2A6*11 (T670C) variants, without significant change in Vmax values. Thus, a moderate decrease in catalytic activity was observed for these CYP2A6 variants. These results suggest that the Vmax for CYP2A6 activity was drastically decreased in the I471T variant, converting FT enantiomers to 5-FU, and the affinity for FT was decreased in the T670C and R485L variants. Indeed, the CYP2A6*10 containing both I471T and R485L substitution showed the lowest activity for FT metabolism among all the alleles tested. In conclusion, CYP2A6 variants (*7, *8, *10, and *11) resulting in amino acid changes markedly altered Km or Vmax for the conversion of both FT enantiomers to 5-FU, leading to a reduction in the catalytic activity, whereas the effects of these CYP2A6 variants on FT metabolism did not depend on the enantiomer.

P110. EXPRESSION AND CHARACTERIZATION OF CYNOMOLGUS CYTOCHROME P450 3A4 Murali Subramanian1, Priyadeep Bhutani1, Sindhuja Selvakumar2, Kaushik Ghosh2, Prasad Krishnamurthy2, Bhadresh Rami2, Sanjith Kallipatti2, Sunil Sukrutharaj2, Sabariya Selvam2, Vivek Halan2, Manjunath Ramarao2 and Sandhya Mandlekar1 1 Optimization, BBRC, Banalore, India, 2Applied Biotechnology, BBRC, Banalore, India Cynomolgus monkeys, given their high genetic similarity with humans, are commonly used in preclinical drug discovery to predict PK, efficacy and toxicity. Specifically, the homology between human and cynomolgus cytochrome P450s (CYPs) are, in general, greater than 90%. Despite such high homology, however, monkeys oftentimes show much lower intestinal bioavailability than humans, frequently attributed to the CYP3A family of enzymes. To determine if species differences in the human and cynomolgus 3A4 enzymes were responsible for some of these species differences, we have expressed and characterized cynomolgus 3A4 (c3A4) and compared its kinetics with human 3A4 (h3A4). A novel HEK293-6E suspension system was utilized for the expression of c3A4, and the kinetics of five substrates, midazolam, triazolam, testosterone, nifedipine and terfenadine, were determined with c3A4 and monkey liver microsomes (MkLM). All five compounds were determined to be good substrates of c3A4 with similar kinetic parameters to h3A4, although some differences were observed in the Km values. Additionally, c3A4 showed no activity against non-h3A4 probe substrates except for human CYP2D6 substrate –bufuralol which suggested potential metabolism of human CYP2D6-substrates by c3A4. Inhibitory studies were also performed and it was found that both ketoconazole and troleandomycin showed similar inhibitory potency towards c3A4 and h3A4, while non-h3A4 inhibitors did not inhibit c3A4 activity suggesting similar inhibitory profiles of h3A4 and c3A4. In summary, c3A4 shows similar kinetic and inhibitory properties as h3A4 but is also capable of metabolizing human CYP2D6 substrates such as bufuralol.

P111. COMPARATIVE STUDY OF THE METABOLIC PATHWAY OF NEW ANTHELMINTIC DRUG MONEPANTEL IN HOST AND PARAZITE USING UHPLC/MS TECHNIQUE Lucie Stuchlikova1, Hana Ba´rtı´kova´1, Robert Jirasko2, Martin Vala´t1, Ivan Vokra´l3, Jirı´ Lamka3, Barbora Szota´kova´1 and Lenka Ska´lova´1 1 Biochemical Sciences, Faculty of Pharmacy, Charles University in Prague, Hradec Kralove, Czech Republic, 2Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic,3Pharmacology and Toxicology, Faculty of Pharmacy, Charles University in Prague, Hradec Kralove, Czech Republic Monepantel (MOP), belonging to a new class of anthelmintic drugs, has been approved for use against nematodes in small ruminants, since 2009 in New Zealand as ZolvixÕ . It is now available in Australia and many European and South American countries for helminthosis treatment and prophylaxis. Contrary to other anthelmintics, MOP is also effective against nematodes strains resistant to benzimidazoles, levamisole, morantel and macrocyclic lactones. Although the use of MOP in veterinary practises has increased rapidly, the information about the metabolic pathways of MOP in ruminants and helminths is insufficient. The study of metabolic pathways of anthelmintics is very important for efficacy of therapy and evaluation of risk of drugresistance developments in helminths. Moreover, the ability of parasites to deactivate administered anthelmintics can represent their defence mechanism. The aim of our work was to identify and to compare the metabolic pathways of MOP in Haemonchus contortus, common helminths of ruminants, and in hosts (sheep). For the in vivo study of MOP metabolism in sheep, male castrated lambs (Ovis sp.) were used. MOP was administered orally to lambs. Urine samples were collected 8 and 48 hours after administration and faeces were collected 48, 48–60, 60–72 hours after administration into plastic tubes. Urine was filtrated and evaporated. Faeces were homogenized and homogenates were extracted using liquid-liquid extraction by acetonitrile. For ex vivo study of MOP metabolism in Haemonchus contortus, one susceptible strain ISE (Inbreed susceptible Edinburgh) and one

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resistant strain WR (White river) of H. contorus were used. Freshly isolated living nematodes were cultivated in RPMI medium containing MOP at 38 C under humid atmosphere with 5% CO2 for 24 hours. Nematodes were homogenized and homogenates were extracted using the solid-phase extraction (SPE). The samples were analysed by UHPLC/MS (QqTOF) in negative-ion mode. The results showed that enzymatic systems in hosts are able to biotransform MOP via oxidation, hydroxylation, hydrolysis, glucuronidation and conjugation with acetylcysteine. Eight metabolites of MOP were detected in urine. In faeces, four different and four the same metabolites as in urine were identified. Only first phase biotransformations via oxidation, hydroxylation and hydrolysis were identified in helminths. Based on the obtained results, the schemes of metabolic pathways of MOP in sheep and in helminths were proposed. This project was supported by Czech Science Foundation, grant No P502/10/ 0217 and by Grant Agency of Charles University, grant No 673612/B-CH/2012.

P112. SPECIES SELECTIVITY IN GLUCURONIDATION KINETICS OF AN MTOR INHIBITOR


Loren Berry1, Liu Jingzhou1, Adria Colletti2, Lin Jasmine1, Paul Krolikowski3, Zhao Zhiyang1 and Yohannes Teffera4 1 Pkdm, Amgen, Cambridge, MA, 2Biogen Idec., Cambridge, MA, 3TD, Amgen, Cambridge, MA, 4Pharmacokinetics and Drug Metabolism, Amgen, Cambridge, MA Species selectivity in glucuronidation kinetics of an mTOR inhibitor Loren M. Berry, Jingzhou Liu, Adria Colletti, Jasmine Lin, Paul Krolikowski, Zhiyang Zhao, Yohannes Teffera The mammalian target of rapamycin (mTOR) is a protein kinase that shows key involvement in aging and age-related disease, and promises to be target for treatment of cancer. In the present study the elimination of potent ATP-competitive mTOR inhibitor 3-(6-amino-2-methylpyrimidin-4-yl)-N-(1H-pyrazol-3-yl)imidazo[1,2b]pyridazin-2-amine (compound 1) is studied in bile duct cannulated rats, and the metabolism of compound 1 in liver microsomes is compared across species. Compound 1 was shown to undergo extensive N-glucuronidation in BDC rats. N-glucuronides were detected on positions N1 (M2) and N2 (M1) of the pyrazole moiety as well as on the primary amine (M3). All three N-glucuronide metabolites were detected in liver microsomes of the rat, dog and human, while primary amine glucuronidation was not detected in monkeys. In addition N1 and N2-glucuronidation showed strong species selectivity in vitro, with rats, dogs and humans favoring N2-glucuronidation and monkeys favoring N1-glucuronide formation. Formation of M1 in monkey liver microsomes also followed sigmoidal kinetics, singling out monkey as unique among the species as far as compound 1 N-glucuronidation. In this respect, monkey might not always be the best animal model for N-glucuronidation of UGT1A9 or UGT1A1 substrates in humans. Impact, of N-glucuronidation of compound 1 could be more pronounced in higher species such as monkey and human, leading to high clearance in these species. While, compound 1 shows promise as a candidate for investigating the impact of pan-mTOR inhibition in vivo, opportunities may exists through medicinal chemistry effort to reduce metabolic liability with the goal of improving systemic exposure.

P113. CLOZAPINE BIOACTIVATION AND BIOINACTIVATION IN HUMANS: ROLE OF GST NULL GENOTYPES AS RISK FACTOR FOR IDIOSYNCRATIC DRUG REACTIONS? Sanja Dragovic1, Tahar van der Straaten2, Jan P.A.M Bogers3, Mackenzie Hadi4, Geny Groothuis5, Nico Vermeulen1 and Jan Commandeur1 1 Division of Molecular Toxicology, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 2Department of Clinical Pharmacology and Toxicology, Leiden University Medical Center, Leiden, Netherlands, 3Onderdeel van Rivierduinen, High Care van GGZ Haagstreek, Leidschendam, Netherlands, 4Pharmacokinetics, Toxicology and Targeting, University of Groningen, Groningen, Netherlands, 5Pharmacy, Pharmacokinetics Toxicology and Targeting group, Groningen, Netherlands Clozapine is an atypical antipsychotic drug associated with idiosyncratic agranulocytosis and hepatotoxicity. Although the exact mechanism is not known yet, formation of a reactive nitrenium ion by myeloperoxidase and/or P450s has been proposed as a possible explanation for these adverse drug reactions. Recently, we have shown that the wide variability in bioactivation of clozapine by human liver microsomes from 100 different individuals is mainly determined by interindividual differences and drug-drug interactions at the level of CYP3A4.1 The susceptibility of patients to adverse drug events might also be determined by a low activity of enzymes involved in inactivation of the reactive drug metabolites. In line with this hypothesis, we showed that GSH dependent detoxification of the nitrenium ion is catalysed by human glutathione S-transferases (hGSTs). Two of the six identified GSH-conjugates of clozapine were only formed in the presence of hGSTs.2 So far, the excretion of GSH-conjugate related metabolites of clozapine in human urine has been poorly characterized. In the present study, we describe the identification of GSH-related metabolites of clozapine in urine patient samples as well as in human liver slice incubations. Both methylthio- and cysteine-conjugates were detected by LC/MS/MS analysis. The anticipated N-acetylcysteine conjugates, however, could not be found. Based on collision-induced fragmentation patterns of the MHþ ions, and by comparison with those of reference compounds obtained by biosynthesis, the presence of GST-dependent conjugates could be demonstrated. Therefore, hGSTs appear to play a role in the protection against reactive clozapine metabolites in humans. A preliminary genotyping study of the patients suggests that the GSTT1/GSTM1 double-null genotype might be a risk factor for clozapine-induced agranulocytosis.

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References 1. Dragovic S, Gunness P, Ingelman-Sundberg M, Vermeulen NP, Commandeur JN (2013) Characterization of human cytochrome P450s involved in the bioactivation of clozapine. Drug Metab Dispos 41(3): 651–8. 2. Dragovic S, Boerma JS, van Bergen L, Vermeulen NP, Commandeur JN (2010) Role of human glutathione S-transferases in the inactivation of reactive metabolites of clozapine. Chem Res Toxicol 23(9): 1467–76.

P114. COMPARISON OF ENZYMES AND TRANSPORTERS OF THE TRIPLE KO FRG (FRGN) MOUSE AND THE FRGN MOUSE (MFRGN) REPOPULATED WITH MOUSE HEPATOCYTES: GENE PROFILING AND LIVER PERFUSION STUDIES


Edwin C.Y. Chow1, Hui Tang1, Ricky Ng1, Susana Hon1, David C. Evans2, Jose Silva2 and K. Sandy Pang1 1 Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada, 2Drug Metabolism & Pharmacokinetics, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Spring House, PA One of the biggest limitations in drug discovery is the definition of metabolism/toxicity of new drug entities in humans and means for assessing efficacy in appropriate disease models. Recently, FRGN [FRG on NOD background with genes knockout of FAH, Rag2, and IL-2rg] mice have been developed via repopulating the liver with human hepatocytes, with the expectation that these mice now contain the entire complement of human transporters and enzymes for ADME or toxicity studies. Different conditions were used for housing [duration of Nitinisone to prevent toxicity in liver and kidney and cycling of antibiotics, in drinking water] between control FRGN and hepatocyte-repopulated mice. To address issues of surgery and housing, we compared murine gene profiles of FRGN and mFRGN (mouse hepatocyte-repopulated) and harmol metabolism in liver perfusion studies. No major difference in mRNA expression was found for enzymes and SLC or ATP transporters for perfused vs. non-perfused livers. Comparison of the pooled (perfused and nonperfused) FRGN and mFRGN liver genes revealed a higher mRNA expression of Cyp3a11, Cyp7a1, Cyp2e1 and Cyp2b10, Sult1a1 (3.8-fold), Sult2a1 (48-fold), Gsta3-3, Bsep (2.8-fold), Oatp1a1, and Oatp1a4 (4.2-fold). There was no difference in hepatic nuclear receptor levels except for CAR, which was considerably higher, and FXR, which was slightly reduced. When liver perfusion studies (2.5 ml/min) were conducted with 50 mM harmol in blood perfusate (20% RBC, 1% albumin in KHB) during single-pass consecutive prograde (P) (from portal vein to hepatic vein; no hepatic arterial flow) then retrograde (R) flow (from hepatic vein to portal vein; no hepatic arterial), or in reversed order, zonation patterns became apparent. FRGN (n ¼ 6)

a

E Sulfation Rate (HS)b Glcuronidation Rate (HG)b HS/HG ratio Bile flow rate (ml/min/g)

mFRGN (n ¼ 3)

Prograde

Retrograde

Prograde

Retrograde

0.699 0.060 4.0 0.6% 68.8 7.4% 0.057 0.008 0.61 0.20

0.774 0.047* 2.6 0.6%* 74.8 6.0% 0.034 0.007* 0.51 0.10

0.693 0.027 11.3 0.1%# 57.3 1.6%# 0.205 0.012# 0.67 0.17

0.671 0.024y 9.4 1.6%y 57.3 0.3%y 0.174 0.038y 0.53 0.19

Data expresses as mean SD p50.05, *P flow vs. R flow, FRGN; # FRGN vs. mFRGN, P flow; y, mFRGN vs. FRGN, R flow a steady-state extraction ratio of harmol b rate, expressed as percent of total rate in (flow x steady-state input concentration, CIn)

The results (see table) revealed a lower steady-state ratio of output rate (bile þ perfusate rate out) of HS/HG during R vs. P), showing intact enzyme zonation, reflective of an anterior distribution of Sult vs. Ugt activities for FRGN mice. A higher %sulfation was observed in mFRGN livers, consistent with increased Sult1a1 activity, thereby reducing %glucuronidation (from 70 to 57%) and a higher HS/HG ratio over that for FRGN mice. The extraction ratio of harmol (E) for FRGN mice was lower with P flow than for R flow due to a higher degree of saturation of Sult1a1. These discrepancies in HS/HG ratio and E were not observed for mFRGN (same HS/HG or E), suggesting loss of enzyme zonation in the mouse-hepatocyte repopulated liver. This study clearly demonstrates that enzyme distribution of Sult/Ugt is lost and enzymatic/transporter activities are higher in mFRGN liver.

P115. GENERATION AND USE OF MULTIPLE CYTOCHROME P450 HUMANIZED AND KNOCKOUT MOUSE MODELS FOR DRUG-DRUG INTERACTION, PHARMACOKINETIC AND SAFETY STUDIES Nico Scheer1, Yury Kapelyukh2, Lesley A. McLaughlin2, Mike McMahon2, Anja Rode1, Colin Henderson2 and C.Roland Wolf2 1 TaconicArtemis, Cologne, Germany, 2University of Dundee, Dundee, United Kingdom Humanized and knockout mice for xenobiotic receptors, drug metabolizing enzymes and transporters are gaining increasing traction as superior models for predicting human responses and many such models have been developed and used in the recent

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past. An obstacle for their wider application in drug discovery and development is the limited number of genetic manipulations in any given model described to date. As a consequence, only selected pathways of drug metabolism and disposition could be studied separately in different mouse lines. In order to overcome this limitation, we have started to combine various single genetic alterations into complex, multiple humanized and knockout mouse models. Here we described the generation and first characterization of a mouse model with deletions of the murine Cyp3a, Cyp2d and Cyp2c gene clusters, as well as a humanized model with replacements of the mouse pregnane X receptor (Pxr), constitutive androstane receptor (Car), Cyp3a, Cyp2d and Cyp2c gene clusters with human PXR, CAR, CYP3A4, CYP2D6 and CYP2C9. The basic characterization of these models as well as their responses to different drugs will be described. We discuss our approach of combining additional modifications with these lines, aiming for the humanization of the major pathways of drug metabolism and disposition in a single mouse model. We expect that these multiple humanized and knockout mouse lines will be useful tools to better predict the PK, DDI and safety of compounds in humans.


P116. SPECIES DIFFERENCE OF WARFARIN SENSITIVITY AMONG BIRDS CAN BE EXPLAINED BY THE TWO ENZYMES, CYTOCHROME P450 AND VITAMIN K EPOXIDE REDUCTASE Kensuke P. Watanabe, Yusuke K. Kawai, Minami Kawata, Shouta M. M. Nakayama, Yoshinori Ikenaka and Mayumi Ishizuka Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan Warfarin is an anticoagulant rodenticide used all over the world, which has becoming a serious problem by causing secondary poisoning incidents in wild birds. However, bird species were believed to be relatively resistant to warfarin because the LD50 of warfarin for experimental bird species were reportedly very high (such as 942 mg/kg in chicken). Therefore, wide species difference in sensitivity to warfarin among bird species can be expected. In human, warfarin sensitivity is determined mainly by the two enzymes, i.e. cytochrome P450 (CYP, especially CYP2C9) and vitamin K epoxide reductase (VKOR, a target enzyme of warfarin). In this study, we focused on avian CYP and VKOR, to clarify the species difference of the sensitivity to warfarin. Totally 11 avian species were examined in this study, such as chicken, ostrich, mallard and owls. We measured warfarin metabolic activity with liver microsomes of them. Chicken had the highest metabolic activity of warfarin than the other species examined, and the activity was as much as about 60 fold of that of owl. Following pharmacokinetic analysis showed the half life of warfarin in chicken was about 40 hours, while that of rat was reportedly about 10 hours. The high metabolic activity of chicken in vitro suggested that the half life of warfarin in the other birds were even longer than that of chicken. Further, we focused on avian CYP2C isoform as a candidate of warfarin metabolizing enzyme, because CYP2Cs play dominant role in warfarin metabolism in human and rat, and also, we had found CYP2C genes are dominantly expressed in chicken liver compared to other isoforms. We cloned CYP2C23 genes of eight bird species and compared the protein expression levels by utilizing a synthesized antibody specific to bird CYP2C23. The protein expression of CYP2C23 showed less than 4 fold difference between chicken and owls. For VKOR inhibition by warfarin, chicken had significantly high inhibition constants, Ki, compared to rat and ostrich, while ostrich Ki was similar to that of rat. In the alignment comparison of putative warfarin binding site of VKORC1 protein, chicken had a sequence of TYV, while TYA is a commonly conserved sequence among most mammalian species. This amino acid residue was suggested to be responsible for the high Ki of chicken. In conclusion, high metabolic ability of warfarin and high Ki of VKOR of chicken were suggested to lead to shorter residue of warfarin in the body and less inhibition effect on VKOR, resulting in high LD50 in vivo. Meanwhile, other birds had less ability of warfarin metabolism compared to chicken, likely resulting in longer half life and residual term. In addition, the possibility was suggested that some bird species have VKOR with low Ki like ostrich, which leads to the high sensitivity of VKOR to warfarin.

P117. EXPOSURE AND METABOLIC DIFFERENCES AFTER LOXAPINE INHALATION ADMINISTRATION TO MALE AND FEMALE JUVENILE AND ADULT RATS Keith Huie1, Rommel Matheson2, James V. Cassella3 and Lori H. Takahashi4 1 PK/ADME, Alexza Pharmaceuticals Inc, Mountain View, CA, 2Charles River Laboratories, Preclinical Services, Montreal (PCS-MTL), Senneville, QC, Canada, 3Alexza Pharmaceuticals Inc, Mountain View, CA, 4Nonclinical Development, Alexza Pharmaceuticals Inc, Mountain View, CA Loxapine, a dibenzoxazepine compound, is a dopamine antagonist and also a serotonin 5-HT2 blocker. Male and female Sprague-Dawley rats at 28, 42 and 60 to 63 days of age were administered aerosolized loxapine at single doses ranging from 0.26 to 2.4 mg/kg. Loxapine and 7-OH-loxapine were quantified by LC/MS/MS using validated methods. Loxapine exposure was highest in 28-day old rats. Except for the low dose group of the 42-day old rats where the females had higher exposure of loxapine, no consistent sex differences in loxapine exposure for all age and dose groups were observed. In contrast, gender differences were observed for 7-OH-loxapine. Female rats had higher exposure of 7-OH-loxapine for 28-day old rats only in the low dose group, but females had increasingly higher exposure than males as the age increased from 42 to 60–63 days.

DOI: 10.3109/03602532.2013.868114

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7-OH-loxapine exposure was also highest for 28-day old rats. The ratio of metabolite-to-parent exposure was between 2 and 4fold for 28-day and 42-day old rats. For the 60 to 63-day old rats, the ratio was close to 1 for the males and approximately 3 for females. In humans, loxapine is metabolized primarily by CYPs 1A2, 3A4 and 2D6 as well as FMOs. The observed decreases in dose normalized AUC of both loxapine and 7-OH-loxapine with age are hypothesized to be due to cytochrome P450 maturation. The differences for the age-related gender differences in the metabolite ratios could be due to some of the metabolizing enzymes being under hormonal control.

P118. METABOLISM OF THE ANTI-HIV DRUG EFAVIRENZ AND PROTEOMIC ANALYSIS OF CYTOCHROME P450 EXPRESSION IN MURINE LIVER AND BRAIN


Elisabeth M. Hersman1 and Namandje´ N. Bumpus2 1 Department of Pharmacology and Molecular Biology, Johns Hopkins University, School of Medicine, Baltimore, MD, 2 Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD Efavirenz (EFV; (4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one) is one of the most widely prescribed anti-HIV drugs. In order to help establish mice as an animal model for investigating EFV pharmacology/toxicology, we characterized the metabolism of EFV in this species. Using liver microsomes isolated from C57Bl/6 and Balb/c mice, two previously unreported monohydroxylated metabolites of EFV were identified via ultra performance liquid chromatography-mass spectrometry. In humans, 7-hydroxyEFV and 8-hydroxyEFV are the only monooxygenated metabolites formed; however, in mice it was found that in addition to forming the 8-hydroxyEFV metabolite, products proposed to result from monooxygenation at the 5-position and 14-position of EFV were also detected. The formation of a 5-hyrdoxyEFV and 14-hydroxyEFV metabolite were supported by fragment ions with mass-to-charge ratios of 225 (proposed to correspond to the loss of C6H2O2) and 260 (proposed to result from the loss of C3H3O2), respectively, that were unique when compared to EFV and synthetic 7-hydroxyEFV and 8-hydroxyEFV. None of these metabolites were formed using murine brain microsomes. To profile the cytochromes P450 (P450) expressed in C57Bl/6 mouse liver and brain high resolution data-dependent mass spectrometry was used and revealed that 24 murine P450s were detectable in liver microsomes including P450s from subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 2F, 2J, 2U, 3A, 4A, 4F, and 4V. In contrast, P450 2D26 was the only P450 isozyme detectable in brain microsomes. Collectively, these data provide a foundation for the use of mice as a preclinical model for understanding EFV pharmacology/toxicology and contributes to a wider understanding of mouse xenobiotic metabolism.

P119. EXPLORING COMPARATIVE PHARMACOKINETICS FOR SCREENING-LEVEL BIOACCUMULATION ASSESSMENT: IN VITRO PHARMACEUTICAL BIOTRANSFORMATION WITH FATHEAD MINNOW (PIMEPHALES PROMELAS) AND RAINBOW TROUT (ONCORHYNCHUS MYKISS) Kristin Connors1, Bowen Du1, Patrick Fitzsimmons2, Kevin Chambliss1, John Nichols2 and Bryan Brooks1 1 Baylor University, Waco, TX, 2U.S. Environmental Protection Agency, Duluth, MN The occurrence of pharmaceuticals in the environment presents a challenge of growing concern. In contrast to many industrial compounds, pharmaceuticals undergo extensive testing prior to their introduction to the environment. In principal, therefore, it may be possible to employ existing pharmacological safety data using biological ‘‘read-across’’ methods to support screening-level bioaccumulation environmental risk assessment. However, few approaches and robust empirical datasets exist, particularly for comparative pharmacokinetic applications. For many pharmaceuticals, the primary cytochrome P450 (CYP) enzymes responsible for their metabolism have been identified in humans. Previous metabolism studies by our lab suggest that substrate specific metabolism patterns and enzyme generalizations between humans and fish may be difficult to make (Connors et al ETC 2013). Specifically, pharmaceuticals that were either substrates for CYP2D6 or general CYP substrates in humans demonstrated low rates of clearance in rainbow trout liver S9. Additionally, no significant metabolism was observed in CYP3A4 and CYP2C9 substrates. Given the high degree of metabolic enzyme conservation among fish, cross species extrapolations may still be possible. Finding a way to extrapolate metabolism data among fish species would be ideal, however the limits of this approach still remains relatively unknown. As a step towards this goal, we employed a comparative metabolism approach using standard substrate depletion assays to contrast the metabolic capabilities between rainbow trout and fathead minnow liver S9. The results indicate that fathead minnows are able to biotransform study pharmaceuticals at a rate approximately 4 to 34 times lower than clearances rates previously measured in rainbow trout. Contrary to observations in rainbow trout, fathead minnows are capable of modest diphenhydramine depletion. Further, enantiomer-specific clearance rates of propranolol were not consistent between fish species. This work demonstrates that relative clearance rates and biotransformation of racemic mixtures in fish were not predicted from human pharmacokinetic data. Additional research describing species differences and exploring tools for species extrapolation in biomedical and environmental studies is needed.

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P120. CONSIDERATIONS TO THE STUDY DESIGN AND INTERPRETATION WHEN CONDUCTING AN INHALED ADME STUDY


Colin Webber1, Simon A Moore2 and Brian A John3 1 Drug Metabolism, Huntingdon Life Sciences, Huntingdon, United Kingdom, 2Aerosol technology, Huntingdon Life Sciences, Huntingdon, United Kingdom, 3Dmpk, Huntingdon Life Sciences, Huntingdon, United Kingdom Huntingdon Life Sciences is one of the leading contract facilities for respiratory safety assessment, a service for which there is currently increasing demand. This presentation focuses specifically on recently conducted inhaled dose ADME studies and seeks to address some common questions about such work. The review includes data from both rodent and non-rodent studies, in which aerosols of radiolabelled test compounds have been generated from dry powders, solutions or MDIs. Reproducing the aerosol characteristics of non-radiolabelled test compounds for the much smaller amounts of radiolabelled compound usually available is a key challenge when using dry powder aerosols. In our laboratories, sub-gram amounts of radiolabelled dry powders have been micronised by ball-milling (ambient or cryogenic) to routinely produce material with MMAD52 microns and inhalable fraction 490% and consistent with larger-scale non-radiolabelled batches. Inter-animal variability in inhalation dosing is also a major consideration in ADME study design and data interpretation. Achieved radioactive dose levels provide an insight on the variability that is likely to be encountered during toxicology studies where achieved dose levels cannot be directly measured. Dose quantification for rodents in snout-only exposure chambers is achieved by radioactivity analysis of a small number of animals killed immediately after exposure, the mean total amount measured being taken to represent the dose received by each of the other animals on the chamber. This includes radioactivity deposited on the body surface, mainly around the snout, which typically represents about 25% of the total and will subsequently be ingested during preening. For non-rodents, dose quantification is achieved by rigorous analysis of the dose utensils used for each animal, subtracting residual radioactivity from the amount dispensed during exposure. This approach results in a more accurate individual dose assessment such that excretion recoveries usually show little variation. This poster includes specimen data from rats and dogs and reviews ways in which these studies may be designed and interpreted to ensure that conclusions are scientifically robust.

P121. REGIONAL AND SYSTEMIC DISTRIBUTION OF TEMOZOLOMIDE FROM A POLYMER IMPLANT IN RAT BRAIN Jingkai Gu1, Yunhui Zhang1, Meiyun Shi1, Mengliang Zhang1, Longmei Cheng1, Heping Sun1, Can Wang1, Yantong Sun2 and Yan Yang1 1 Collage of Life Science, Research Centre for Drug Metabolism, Jilin University, Changchun, China, 2School of Pharmaceutical Sciences, Jilin University, Changchun, China Background: Temozolomide (TMZ) is a highly permeable alkylating agent for the treatment of malignant gliomas. While it penetrates blood-brain barrier and expose in brain tumor to some extent (brain/plasma AUC ratio was 0.35 0.39 after oral administration in rats), it is also extensively distributed in the body and exerts off-target adverse effects, such as marrow depression. In order to increase its exposure in brain tumor and decrease the exposure as well as adverse effects in non-target tissues, TMZ was loaded in polifeprosan 20 as a potential intracerebral chemotherapy. It has been known that malignant gliomas relapse within several centimeters from the excision site in post-surgery patients, and therefore chemotherapeutics delivered by biodegradable polymers should penetrate sufficient distance from implant center; meanwhile, it should not transport too distant away and be highly exposed to non-malignant brain tissue. Purpose: This non-clinical pharmacokinetics study was conducted to evaluate: a) the extent of TMZ distribution in brain from implant center (regional distribution) and b) the absolute bioavailability, brain exposure and body distribution of TMZ after intracerebral administration (systemic distribution). Methods: For regional distribution study, rat brains were collected after TMZ implantation (10 mg/kg body weight), and each cerebral hemisphere was cut into 2 mm-thick slices along coronal or sagittal plane. Absolute bioavailability was studied by plasma AUCs after intracerebral and intravenous administrations. Brain exposure was evaluated using cerebrum AUC and plasma AUC after intracerebral. Extracranial tissues (such as heart, liver, kidney etc), cerebrum, cerebellum, hippocampal gyrus, hypothalamus, corpus striatum and cerebral cortex were collected after intracerebral TMZ administration. TMZ concentrations in brain slides, plasma or tissue were determined by LC-MS/MS. Results: TMZ was predominantly distributed in left hemisphere where polymer implant was inserted in. TMZ penetrated 4 mm along coronal plane 0.5 h after administration and 8 mm afterwards. In left hemisphere, TMZ concentration was approximately normally distributed from implant center along sagittal plane. Brain/ plasma AUC ratio was 26 after intracerebral administrations and absolute bioavailability (plasma) was 70%. In each functional section of brain, TMZ was mainly distributed in corpus striatum and cerebral cortex, while TMZ concentrations in cerebellum, hippocampal gyrus, hypothalamus were ten-fold to hundreds-fold lower than the formers. Intracerebral delivered TMZ was extensively distributed in extracranial tissues; however, TMZ concentrations in extracranial tissues were at the similar or lower level than those in cerebellum. Conclusion: The results indicated that the brain TMZ exposure produced by polymer implant was 74-fold higher than that produced by oral administration and systemic exposure was decreased by 30%. TMZ delivered by polymer implant penetrated a few millimeters from implant center in rat brain. The regional distribution should be further studied in primates to understand it pharmacokinetics basis in human.

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P122. DISPOSITION OF PRADIGASTAT (LCQ908) IN HUMANS: IN VIVO AND IN VITRO INVESTIGATIONS


Alana Upthagrove1, Heidi Einolf1, Imad Hanna1, Angela Bretz1, Lai Wang1, Lana Peng1, Bing Zhu1, Arpine Vapurcuyan1, Safet Palamar1, Tapan Majumdar1, Kirk Bordeaux1, Tapan Ray1, Phi Tran1, Charles Meyers2 and Jin Chen1 1 Novartis Institutes for BioMedical Research, East Hanover, NJ, 2Novartis Institutes for BioMedical Research, Cambridge, MA Pradigastat (LCQ908) is a potent and specific DGAT1 inhibitor which is under development for treatment of patients with familial chylomicronemia syndrome. In order to understand the disposition of pradigastat in humans, a number of in vitro studies as well as an in vivo study have been conducted. In human hepatocytes in vitro, pradigastat was metabolized mainly to the direct glucuronide M18.4. Two trace level oxidation products were also found, including M20. The UGT enzyme contributions to the formation of M18.4 were assessed using recombinant human UGT enzyme kinetic analysis and scaling of the contributions to hepatic intrinsic clearance. The scaling of the kinetic parameters was made possible with the absolute quantification of the specific UGT protein content of recombinant UGT-enzymes and in human liver microsomes by LC-MS. The scaled kinetic parameters estimated similar contributions of UGT1A1 (44%) and UGT1A3 (39%) to the pradigastat glucuronidation activity in human liver microsomes, followed by UGT2B7 (17%). Additional investigations were conducted to confirm the structure of M18.4, and to assess its potential instability toward hydrolysis and acyl rearrangement. To study metabolism and excretion of pradigastat in vivo, four healthy male subjects received a single oral dose of 10 mg (60 mCi) of [14C]pradigastat followed by blood, urine, and feces collection for up to 28 days post-dose. Pradigastat was absorbed slowly. The maximum concentrations (mean Cmax 105 ng/mL) were reached with a medianTmax of 15 hours. The elimination of pradigastat was slow, with a mean terminal T1/2 of 143 h. The mean systemic exposure (AUCinf) was 21200 ng/mL*h, with apparent oral clearance (CL/F) of 0.5 L/hr and apparent volume of distribution (Vz/F) of 102.6 L. Mean 92% recovery of the radioactive dose in excreta was achieved within 28 days post-dose. Essentially all of the [14C]pradigastat dose was recovered in the feces, with negligible radioactivity in urine. Metabolites M18.4 and M20 were detected in plasma, but in all of the subjects, the plasma radioactivity consisted mainly of unchanged pradigastat. M20 constituted a negligible fraction of the dose recovered in excreta, confirming that oxidative metabolism makes a minimal contribution to elimination of pradigastat in humans. M18.4 was not detected in the excreta. However, because of the inherent instability of the glucuronide toward hydrolysis in the gastrointestinal tract, the extent to which pradigastat was eliminated via the glucuronide could not be determined. From the in vivo and in vitro data together, however, it can be concluded that in humans, pradigastat is metabolized mainly to the acyl glucuronide M18.4. UGT1A1, UGT1A3, and UGT2B7 contribute to the formation of M18.4 in the liver. Systemic exposure to M18.4 is very low, and moreover, it has shown low potential for acyl rearrangement. Renal excretion does not contribute significantly to pradigastat elimination. However, because of the instability of the glucuronide in the intestinal tract, the relative contribution of metabolism vs. biliary excretion of unchanged pradigastat has not been established.

P123. THE IMPORTANCE OF VILLOUS PHYSIOLOGY AND MORPHOLOGY IN MECHANISTIC PBPK MODELS Emile Chen, Guoying Tai and Harma Ellens Drug metabolism and Pharmacokinetics, GlaxoSmithKline, King of Prussia, PA Existing PBPK models incorporating intestinal first-pass metabolism account for effect of drug permeability on accessible absorption surface area by use of ‘‘effective’’ permeability, Peff, without adjusting number of enterocytes involved in absorption or proportion of intestinal CYP3A involved in first-pass metabolism. The current model expands on existing models by accounting for these factors. The PBPK model was developed using Saam II. Midazolam clinical data was generated at GlaxoSmithKline. The model simultaneously captures human midazolam blood concentration profile and previously reported intestinal availability, using values for CYP3A CLint, permeability and accessible surface area comparable to literature data. Simulations show: (1) for highly permeable drugs absorbed through a small portion of villous surface, failure to distinguish enterocytes involved in absorption from those that are not, results in overestimation of intestinal metabolism; (2) first-pass extraction of poorly permeable drugs occurs primarily in enterocytes, drugs with higher permeability are extracted by enterocytes and hepatocytes; (3) differences in permeability of perpetrator and victim drugs results in their spatial separation along the villous axis and intestinal length, diminishing drug-drug interaction magnitude. The model provides a useful tool to interrogate intestinal absorption/metabolism of candidate drugs and factors controlling this.

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P124. COMPARATIVE IN-VITRO DERMAL PENETRATION STUDIES WITH [14C]-ANILINE USING STRAT-M MEMBRANE AND DERMATOMED HUMAN SKIN


Stuart G. Wood1, Gill Ford1, Hayley Leech2, Karen Wilson2 and Andrew McEwen1 1 Quotient Bioresearch Limited, Rushden, United Kingdom, 2Metabolic Chemistry, Quotient Bioresearch Limited, Rushden, United Kingdom Purpose: The gold standard for in-vitro dermal penetration studies requires the use of radiolabelled test compound and dermatomed human skin. Whilst this provides valuable information for use in risk assessment studies for regulatory submission, the search for alternatives for use in the discovery phase of development continues. Often what is required is a comparative assessment of penetration between alternative drug products or drug formulations prior to selection of the lead compound. In this case the use of human skin is not required and decisions can be made based on permeation through a standard membrane. Methods: In this study we report a comparative evaluation of the permeation of [14C]-aniline through Strat-M membrane and human dermatomed skin. Skin preparations were mounted in Franz static dermal penetration cells with the stratum corneum uppermost. The receptor fluid was filled with physiological saline with 5% bovine serum albumin (12 mL) and placed into a V9 magnetic stirrer block. The receptor fluid was maintained at a constant temperature of 32 2 C using heated water jackets. Prior to use the integrity of each sample (skin or membrane) was determined using tritiated water. The test compound was applied in aqueous solution as a single finite dermal application for a maximum of 24 hours. Concentrations tested were in the range 100– 500 mg/mL. All cells were occluded following dosing. Receptor fluid was analysed for concentrations of radioactivity. To help with the interpretation of the results additional experiments were performed using [14C]-testosterone, one of the OECD reference compounds, for comparative purposes. Results: Permeation constants (Kp) were calculated from the data obtained. For aniline the permeation constants were in the ranges 3.82E-04 to 1.47E-03 and 1.74E-04 to 8.55E-04 for skin and Strat-M membrane respectively. Permeation rates were general higher using Strat-M membrane than for human skin. For testosterone the permeation constants were 3.08E-04 and 2.79E-0 for skin and Strat-M membrane respectively. Conclusions: The permeation of aniline through human skin and Strat-M membrane has been investigated. Permeation rates whilst similar were generally higher in the membrane than observed in the skin. The use of a membrane could enable compounds to be ranked in a standard system. For testosterone the permeation rates were also found to be similar in both human skin and artificial membrane.

P125. COMPARISON OF THE DRUG CONCENTRATIONS IN THE BRAIN INTERSTITIAL AND CEREBROSPINAL FLUIDS IN CYNOMOLGUS MONKEYS Yoko Nagaya1, Yoshitane Nozaki1, Osamu Takenaka1, Hiroyuki Kusuhara2, Kazutomi Kusano1 and Tsutomu Yoshimura1 1 Drug Metabolism and Pharmacokinetics, Eisai Co., Ltd., Tsukuba, Japan, 2Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Purpose: Understanding of central nervous system (CNS) penetration of drug candidates is crucial for CNS drug discovery. Cerebrospinal fluid (CSF) concentration (CCSF) is widely used as a surrogate of unbound brain concentration assuming that lipophilic compounds freely diffuse between CSF and interstitial fluid (ISF) compartments although it has been suggested that CCSF overestimates the unbound brain concentration for P-glycoprotein (P-gp) substrates in rodents because of active efflux at the blood-brain barrier. However, a species difference in the expression of P-gp at the blood-brain barriers has been reported; higher in rodents than in humans, suggesting that the impact of the active efflux on the prediction of ISF concentration (CISF) from CCSF is less remarkable in humans. This study was designed to evaluate the utility of CCSF as surrogate of CISF by comparing these concentrations of twelve compounds including P-gp substrates in cynomolgus monkeys. Methods: Dialysate, CSF and blood were simultaneously and serially collected from a microdialysis probe implanted in the cerebral cortex, an indwelling cannula in the cisterna magna and the saphenous vein, respectively, under the constant intravenous infusion following a priming dose to achieve steady state in cynomolgus monkey. CISF was calculated from the dialysate concentrations following a retrodialysis method. At the end of the experiment, the animals were sacrificed, and the brain and blood samples were obtained to determine brain (Cb) and plasma concentration (Cp), respectively. The in vitro unbound fractions in brain homogenate and plasma were determined by an equilibrium dialysis to estimate unbound brain (Cb,u,homogenate) and plasma concentration (Cp,u), respectively. Results and Discussion: CISF, CCSF, and Cp,u reached steady state within 3 or 4 hours after intravenous infusion for all compounds. Although CISF, CCSF, and Cp,u were similar for non–P-gp substrates (antipyrine and carbamazepine), typical P-gp substrates (desloratadine, risperidone, verapamil and quinidine) showed CISF/Cp,u and CCSF/Cp,u quite lower than unity, suggesting that P-gp-mediated active efflux was a major determinant of the CNS penetration at least for this compound set. Although CCSF of typical P-gp substrates, desloratadine, verapamil and quinidine showed a trend to overestimation of CISF, CCSF well predicted CISF to within 3-fold. Cb,u,homogenate was also correlated with CISF and CCSF. However, Cb,u,homogenate showed a tendency toward overestimating CISFand CCSF , where those of desloratadine and risperidone were 6.7- to 14-fold and 5.1- to 5.4fold overestimated by the Cb,u,homogenate. Conclusion: Although CCSF of typical P-gp substrates showed a trend toward overestimating CISF, the difference was less than 3-fold. Therefore, CCSF could be a good surrogate of CISF, even for P-gp substrates in nonhuman primates.

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P126. FORMALDEHYDE INFLUENCE DNA DAMAGE REPAIR PROCESS THROUGH PARP-1 PATHWAY IN THE HUMAN BRONCHIAL EPITHELIAL CELL LINES


Xiaowei Jia and Yuxin Zheng Key laboratory of Chemical Safety and Health, National Institute for Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China Formaldehyde is a well-known toxicity and carcinogenicity and wide spread human exposure has raised public concern over its safety. It is classified as carcinogenicity in humans (group1). The results of in vitro test, laboratory animals test and in exposed humans studies shown formaldehyde is a genotoxic agent. The genotoxicity are considered to play important roles in the carcinogenesis of formaldehyde in nasal tissues. The goal of this study is to investigate the cellular pathways or proteins involved in DNA damage repair induced by low concentration formaldehyde in Human Bronchial Epithelial (HBE) cells. The cell counting Kit-8 was used to measure the cytotoxic effects of formaldehyde on HBE cells. An alkaline COMET assay was conducted to test DNA damage by formaldehyde. The protein expression level was assessed by Western Blot. We used RNA interference cell to test the role of ATM in formaldehyde -induced DNA damage repair and NBS1 and p-p53 expression. In the results, we found the formaldehyde cloud induce the single-strand breaks (SSBs) in low concentration of 5 mM. Interestingly, the SSB is accompanied with increased expression of ataxia telangiectasia mutated (ATM), Nbs1 and p-p53. formaldehyde -induced SSBs were quickly repaired in ATM siRNA depleted cells, demonstrating that ATM is not required for efficient completion of SSBs. Moreover, consistent with knocking down of ATM, expressions of Nbs1 were significantly decreased, and p-p53 expression was showed a slight decrease. Formaldehyde was also found to trigger up-regulation of poly(ADP-ribose) polymerase (PARP). Furthermore, pretreatment of cells with PJ34, a specific PARP-1inhibitor, we found a high level of DNA damage and delayed repair in PARP-1 inhibited cells, as reflected by a significant increase of the three COMET parameters compared with controls. In summary, our results indicate that low concentration FA-induced DNA damage repair may be through PARP-1 pathway, but not ATM pathway. These findings will help us to understand the signal transduction mechanisms involved in the carcinogenic effects of FA at DNA damage repair process.

P127. INVESTIGATING THE ADME PROPERTIES OF AN IONIC LIQUID SALT OF SULFASALZINE, A NOVEL APPROACH TO IMPROVE DRUG EXPOSURE Mohammad Shadid1, Gabriela Gurau2, Bei-Ching Chuang1, Emily Guan3, Mingxiang Liao1, Swapan Chowdhury1, Jing-Tao Wu1, Syed A. A. Rizvi4, Robin D. Rogers2 and Robert J. Griffin1 1 Drug Metabolism and Pharmacokinetics, Takeda Cambridge US, Cambridge, MA, 2Center for Green Manufacturing and Department of Chemistry, The University of Alabama, Tuscaloosa, AL, 3Drug Safety Evaluation, Takeda Cambridge US, Cambridge, MA, 4Department of Pharmaceutical Sciences, Nova Southeastern University, Fort Lauderdale, FL Ionic liquids are generally defined as salts with melting points below 100 C. They possess unique properties and often exhibit better solubility than standard salts. As of the writing of this abstract, the search of ‘‘ionic liquid’’ in SciFinder search engine resulted in over 18,000 publications. Ionic liquids have demonstrated utility as solvents, lubricants, thermal fluids, optical fluids and in electrochemical applications. Although ionic liquid forms of active pharmaceutical ingredients (IL-APIs) have been reported in the literature, much work to be done in the development of new IL-APIs with improved solubility and drug delivery profiles. The purpose of this investigation was to examine the ADME properties of an ionic liquid using a poorly soluble BCS class 4 compound; sulfasalazine as an example. Sulfasalazine-choline ionic liquid exhibited 1 106 folds improvement in saline solubility over sulfasalazine to yield concentrations of active sulfasalazine of 38.8 mg/mL and 0.0000346 mg/mL respectively. Upon administration to rats, sulfasalazine and sulfasalazine-choline ionic liquid had equivalent intravenous (IV) exposures at a low solution dose of 0.5 mg/ kg. However, the superior solubility of the ionic liquid enabled administration of much higher intravenous doses (6, 12, and 24 mg/kg, dissolved directly in saline) which resulted in dose proportional increases in exposure. These IV doses have never been reported to sulfasalazine due to solubility limitations. To compare bioavailability, 50 mg/kg sulfasalazine suspension in 0.5% HPMC/0.2% Tween and 50 mg/kg sulfasalazine-choline ionic liquid dissolved directly in saline were given to rats via gavage. There was a 2.5-fold increase in the absolute bioavailability for the sulfasalazine-choline compared to sulfasalazine. The in vitro metabolic stability and permeability assays revealed no statistical differences between sulfasalazine and sulfasalazine-choline ionic liquid. Both forms of sulfasalazine were fairly stable in S9 incubations and exhibited low permeability based in a CACO-2 assay indicating the oral exposure effects were not due to the choline counter-ion altering the permeability or metabolism of sulfasalazine. In conclusion, ionic liquids may provide an attractive approach to address two of the problems commonly encountered in drug development. Solubility limitations on the dose that can be administered as a parenteral solution and solubility limited bioavailability may both be improved through preparation of the drug substance as an ionic liquid.

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P128. SONODYNAMICALLY INDUCED ANTICANCER EFFECTS BY FUNCTIONALIZED FULLERENES


Yumiko Iwase1, Nagahiko Yumita1, Takahiro Imaizumi1, Ai Sakurazawa1, Yuuka Kaya1, Koji Nishi1, Shin-ichiro Umemura2, Fu-Shih Chen3, Yasunori Momose4 and Toshihiko Ikeda1 1 School of Pharmacy, Yokohama College of Pharmacy, Yokohama, Japan, 2Department of Electrical and Communication Engineering, Tohoku University, Sendai, Japan, 3Department of Physical Education and Kinesiology, National Dong Hwa University, Taiwan, 4School of Parmaceutical Sciences, Toho University, Funabashi, Japan Background: Functionalized fullerenes, such as polyhydroxy fullerene (PHF), have attracted particular attention due to their water solubility and their potential application in tumor imaging and therapy as carbon nanomaterials. Recently, we found that photochemically active porphyrins such as hematoporphyrin and porfimer sodium can induce significant cell damage when activated by ultrasoundIn this study, the sonodynamically induced antitumor effect of PHF was investigated. Material and Methods: Sonodynamically induced in vitro antitumor effects of PHF in combination with ultrasound were investigated using isolated sarcoma 180 cells and solid tumor from colon 26 carcinoma cells. The viability of the isolated cells was determined by staining of the cells with trypan blue dye. ESR spectrometry and spin trapping technique were used to measure reactive oxygen species. The in vivo antitumor effects were evaluated by the histological observation and tumor growth after the exposure. Results: The cell damage induced by sonication was enhanced by two-fold in the presence of 80 mM PHF. In addition, the combination of PHF and ultrasound enhanced nitroxide generation in the same acoustical arrangement. Histidine significantly inhibited these enhancement. The combined treatment of ultrasonic exposure with PHF suppressed the growth of implanted colon 26 tumors. The destruction of tumor tissue was observed with the ultrasonic treatment in combination with PHF, while neither the treatment with PHF alone nor that with ultrasound alone caused any necrosis. Discussion: The significant reduction by histidine in the number of sonodynamically induced cell damage and nitroxide generation suggests that ultrasonically generated reactive oxygen species such as singlet oxygen is an important mediator of sonodynamically induced apoptosis. These results suggest that PHF is a potential sonosensitizer for sonodynamic treatment of solid tumors. Conclusion: We believe that nanotechnology in medicine might be able to improve sonodynamic anticancer therapeutic approach.

P129. PRE AND POST DERIVATIZATION STABILITY OF MONOAMINES IN RAT BRAIN MICRODIALYSATES IN ABSENCE OF STABILIZING AGENTS Ranjith kumar Ponnamaneni, Arun kumar Manoharan, Rajesh Kumar Boggavarapu, Vijay Benade, Saivishal Daripelli, Imran Khan, Vishwottam Kandikere and Ramakrishna Nirogi Discovery Research, Suven Life Sciences Ltd, Hyderabad, India Monoamines are small and polar in nature they help in signal transmission between neurons and other cells around the synapse. A dysfunction in neurotransmitters eventually leads to many neurological disorders like Alzheimer’s, Parkinson’s disease, depression, obsessive compulsive disorder. So monitoring these levels in brain regions in case of CNS disorders help in understanding disease mechanisms, and in turn makes possible for new therapeutic approaches or in designing new drugs. In spite of having sensitive analytical technique it is reasonably important to understand the stability of these monoamines in CSF samples which otherwise would leads to wrong interpretation of the results. In addition to the existing limitations of microdialysis technique like low sample volumes, low levels with high inorganic salts and sample collection intervals at different times, the stability of monoamines pose many problems in sample handling. The stability in biological matrices is a critical consideration in the selection of an appropriate method to prepare the samples for analysis. Establishing and ensuring the stability of monoamines from collection phase through storage, sample processing and analysis and also designing the experimental conditions as per the established stability of these monoamines is important. The stability of neurotransmitters in absence of stabilizing agents in CSF of brain regions at different times as per the required sample collection duration is presented by analyzing the samples with LC-MS/MS system. Sample handling and derivatization procedure is discussed for dopamine, norepinephrine and serotonin in CSF samples of brain regions of study requiring sample collection for longer duration (24 hr).

P130. METABOLITE IDENTIFICATION IN DRUG DISCOVERY. NEW TECHNIQUES TO EXPLORE THE HRMS Ismael Zamora1, Fabien Fontaine1, Blanca Serra1, Xavier Pascual1 and Luca Morettoni2 1 Lead Molecular Design, S.L., San Cugat del Valle´s, Spain, 2Molecular Discovery, London, United Kingdom One of the key factors in the discovery process of a new chemical entities is the metabolic fade of the lead compounds. Nowadays, the in-vitro estimation of metabolic clearance is a typical tier 1 assay, which end point is a number that indicates if the compound is slowly or quickly metabolized, measuring the parent compound disappearance in a Trple Quad Mass spectrometer by nominal mass multiple reaction monitoring. Nevertheless, high resolution mass spectrometers linked to Liquid chromatography (LC-HRMS) has advanced in the recent years, and it is possible not only to follow the parent disappearance but

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also the metabolite formation in the same experiment, delivering to the discovery teams not only a number as end-point but also the probable chemical structure of the metabolites. The amount of data that is produced by these new instruments supersedes the actual capacity to analyze the data. In this article we are presenting a new software technology that can process the LC-HRMS data to obtain suggestions for metabolite structures, being able to process, archive, share and communicate the results across the different units of Drug Discovery, increasing the efficient of the metabolite identification process to a extend that the identification of the major metabolites (soft spot ID) can match the speed of clearance measurement.

P131. SIMULTANEOUS PK ANALYSIS AND METABOLITE IDENTIFICATION USING NOVEL MASS SPECTROMETRY ACQUISITION TECHNIQUES


James Ferguson1, Suma Ramagiri2, Loren Olson3, Eva Duchoslav2, Gary Impey2 and Ragu Ramanathan4 1 AB SCIEX, Framingham, MA, 2AB SCIEX, Concord, ON, Canada, 3AB SCIEX, San Jose, CA, 4Dmpk, QPS, LLC, Newark, DE Introduction: Identifying, characterizing, and quantifying drugs and their metabolites in biological matrix is a major part of the drug development process. High-resolution mass spectrometry (HRMS) combined with ultrahigh-pressure liquid chromatography (UHPLC) and full scan acquisition is gaining importance as a potential streamlined method for simultaneous PK analysis and metabolite ID. Here, we discuss, a unique data independent HRMS acquisition approach called Sequential Window acquisition of All THeoretical fragment ion spectra (SWATH) for simultaneous Qual/Quant analysis. We also investigate differential mobility spectroscopy (DMS) for separation of isobaric metabolites like positional isomers. We explore two major advantages of these novel mass spectrometry strategies in analytical drug development: 1. SWATH, an easy to use generic MS method for digital archive of met ID information and retrospective data mining capability and 2. Unique orthogonal DMS separation of positional isomers like hydroxyl metabolites without a lengthy chromatographic run times or expensive chiral columns. Methods: In vivo samples for Loratadine and Nefazadone were analyzed using SWATH data independent acquisition strategy with a generic LC gradient. For SWATH data analysis, blood samples were collected at pre-dose and at post dose at 0.5, 1, 2, 4, 8, 12, 24, and 48 hours. The MS method was developed on a TripleTOFÔ system. A 50 mL aliquot of plasma from each time point was diluted with 50 mL of internal standard solution containing 100 ng/mL of d4-desloratadine in acetonitrile. For DMS analysis, Synthetic oxidative metabolites of carbamazepine—CBZ (2-hydroxy CBZ, 3-hydroxyCBZ and 10,11-CBZ epoxide and two verapamil (VERA) demethylations—Norverapamil and O-desmethyl verapamil from Toronto Research Chemicals were used to assess DMS separation of isobaric metabolites on TripleTOF system equipped with SelexIONÔ Technology. Preliminary data: SWATH involves the use of multiple Q1 precursor windows ranging from 15–50 Da and performing MS/MS scan while stepping across windows. For all compounds investigated, the lower limit of quantification (LLOQ) was 0.1 ng/mL. All calibration curves exhibited a linear dynamic range of 3.5 to 4.0 orders of magnitude. The % CV was within 15% and the accuracy ranged from 85–115% for all compounds. UHPLC-HRMS integrated Qual/Quant workflows gave the benefit of known and unknown metabolite quantification and identification. For DMS analysis, 2- hydroxy carbamazepine and 3-hydroxy carbamazepine were separated in the mobility cell under 2 compensation voltages. Calibration range tested for 2-OH CBZ in a mixture of all three CBZ oxidations was 1 to 100 ng/mL; acceptable % CV and accuracies were achieved. Conclusions: SWATH based non-targeted MS/MS mode of quantification showed improved sensitivity and signal-to-noise (S/N) ratio by combining TOF-MS and MS/MS responses of Loratadine and its metabolites while employing a non-targeted generic method. DMS has been shown to be an additional orthogonal separation technique in addition to those currently available in LC-MS and LC-MS/MS. Very early work illustrating the potential for separation of isobaric positional isomers and also provide a cleaner background, possibly with more signal due to suppression of interferences resulting from PEG formulation.

P132. METABOLITE IDENTIFICATION USING DIFFERENTIAL ION MOBILITY SPECTROMETRY COUPLE TO MASS SPECTROMETRY Carmai Seto, Takeo Sakuma and Pauline Vollmerhaus AB SCIEX, Concord, ON, Canada Characterizing and identifying drugs and their metabolites in biological samples such as plasma, urine or bile can be challenging because of isobaric interferences and high background. Characterizing metabolites in these samples are especially difficult because metabolites may be circulating at much lower concentrations than the parent drug. In this study, the use of differential ion mobility spectrometry (DMS) couple to a high resolution accurate mass system in the analysis drugs and their metabolites in urine samples was investigated. DMS has been shown to reduce background noise as well provide additional selectivity needed for analysis of complex samples. Several drugs and their metabolites were studied. Saliva and urine samples were collected from patients taking several different drugs, lansprazole, clarithromycin and amoxicillin, at fairly high doses. The samples were diluted/extracted prior to analysis. Each drug was incubated in rat microsomes under oxidative conditions in order to develop LC

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conditions for each drug and their respective metabolites. A mass spectrometer with a differential ion mobility device installed coupled was used to analyze the samples. DMS parameters were optimized for each drug studied. Metabolites were identified and characterized for several different compounds in human urine and rat microsomal incubations using TripleTOFÕ 5600þ systems with SelexIONÔ DMS technology. For example, oxidation, di-oxidation, glucoronide and sulfate metabolites of lansprazole were detected and characterized in the human urine samples. Analysis of drugs and their metabolites in urine can be difficult due to matrix interferences. However, analysis in this study was made more difficult because the patient was taking three different medications simultaneously. However, with the use of high resolution mass spectrometry along DMS, selective analysis can be achieved. The DMS can selectively filter compounds that have different mobility and therefore simplify the data acquired.


P133. THE RELEVANCE OF SOLVENT SELECTION IN METABOLISM STUDIES: ANSAMYCIN DEGRADATION CASE STUDY Sherri Smith1, Kris Depew2, James Porter3, Brendan Arsenault4, Chungang Gu5, Kelly Slocum6, Randall Press7 and Mark Gohdes8 1 Dmpk, Infinity Pharmaceuticals, Inc., Cambridge, MA, 2Process Chemistry, Infinity Pharmaceuticals, Inc., Cambridge, MA, 3 Product Development, Infinity Pharmaceuticals, Inc., Cambridge, MA, 4Pharmaceutical Development, Infinity Pharmaceuticals, Inc., Cambridge, MA, 5Oncology DMPK, AstraZeneca Pharmaceutical LP, Waltham, MA, 6Oncology Pharmacology, Novartis Institute for Biomedical Research, Inc., Cambridge, MA, 7Drug Metabolism, Covance Laboratories, Inc, Madison, WI, 8Drug Metabolism, Covance Laboratories Inc., Madison, WI It can be challenging to distinguish metabolites from degradation products when conducting metabolism studies. The data in this presentation show how IPI-504 degradation products resulting from solvent selection can be erroneously attributed to metabolites. IPI-504 (17-allylamino-17-demethyoxygeldanmycin hydroquinone hydrochloride or retaspimycin HCl) is an intravenously administered, potent and selective heat shock protein 90 (Hsp90) inhibitor currently under evaluation in two nonsmall cell lung cancer clinical trials. The hydroquinone HCl salt IPI-504 readily converts to the quinone 17-AAG under ambient conditions; the hydroquinone salt is deprotonated and readily oxidizes to the quinone within minutes, presumably via oxygenmediated transformation. An apparent redox equilibrium between the quinone/hydroquinone pair is established soon thereafter in plasma due to enzymatic reduction and oxidation reactions. During a mouse mass balance study using [14C]-IPI-504, an abundant unexpected product was detected and assigned as a dioxidation metabolite (M27) to 17-AAG. Previous in vitro experiments in liver microsomes had not predicted this in vivo metabolite. To aid in structural elucidation of M27, a Thermo Scientific LTQ Orbitrap high-resolution mass spectrometer was employed. Using both accurate mass and unique fragment ions, M27 was identified as a methanol adduct of 17-AAG and later confirmed by NMR, TLC and chemical synthesis. Initial metabolite profiling/identification studies using a low-resolution mass spectrometer did not differentiate between a dioxidation metabolite and methanol adduct, since both products share the same nominal mass, multiple common fragment ions, and retention time. The ansamycin ring is susceptible to methanolysis in a time- and temperature-dependent manner so seemingly small changes in preparation and analysis could lead to changes in metabolite/degradant pattern and abundance. Additional experiments were conducted to further characterize this phenomenon. Unlabeled IPI-504 was administered to mice and plasma was collected and precipitated separately in three solvents: acetonitrile, methanol, and ethyl acetate. While the analyte peak responses change slightly in various solvents, three unique peaks were detected in the samples prepared with methanol, all characterized as methanol adducts. Increasing the LC gradient to two hours also enabled separation of a dioxide metabolite and methoxide degradation product; however, this time-consuming approach is not always practical. Spiking naı¨ve mouse plasma with [14C]-IPI-504 and subsequent precipitation with either acetonitrile or acetonitrile:methanol mixture also resulted in significantly different profiles, the latter showing M27 and relatively lower abundance of 17-AAG. These combined experiments support the conclusion that the ansamycin ring of IPI-504 undergoes hydrolysis with exposure to methanol. The importance of understanding drug stability in solvents prior to initiation of development studies is fundamental.

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P134. BIOACTIVATION STUDIES OF ANTIMYCOBACTERIAL 4-ARYL-2-AMINOTHIAZOLES


Mathew Njoroge, Faith Mjambili and Kelly Chibale Department of Chemistry, University of Cape Town, Cape Town, South Africa 4-Aryl-2-aminothiazoles have recently been described as potent anti-mycobacterial agents1. However, a series of compounds synthesised from this scaffold were found to have varying degrees of in vitro cytotoxicity. Since toxicity of aminothiazoles has previously been linked to their metabolic bioactivation2, the metabolism of these compounds was studied to understand their toxicity. The studies were conducted using human, rat and mouse microsomal and S9 fractions using glutathione as a trapping agent. The incubations were analysed on an ABSciex 4000QTrapÕ system coupled to an Agilent 1200 HPLC. Metabolites were detected and tentatively identified using a combination of full scan and selective scan electrospray ionisation methods coupled through information dependent acquisition to enhanced product ion scans. Further experiments, including H/D exchange and MS3 scans were performed to further confirm the identity of the metabolites. Several metabolites were detected and tentatively identified, including glutathione adducts of the aminothiazole nucleus. The elucidation of the metabolic pathways leading to formation of the glutathione adducts suggests that an epoxide intermediate is formed in vitro by enzymatic processes. This epoxide intermediate could contribute to toxicity by covalent binding of biomolecules. However, the results also suggest that the bioactivation mechanism is not tied to their antimycobacterial activity. These findings will contribute to the design of 4-aryl-2-aminothiazoles with potent antimycobacterial activity and reduced bioactivation potential.

References (1) Ananthan, S.; Faaleolea, E. R.; Goldman, R. C.; Hobrath, J. V; Kwong, C. D.; Laughon, B. E.; Maddry, J. a; Mehta, A.; Rasmussen, L.; Reynolds, R. C.; Secrist, J. a; Shindo, N.; Showe, D. N.; Sosa, M. I.; Suling, W. J.; White, E. L. Highthroughput screening for inhibitors of Mycobacterium tuberculosis H37Rv. Tuberculosis (Edinb) 2009, 89, 334–53. (2) Subramanian, R.; Lee, M. R.; Allen, J. G.; Bourbeau, M. P.; Fotsch, C.; Hong, F.-T.; Tadesse, S.; Yao, G.; Yuan, C. C.; Surapaneni, S.; Skiles, G. L.; Wang, X.; Wohlhieter, G. E.; Zeng, Q.; Zhou, Y.; Zhu, X.; Li, C. Cytochrome P450-mediated epoxidation of 2-aminothiazole-based AKT inhibitors: identification of novel GSH adducts and reduction of metabolic activation through structural changes guided by in silico and in vitro screening. Chem Res Toxicol 2010, 23, 653–63.

P135. IMPROVEMENT IN PREDICTION OF HUMAN PHARMACOKINETICS OF BIOTHERAPEUTICS Yoichi Naritomi, Kenji Tabata and Toshio Teramura Astellas Pharma. Inc., Tsukuba-shi, Japan The prediction of human pharmacokinetics (PK) is important in estimating clinical dose and conducting PK/pharmacodynamics (PD) analysis of biotherapeutics such as monoclonal antibodies (mAbs), PEGylated Fab fragments, and Fc fusion proteins. In the intravenous administration of biotherapeutics, the prediction of human total clearance (CLt) and distribution volume at steady state (Vdss) using allometric scaling methods has been reported, but relatively few biotherapeutics have been tested in these reports. In the subcutaneous administration of biotherapeutics, bioavailability (F) and absorption rate constant (ka) are important in the prediction of human PK, but the prediction of these parameters has not been sufficiently evaluated and the number of reports is limited. Here, we improved the prediction of human CLt and Vdss after intravenous administration using PK data of 48 biotherapeutics and F and ka after subcutaneous administration using PK data of 28 biotherapeutics. CLt values tended to be higher for Fc fusion proteins and mAbs targeting membrane-bound antigens compared to those of mAbs targeting soluble antigens. The value of Vdss for the majority of biotherapeutics was approximately 0.1 L/kg. The mean scaling exponent of each type of biotherapeutic for CLt and Vdss was calculated from monkey and human PK data. The mean scaling exponent of each type of biotherapeutic for CLt ranged from 0.87 to 1.01, and was higher than the common scaling exponent value of 0.75. In contrast, the mean scaling exponent of each type of biotherapeutic for Vdss was approximately 1. Successful predictions for human CLt and Vdss were obtained using the mean scaling exponent for each type of biotherapeutic. We observed large differences between monkeys and humans in F and ka. However, most biotherapeutics exhibited bioavailability ranging from 40% to 100%. We also observed that the range of ka values of mAbs targeting membrane-bound or soluble antigen was different from that of Fc fusion proteins. These results provide an improved prediction of human PK for biotherapeutics following intravenous or subcutaneous administration that will facilitate future drug discovery.

P136. PHARMACOKINETICS, DISTRIBUTION, METABOLISM AND EXCRETION OF TEMOZOLOMIDE ESTER, A NEW ALKYLATING AGENT FOR CANCER THEARAPY Yunhui Zhang1, Jingkai Gu1, Qun He1, Lei Yin2, Tingting Wang2, Meiyun Shi1, Yantong Sun3 and Yan Yang1 1 Collage of Life Science, Research Centre for Drug Metabolism, Jilin University, Changchun, China, 2Clinical Pharmacology Center, Research Institute of Translational Medicine, The First Bethune Hospital of Jilin University, Changchun, China, 3School of Pharmaceutical Sciences, Jilin University, Changchun, China


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Temozolomide is a widely used alkylating agent for the treatment of glioblastoma and melanoma. In order to improve its permeability across mucus membrane for topical application, temozolomide ester (hexyl 3-methyl-4-oxo-3H, 4H-imidazo[4,3d][1,2,3,5]tetrazine-8-carboxylate, TMZA-HE) was designed[1] and prepared in vaginal suppositories for the therapy of cervix cancer. The pharmacokinetics, distribution, metabolism and excretion of its hydrolysate, TMZA, were studied after intravaginal TMZA-HE administration. Absorption and pharmacokinetics: After intravaginal administration of TMZA-HE in Wistar rats, multiple peaks of the plasma TMZA concentration-time profiles were observed. The first concentration peaks were reached within 1 h and half-lives were 3.1 5.7 h. Nonlinear pharmacokinetics was observed based on proportionality of AUC (1:4.25:6.22) and Cmax (1:1.67:1.98) after three doses of TMZA-HE (1:2:4). The absolute bioavailability of TMZA after intravaginal administration was 26%. The investigated pharmacokinetic parameters were not significantly different (p40.05) after single and multiple intravaginal administrations. The pharmacokinetics in Beagle dogs was similar to that in Wistar rats. Distribution: TMZA was extensively distributed after intravaginal TMZA-HE administrations in rats. High TMZA concentrations were found in digestive tract and urinary organs. These results indicated TMZA underwent enterohepatic recirculation and was excreted predominantly in urine. Plasma binding was 50.52 58.16%. The efflux ratio of TMZA on Caco2 and MDCK-MDR1 cell monolayers were 2.63 and 3.41, respectively, indicating TMZA was a substrate of P-gp. Metabolism: The fate of TMZA-HE was proposed with five metabolites identified by MIM-MRM-EPI scan: TMZA-HE was hydrolyzed to TMZA by esterase, followed by pH-dependent degradation of TMZA as a similar way of temozolomide. An esterified ringopening product was also identified. The hydrolysis activity of topical esterase was studied using in vitro incubation of TMZAHE in vagina and uterus homogenates, where a Km of 20.74 mM was determined. Excretion: The recovered dose (23.53%) was predominantly excreted in urine (21.58%), and a small amount was excreted in feces. The difference of amount recovered in bile (10.31%) and in feces (1.95%) demonstrated enterohepatic recirculation.

P137. A NOVEL APPROACH TO AUTOMATED GLUCOSE MONITORING IN AWAKE AND FREELY MOVING ANIMALS Brad Gien1, Scott Peters1, Candace Rohde-Johnson1, Daniel Aillon2, Peter Patillo2 and Jessica Squires1 1 BASi, West Lafayette, IN, 2Pinnacle Technology Inc., Lawrence, KS With the staggering increase in the incidence of diabetes in the United States, it is becoming ever more important to have useful tools for exploring the disease and associated therapies. One key endpoint in diabetic research is the monitoring of glucose levels, and current techniques cause significant stress to the animals, potentially causing unreliable results. The ability to record glucose concentration data from undisturbed animals would be a valuable tool in diabetes research. In pursuit of this goal, we adapted an implantable neural glucose sensor to the flow path of an automated blood sampling system. This combination allowed the measurement of glucose concentrations without causing disruption or stress to the animal and has the potential to provide accurate and reliable data for diabetes research. In order to adapt the implantable neural glucose sensor to the sampling system, a sensor interface was created within the blood flow path. This interface made it possible to draw samples of blood through the sensor and provide instant glucose concentration levels. Minor adjustments were made to the glucose sensor and interface over the course of several studies. With this configuration, it was possible to either continue collecting the blood into a refrigerated vial for pharmacokinetic drug concentration analysis or to return the whole blood sample to the animal with no net blood loss. To verify the method, n ¼ 20 male Sprague-Dawley rats received a PO administration of glucose. Blood samples were simultaneously compared with data from commercially available external glucose monitors and demonstrated the flow-through glucose sensor to be reliable and accurate. This method has the potential to both reduce animal numbers and to improve the quality of data when conducting diabetes research. We also believe that the combined approach used in this study has additional applications in therapeutic areas other than diabetes.

P138. DESIGN, EVALUATION AND SYNTHESIS OF NOVEL ANTICANCER DRUGS TARGETING THE DIMERIZATION ARM OF EGFR Zechariah Marting, David Edward Hibbs, Paul Groundwater and Thomas Grewal Faculty of Pharmacy, University of Sydney, Sydney, Australia The global fight against cancer from all fronts is age long, though it has been slowly and steadily, especially before our modern era when it is becoming more refined. Despite the obvious advances on record cancer has remained a global health burden. The fact that 13.1 million humans are likely going to die by 2030 as a result of the disease as predicted by WHO is perplexing considering our present endeavors. Our current tasks are fully linked to the emerging molecular information, which sometimes takes a while to be uniformly accepted, still remains dare to drug discovery and development, being that chemotherapy plays a vital part to the overall cancer effective management. The epidermal growth factor receptor is one singular cell surface or single transmembrane receptor, among others, that has received, hitherto, adequate research attention since the discovery of its ligand(epidermal growth factor, EGF) over 50 years ago. The fair achievements recorded thus far in the treatment of certain recalcitrant cancer types, except for the evolution of the drug resistance in addition to side effects associated with drugs that

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target the receptor and its ligand, is appreciative. The roles of the EGFRs in normal and neurodegenerative states are not only well reported in many reviews but also their elaborate implication in several types of cancer too. EGFRs, in normal physiological disposition, significantly express their title role in the proliferation, migration, apoptosis and cell growth in the absence of any aberration. Dimerization, which has been bountifully defined as the formation of a functional protein complex of two units, is an essential and well documented initial step for the subsequent cascades that follow when the ligand(EGF, TGF-a, etc.) is bound to EGFR. At the moment only a few of the anticancer agents inhibiting dimerization are being investigated. The need for innovative approaches leading to novel developments stand out to be a need without wavering. We report here that, using the Schrodinger Suite, not only the dimerization region of a dimer EGFR has been exposed through site-mapping for drug targeting but also the monomeric arm had been docked with ligands generated from the curated ZINC Chemical Database. Close to 3 million compounds from the ZINC and Ambinter Chemical Databases were screened using the Suite and the protocols defined. The potentially 11 active compounds that emerged were not just based on ‘good’ Gscores but a few other drug parameters had to be carefully considered using the same Suite. Indeed, there are known tyrosine kinase inhibitors that act within the cell cytosol which are known to inhibit the cascades at some stages leading up to terminating the physiological part initiated by the EGFREGF complex, but far fewer have noted to prevent dimerization through the monomeric arm. These potentially active compounds are currently undergoing further work.

P139. A POTENT MOLECULAR PATHWAY INHIBITOR THAT INHIBITS CANCER CELL PROLIFERATION John Cashman1, Karl Okolotowicz1, Mary Dwyer1, Sigeng Chen1 and Mark Mercola2 1 Human BioMolecular Research Institute, San Diego, CA, 2Sanford Burnham Research Institute, La Jolla, CA The need for effective anti-cancer agents in the United States is immense. The situation world-wide is similar. The goal of our work is to characterize and refine small molecules that suppress colon, prostate, breast and pancreatic cancer progression. The long term goal of this work is to develop a drug that is useful to stop human cancer. The hypothesis is that the compounds work at a critical signaling pathway site common to a number of cancer cells to inhibit cancer cell proliferation. The lead compounds potently increased apoptosis in human colon, prostate, breast and pancreatic cancer cell lines. The lead compound also decreased proliferation and viability of colon, prostate, breast and pancreatic cancer cancer cell lines in vitro. However, the lead compounds do not indiscriminately kill cancer cells. Xenograft studies employing either a colon or a prostate cancer cell line showed that the lead compound inhibited tumor growth 480% as compared to vehicle-treated mice. The lead compounds are non-toxic, pharmacologically suitable, drug-like compounds with excellent physiochemical properties. Acute administration of the lead compound (200 mg/kg) or chronic administration (20 mg/kg 28 days) did not show any abnormal serum clinical chemical values or abnormal pathology. Pharmacokinetic studies showed that the lead compounds possessed reasonable bioavailability. It appears the lead compounds are acting selectively on dividing tumor cells and not on normal or non-dividing cells and this may at least in part explain the lack of toxicity of this class of compound. Based on our extensive in vitro and in vivo preliminary data using colon, prostate, breast and pancreatic cancers, we have strong support that the lead compound will likely inhibit proliferation of other cancer types.

P140. IN VITRO CHARACTERIZATION OF DP44MT AND BP4ET METABOLIC PRODUCTS Eliska Mackova - Potuckova1, Jaroslav Roh1, Jan Stariat1, Vit Sestak1, Pavlina Haskova1, Petra Kovarikova1, Des R. Richardson2 and Tomas Simunek1 1 Charles University in Prague, Faculty of Pharmacy, Hradec Kralove, Czech Republic, 2Iron Metabolism and Chelation Program, Bosch Institute and Department of Pathology, University of Sydney, Sydney, Australia Di-2-pyridyl ketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and 2-benzoylpirydine 4-ethyl-3-thiosemicarbazone (Bp4eT) are potent thiosemicarbazone iron and copper chelators. Their antineoplastic activities were described by in vitro studies on cellular models, and in case of Dp44mT it was confirmed by in vivo studies on tumor xerografts in nude mice. Recently, the metabolites of Dp44mT and Bp4eT chelators were identified after their incubation with microsomal fractions from human and rat livers. The same metabolic products were also found in rat plasma, urine and feces after i.v. administration of each chelator to rats [1]. Dp44mT was demethylated to di-2-pyridyl ketone 4-methyl-3-thiosemicarbazone (Dp4mT) and cleaved to basic di-2pyridyl ketone. Bp4eT was oxidized to 2-benzoylpirydine 4-ethyl-3-semicarbazone and to N3-ethyl-N1-[phenyl(pyridin-2yl)methylene] formamidrazone. The aim of this study was to characterize the antiproliferative activities of Dp44mT and Bp4eT metabolites on both cancer and non-cancerous cell lines and to estimate their abilities to mobilize iron from cells. Antiproliferative activities were studied on HL-60 human promyelocytic leukemia cell line, MCF-7 human breast adenocarcinoma cells, A549 human lung adenocarcinoma cells and HCT116 human colon carcinoma cells after 72 hour incubations. Toxicity of the metabolites was studied on H9c2 neonatal rat-derived cardiomyoblasts and 3T3 mouse embryo fibroblasts after the same 72 hour long incubation. All proliferation and toxicity studies were evaluated by MTT assay. The 59Fe mobilization and the prevention of 59Fe uptake from 59Fe labeled human holotransferin were performed on MCF-7 cells. All the

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studied metabolites showed at least 100 times lower antiproliferative activities towards all studied cancer cell lines and also decreased toxicity towards both non-cancerous cell lines than their parent substances. With the exception of Dp44mT all the metabolites almost lost their 59Fe mobilizing activity and at the same time they almost did not prevent the 59Fe uptake from 59Fe labeled holotransferin. Hence, this study demonstrates that iron and copper chelators Dp44mT and Bp4eT are metabolized to non-toxic products or to less biological active substances than the parent chelators. This study was supported by the Charles University (grant 299511) and Internal Grant Agency of the Ministry of Health of the Czech Republic (project No. NT 12403-3/2011).

Reference


[1] Stariat J, Sestak V, Vavrova K, Nobilis M, Kollarova Z, Klimes J, Kalinowski DS, Richardson DR, Kovarikova P. LC-MS/ MS identification of the principal in vitro and in vivo phase I metabolites of the novel thiosemicarbazone anti-cancer drug, Bp4eT. Anal Bioanal Chem. 2012; 403(1):309–21.

P141. NOVEL ZINC PHTHALOCYANINES WITH HIGH ANTITUMOR PHOTODYNAMIC ACTIVITY AND LOW DARK TOXICITY Miloslav Machacek1, Saad Makhseed2, Waleed Alfadly2, Antonin Cidlina3, Veronika Novakova4, Petr Zimcik3, Pavlina Haskova1 and Tomas Simunek1 1 Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy, Hradec Kra´love´, Czech Republic, 2 Department of Chemistry, Kuwait University, Kuwait, 3Department of Pharmaceutical Chemistry and Drug Control, Charles University in Prague, Faculty of Pharmacy, Hradec Kra´love´, Czech Republic, 4Department of Biophysics and Physical Chemistry, Charles University in Prague, Faculty of Pharmacy, Hradec Kra´love´, Czech Republic Photodynamic therapy is relatively novel approach in the treatment of localized tumors using specific compounds photosensitizers. This technique includes two distinct steps: uptake of the compound into malignant cells with subsequent irradiation by activating light. Photosensitizer produces cytotoxic and highly reactive oxygen species (ROS) damaging and destroying the cancer cells through damage of important macromolecules. Additionally, the generated ROS may trigger the activation of signal pathways leading to induction of apoptosis. Several phthalocyanine (Pc) photosensitizers were studied in this work. In general, Pcs are often hydrophobic compounds with poor solubility in aqueous media. However, all studied compounds bear cationic peripheral or non-peripheral substituents that render them highly hydrophilic. Mouse fibroblasts cell line (3T3) was used to evaluate inherent toxicity of studied Pcs in dark. All compounds showed low dark toxicities with TC50 values in »104 M range. Photodynamic treatment experiments were performed mainly on human cervix carcinoma cell line (HeLa), but melanoma (SK-MEL-28) and colorectal carcinoma (HCT 116) cell lines were also used to establish the ability of compounds to damage different tumor cell lines. Irradiation of the cells with red light (4570 nm, 12.4 mW/cm2, 11.2 J/cm2) induced strong photodynamic effect with EC50 values in 108 M range. Epifluorescence microscopy was used for analyses of morphological changes, changes in mitochondrial inner-membrane potential and apoptosis/necrosis during experiment. Cell shrinkage and disintegration that starts even during irradiation itself was observed. Further investigations will focus on particular cell death pathways, intracellular ROS production and localization of compounds within cellular compartments. Study was supported by the Czech Science Foundation (grant No. 13-27761S).

P142. PRECLINICAL METABOLISM AND PHARMACOKINETICS OF BENTYSREPININE (Y101), A NOVEL ANTI-HBV AGENT Huirong Fan1, Yuanyuan Xia1, Xiaoyan Ci1, Shiqi Dong2, Yong Zeng1, Quansheng Li1, Xiulin Yi1, Guangli Wei1, Yazhuo Li1, Duanyun Si1, Guangyi Liang3 and Chang-xiao Liu1 1 State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, China, 2Yantai University, China, 3The Key Laboratory of Chemistry for Natural Products of Guizhou Province, Chinese Academy of Sciences Bentysrepinine (Y101), a novel phenyalanine dipeptide compound, is now an investigated new drug (IND) for treatment of patients with Hepatitis B virus (HBV)-infected hepatitis in China. As pharmacokinetic and metabolic properties play a key role in predicting dosing regimens for clinical studies and avoiding failure in the clinical development phase. The preclinical pharmacokinetics and metabolism of Y101 were therefore performed to investigate the drug-like characteristics of Y101. According to an LC/MS/MS assay developed and validated in our laboratory [1], the concentration of Y101 in rat plasma, tissues, urine, feces, bile and liver microsomes were determined. The resulting pharmacokinetic parameters exhibited favorable pharmacokinetic properties of Y101, for example, rapid absorption (1–2 h), extensive distribution (90–150 L/kg), higher systemic clearance (10–20 L/h/kg), and moderate oral bioavailability (46.0%) in rats. Extensive tissue distribution in rats and

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higher levels in target organ (liver) were associated with potent efficacy and low toxicity. Less than 15.0% of Y101 were excreted from urine, feces and plasma in rats, and the doses recovered were 0.823%, 14.0% and 2.83%, respectively. A quadrupole linear ion trap mass spectrometer system (QTRAP LC/MS/MS) was applied to identify the metabolites in bile, urine, feces and plasma after a single oral administration of 100 mg kg1to rats. Of 33 metabolites detected, 4 were identified by comparing the chromatographic and mass spectrometric behaviors with the authentic standards. The predominant route of metabolism was believed to be hydrolysis of amide to form M8. The other routes of metabolism involved might be hydroxylation, N-demethylation, dehydrogenation, N-oxydization and further converted to glucuronide and glucose conjugates. Enzyme phenotyping were characterized using human liver microsomes and recombinant human cytochrome P450s (CYPs). Multiple CYPs (CYP3A4, CYP1A2 and CYP2D6) that were involved in the metabolism of Y101 exhibited a limited potential for drug-drug interactions. After incubation with five major recombinant human CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4) in the presence of their specific probe substrates, inhibition by Y101 were not observed (IC50420 mM). Additionally, induction assay by Y101 demonstrated that Y101 was not able to induce CYP1A, 2B6 and CYP3A4 in human hepatocytes. Consequently, overall drug-drug interaction potential remained at low level, since Y101 was neither an inhibitor nor an inducer. In summary, favorable preclinical metabolism and pharmacokinetic properties of Y101 supported its clinical development as an IND.

Reference [1] Fan H, Li R, Gu Y, Si D, Liu C. Quantitation of bentysrepinine (Y101) in rat plasma by liquid chromatography tandem mass spectrometry: application to pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci. 2012:15(889– 890):103–109.

P143. PHARMACOKINETICS, TISSUE DISTRIBUTION, METABOLISM AND EXCRETION OF ONO-8815, A PROSTAGLANDIN E2 RECEPTOR TYPE-2 SELECTIVE AGONIST, IN RATS Tetsu Kondo1, Haruo Imawaka2, Kazuki Nakayama1, Kimio Shibakawa1, Junji Komaba1, Ichiro Matsumoto1 and Mikio Ogawa1 1 Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., Ibaraki, Japan, 2Research Promotion, Ono Pharmaceutical Co., Ltd., Osaka, Japan Prostaglandin E2 receptors have been classified into four subtypes, EP1, EP2, EP3, and EP4, which have been studied as potential drug targets. EP2 receptors are widely distributed throughout the body and have important roles in vasorelaxation, bronchodilation, suppression of spontaneous uterine activity, neuroprotection, and bone development. ONO-8815Ly (the lysine salt of ONO-8815) is identified as an EP2 selective agonist (EC50 for mEP2: 2.8 nmol/L). The Phase I clinical studies have demonstrated that ONO-8815Ly markedly inhibits uterine contractions in non-pregnant women and reduces the uterine response to oxytocin injections. A recent study with a rabbit model of traumatic articular cartilage degeneration reported that ONO8815Ly prevents the cartilage degradation in the knees. Based on these findings, we consider that EP2 can be a potential drug target. In drug development, the ICH-M3 (R2) guideline requires the information on pharmacokinetics (absorption, distribution, metabolism, and excretion; i.e., ADME). We utilized the [3H]-labeled ONO-8815 since radio-labeled compounds are particularly useful to obtain metabolite profiles. The purposes of this study were to clarify the ADME of the radioactivity after intravenous administration of [3H]ONO-8815 to rats, to investigate the metabolism prolife using rat hepatocytes, and to compare the gender differences in the ADME characteristics. Following single intravenous administration of [3H]ONO-8815 at 10 mg/kg to male rats, the radioactivity concentrations in the plasma declined with the half-life of 8.2 h. The radioactivity distributed in most of the tissues was eliminated in parallel with that in the plasma. At 24 h postdose, the radioactivity concentrations in all the tissues decreased to 3% or less of respective maximum. The biliary excretion of radioactivity was 54%, which was measured up to 24 h postdose. No gender differences were observed in the comparison of PK profiles between male and female rats. The radio-HPLC analyses detected 18 metabolite peaks but not that of unchanged [3H]ONO-8815 in urine and bile of male rats. The main metabolite peaks in male rat hepatocytes were very similar to those in females. These findings suggest no gender differences in the metabolism. However, gender difference was observed in the excretion study, which was analyzed in the urine and feces up to 120 h after intravenous administration of [3H]ONO-8815. In males, urinary excretion was 42% and fecal excretion was 57%. On the other hand, in females, the urinary excretion was higher by about 15% of the dose than that in males whereas the fecal excretion was lower than that in males. This gender differences in the excretion are considered to be attributed to the excretion processes of the metabolites into urine.

P144. BILE DUCT-CANNULATED RATS IN MASS BALANCE EXCRETION STUDIES AND METABOLITE PROFILING Helen L. Shen1, Bruce Aungst2, Sarah Patterson2, Yvette Warner2, Jaclyn D’Elia-Eckbold2, Alfred Lordi2 and Zamas Lam2 1 QPS, LLC, Newark, DE, 2Dmpk, QPS, LLC, Newark, DE


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Bile duct cannulated (BDC) rats are often used in mass balance excretion (AME) studies, and the bile, urine, feces, and sometimes plasma samples are subsequently used for metabolite profiling and identification. The objective of this study was to review 2013 in-house mass balance excretion studies data from Sprague Dawley (SD) and Wistar Han (WH) rats to compare the WH vs. SD BDC model based on bile flow and collection volumes. SD BDC rats have been widely used in AME studies, but in recent years there has been a trend toward WH BDC rats. In our experience the WH model is less robust than the SD model with respect to tolerating surgical alteration and requires longer post-surgical recovery time. This study compares results from 33 studies (using 95 WH rats in 21 studies and 86 SD rats in 12 studies) performed in a 5month period. Both WH and SD rats were supplied by a single vendor and were prepared with an indwelling bile duct cannula, which was used for bile samples collection and for infusion of bile salt replacement solution through the duodenal loop. Most BDC rats received infusion of taurocholate solution during bile collection, while some animals received electrolyte supplement from their drinking water. Radiolabeled test articles were given as an IV bolus or by oral gavage. Bile, urine, and feces were collected on dry ice at pre-dose and up to 72 h post-dose for WH rats and up to 168 h post-dose for SD rats. The sample weights of bile, urine, and feces were recorded. All samples were analyzed for radioactivity, with feces being homogenized and oxidized. DEBRAÔ v6.1 LIMS system was used during the in-life and sample and data processing. The overall radioactivity recovery from SD rats averaged 92.4%. The overall radioactivity recovery in WH rats averaged 90.9%, with two studies having lower recovery possibly due to the position of the radiolabel. The overall mean volume of 24-h interval bile collection was similar in SD and WH rats: 17.0 g (19% CV) from SD rats and 16.1 g (39% CV) from WH rats. This data provides evidence that SD BDC rats are a more robust model for studies lasting longer than 72 hours. For WH BDC rats, because of the observed higher variability, it would be prudent to have spare rats available for mass balance excretion studies. Results compiled from approximately 200 BDC studies (over a 3-year period) with both rat strains indicated that for approximately two-thirds of the compounds tested, more than 40% of the recovered radioactivity was eliminated in the bile. Maintenance of physiologic bile flow is therefore a critical component of rodent excretion mass balance and metabolite profiling investigations.

P145. EX VIVO PROTEIN BINDING IN HEALTHY DONORS AND DISEASED PATIENTS, AND COMPARISON OF EX VIVO AND IN VITRO RESULTS Helen L. Shen1, Wenbin Liu2, Lata Venkatarangan3, Bruce Aungst2 and Zamas Lam2 1 QPS, Newark, DE, 2Dmpk, QPS, Newark, DE, 3DMPK-Hepatic Biosciences, QPS, Research Triangle Park, NC The objective of this study is to compare ex vivo plasma protein binding in healthy volunteers and in patients from various disease states. Plasma protein binding is one of the ADME properties of drug candidates characterized during the drug discovery and development process. The protein binding data are generally derived from in vitro experiments during preclinical development and not until Phase II/III clinical trials are ex vivo data generated when the effects of disease state on pharmacokinetics are investigated. In this study plasma samples were collected from various clinical studies and were stored at 70 C. Protein binding for more than 15 NCEs and marketed drugs as well as their metabolites was determined using equilibrium dialysis (ED), rapid equilibrium dialysis (RED), ultrafiltration (UF), or ultracentrifugation (UC). Liquid scintillation counting or LC-MS/MS was used for bioanalysis. The effect of renal impairment on plasma protein binding was examined for seven compounds (with eight metabolites for three of the compounds). No difference was observed between renal impaired and healthy subjects for the compounds or their metabolites. Similarly, the effect of hepatic impairment on plasma protein binding was examined for five compounds, and no significant difference was observed. An effect of disease on protein binding was observed for some compounds in cancer patients, most likely due to change of alpha acid glycoprotein level in these patients. To evaluate method variability during ex vivo protein binding determination, blank plasma spiked with the test compound was prepared for in vitro protein binding assessment in parallel. As a secondary objective, difference in protein binding determined in vitro vs. ex vivo was evaluated. Of the eleven compounds investigated, we found five compounds and five metabolites derived from two of the compounds showed higher free fraction in vitro than ex vivo with two fold or more differences. These examples clearly demonstrated that matching healthy normal control in the same study is necessary for an appropriate comparison of plasma protein binding change due to disease state. The reason for lower binding in vitro than ex vivo in plasma from healthy subjects is not clear, as factors such as concentrations of the drug or metabolites and the chemical functionality of the metabolites cannot adequately explain the observed differences in binding.

P146. PD(II)-CATALYZED DECARBOXYLATIVE ACYLATION OF PHENYLACETAMIDES WITH A´-OXOCARBOXYLIC ACIDS VIA C–H BOND ACTIVATION AND APPLICATION TO THE SYNTHESIS OF 3-ISOCHROMANONE Jihye Park1, Mirim Kim1, Minyoung Kim2 and In Su Kim1 1 School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, South Korea, 2Sungkyunkwan University, Suwon, Gyeonggi-do, South Korea


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The development of new carbon-carbon bond-forming reactions continues to be an essential goal in organic chemistry. Traditional metal-catalyzed cross-coupling reactions between aryl metal reagents and aryl halides are well-established methods for the construction of C–C bonds and synthesis of complex molecules. Recently, transition-metal-catalyzed decarboxylative cross-coupling reactions using aryl carboxylic acids as aryl surrogates have received much attention since such transformations provide new opportunities to use readily available carboxylic acids as starting materials for organic synthesis. Transition-metal-catalyzed oxidative acylation of sp2C–H bonds in aromatic compounds with various directing groups, e.g., pyridines, oximes, acetanilides, and indole, with aldehydes or alcohols were reported. However, decarboxylative C–H bond acylations using a-oxocarboxylic acids as acyl surrogates were relatively unexplored. Goossen first demonstrated a palladium-catalyzed decarboxylative crosscoupling reaction of aryl bromides with a-keto carboxylate salts as acyl anion equivalents to afford diaryl ketones. Ge described elegant studies on a palladium-catalyzed decarboxylative acylation of acetanilides and phenylpyridines with a-oxocarboxylic acids as acyl sources via C–H bond activation. Recently, Guo and Duan described a decarboxylative acylation of cyclic enamides with a-oxocarboxylic acids to provide b-acyl enamides. Herein we described our recent result on a Pd-catalyzed decarboxylative ortho-acylation of O-methyl ketoximes and phenylacetamides with a-keto acids via C–H bond activation. This protocol provides an efficient access to a range of ortho-acyl phenylacetamides, which can be easily converted to 3-isochromanone derivatives.

Reference 1. ‘‘Pd(II)-Catalyzed Decarboxylative Acylation of Phenylacetamides with a-Keto Acids via C-H Bond Activation’’ Park, J.; Kim, M.; Sharma, S.; Park, E.; Kim, A.; Lee, S. H.; Kwak, J. H.; Jung, Y. H.; Kim, I. S. Chem. Commun. 2013, 49, 1654–1656.

P147. PHARMACOKINETICS OF A NOVEL DGAT-1(DIACYLGLYCEROL ACYLTRANSFERASE-1) INHIBITOR, KR-69232 Eun-Young Kwak, So Hee Im, Hyewon Seo, Woon-ki Cho, Zhi Zheng, Sung-Hoon Ahn, Jin Sook Song and Myung Ae Bae Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, Daejon, South Korea A novel diacylglycerol acyltransferase1 (DGAT1) inhibitor, 2-(4-(4-(5-(2-phenyl-5-(trifluoromethyl) oxazole-4-carboxamido)1H-benzo[d]imidazol-2-yl) phenyl) cyclohexyl) acetic acid (KR-69232), has been synthesized for the therapeutic effects against several metabolic disorders such as obesity, insulin resistance and type II diabetes mainly caused by excessive triglyceride (TG) in blood. In vitro and in vivo studies were carried out to characterize the metabolism and pharmacokinetics of KR-69232. Our candidate compound exhibited high stability to oxidative and glucuronic metabolism in human and rat liver microsomes. In Caco-2 cell monolayers, low permeability (1.93 106 cm/s) at 50 mM was shown in the absorptive direction. The fraction of the compound was highly bound to plasma proteins more than 99% in both rat and human. Furthermore the concentration in the intestine of KR-69232 was determined as approximately 10-times that in the plasma, demonstrating the low absorption into blood. After intravenous administration in rats, the linear elimination was observed at tested doses (1–5 mg/kg) with low systemic clearance values ranging from 0.0551 to 0.124 L/h/kg. Following oral administration, it exhibited low oral bioavailability (510%) and slow absorption (Tmax, 3.8–5.2 h) over the dose ranges.

P148. EPOXYEICOSATRIENOIC ACIDS PREVENT CISPLATIN-INDUCED RENAL APOPTOSIS THROUGH A P38 MAPK REGULATED INTRINSIC MITOCHONDRIAL PATHWAY Yingmei Liu1, Deanna Kroetz2, Jean Olson3, Heather Webb4, Xiaodan Lu5 and Sinh Nguyen5 1 Bioengineering and Pharmaceutic Science, University of California San Francisco, San Francisco, CA, 2Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, 3Anatomic Pathology, University of California San Francisco, San Francisco, CA, 4Areˆte Therapeutics, Hayward, 5Bioengineering and Therapeutic Sciences, Areˆte Therapeutics, Hayward Soluble epoxide hydrolase (sEH) catalyzes the conversion of epoxyeicosatrienoic acids into less active eicosanoids and inhibitors of sEH have anti-inflammatory and anti-apoptotic properties. Based on previous observations that sEH inhibition attenuates cisplatin-induced nephrotoxicity by modulating NF-kB signaling, we hypothesized that this strategy would also attenuate cisplatin-induced renal apoptosis. Inhibition of sEH with 1-adamantan-1-yl-3-(1-methylsulfonyl-piperidin-4-yl-urea) (AR9273) reduced cisplatin-induced apoptosis through mechanisms involving mitochondrial apoptotic pathways. Renal mitochondrial Bax induction following cisplatin treatment was significantly decreased by treatment of mice with AR9273 and these anti-apoptotic effects involved p38 MAPK signaling. Moreover, in LLC-PK1 cells, excessive reactive oxygen species (ROS) is associated to cells apoptosis. Cisplatin-induced mitochondrial trafficking of Bax and cytochrome c as well as caspase activation are

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significantly attenuated in the presence of EETs. Collectively, these in vivo and in vitro studies demonstrate a role for EETs in limiting cisplatin-induced renal apoptosis. Inhibition of sEH represents a novel therapeutic strategy for protection against cisplatin-induced renal damage.

P149. IN VIVO DISCOVERY ADME PROPERTIES OF 10 LATE STAGE IRREVERSIBLE INHIBITORS: CAN TRADITIONAL ADME ASSAYS SERVE AS SCREENING GATES?


Ashutosh Kulkarni, Yang Tang, Zhihong O’Brien and Mehran Moghaddam Celgene Corporation, San Diego, CA Purpose: ADME data has always been gating in the drug discovery process. It plays a key role in distinguishing compounds with good drug-like properties from those with poor ones. The resurgence of irreversible inhibitors has been well documented with several companies having very active irreversible inhibitor programs. To implement effective screening paradigms for irreversible inhibitors in discovery DMPK and define passing criteria for DMPK parameters, we evaluated the in vivo properties of 10 late stage or marketed irreversible inhibitors using our traditional DMPK assays. Methods: The 10 irreversible inhibitors included Abiraterone, Afatinib, Boceprevir, Canertinib, Carfilzomib, Dacomitinib, Ibrutinib, Nertinib, Telaprevir, and Vigabatrin. Most of these are orally administered except Carfilzomib which is administered as an IV infusion. We put these 10 compounds through our traditional in vivo discovery screening assays used for screening reversible inhibitors. These comprised of rat pharmacokinetic studies following intravenous dosing at 2 mpk dose to evaluate parameters such as plasma clearance (CL), volume of distribution (Vss), mean residence time (MRT), blood: plasma ratio (BP ratio), and oral dosing at 10 mpk dose to evaluate parameters such as maximal plasma concentration (Cmax), time to reach Cmax (Tmax), and oral bioavailability (F%). The data was analyzed for any potential trends that would assist in defining passing criteria for future compound progression. Results: Plasma clearance for the 10 compounds ranged from slow (11 mL/min/kg) to very rapid (350 mL/min/kg). The Vss ranged from low (0.67 L/kg) to very high (115 L/kg) with most of the rapidly clearing compounds exhibiting high Vss but BP ratios close to 1, suggesting rapid and extensive tissue distribution. MRT’s also ranged from short (0.5 hr) to long (7.4 hr). The oral exposure of these 10 compounds also showed a very broad range with Cmax ranging from 0.01–77 uM and AUC0–inf ranging from 0.03–106 uM*hr. The oral bioavailabilities ranged from 0.23–91%. Conclusion: The large range in in vivo ADME properties of these 10 late stage inhibitors, which have progressed beyond early clinical safety and efficacy criteria, suggest that traditional DMPK screening assays (to measure IV CL, Vss, MRT’s, AUC’s, F% etc) cannot be used in the discovery screening mode for irreversible inhibitors because they won’t help you in effectively screening out poor candidates. Non-traditional assays such as mass modification, protein digests, kinetics of enzyme inhibition and target occupancy, and target turnover in combination with the PK profiles are important considerations for compound progression.

P150. IN VITRO DISCOVERY ADME PROPERTIES OF 10 LATE STAGE IRREVERSIBLE INHIBITORS: CAN TRADITIONAL ADME ASSAYS SERVE AS SCREENING GATES? Ashutosh Kulkarni, Samantha Richardson, April Bai, Yang Tang, Zhihong O’Brien and Mehran Moghaddam Celgene Corporation, San Diego, CA Purpose: ADME data has always been gating in the drug discovery process. It plays a key role in distinguishing compounds with good drug-like properties from those with poor ones. The resurgence of irreversible inhibitors has been well documented with several companies having very active irreversible inhibitor programs. We evaluated the in vitrodiscovery ADME properties of 10 late stage or marketed irreversible inhibitors in order to define passing criteria for compound advancement. Methods: The 10 irreversible inhibitors included Abiraterone, Afatinib, Boceprevir, Canertinib, Carfilzomib, Dacomitinib, Ibrutinib, Nertinib, Telaprevir, and Vigabatrin. Most of these are orally administered except Carfilzomib which is administered as an IV infusion. We put these 10 compounds through our traditional in vitrodiscovery screening assays used for screening reversible inhibitors. These comprised of: rat and human liver S9 stability, rat and human blood stability, CYP3A4 inhibition assay (including timedependant inhibition), Caco-2 permeability assay, and rat and human plasma protein binding assays. We analyzed the data for any potential trends that would assist in defining passing criteria for future compound progression. Results: The rat and human S9 stability for the compounds ranged from 1% to 100%. The blood stability ranged from 30% to 100%. The CYP3A4 IC50 ranged from 0.5 uM to 410 uM with most compounds showing moderate shifts in the IC50 values in the presence of NADPH compared to those in the absence of NADPH. The Caco-2 permeability (Aa`B) ranged from negligible (0.6 106 cm/s) to highly permeable (31 106 cm/s) and so did the efflux ratio (0.2–30). Most of the compounds were highly protein bound in both rat and human with binding 90%. Conclusion: The large range in in vitro ADME properties of these 10 late stage inhibitors, which have progressed beyond early clinical safety and efficacy criteria, suggest that traditional in vitro ADME assays cannot be used in discovery as high throughput screening tools for decision making on compound progression for irreversible inhibitors because they won’t help you in effectively screening out poor candidates. Non-traditional assays such as mass modification, protein

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digests, kinetics of enzyme inhibition and target occupancy, and target turnover in combination with the PK profiles are important considerations for compound progression.

P151. AUTOMATED MULTIPLE CHARGE STATE DETECTION OF PHASE II GLUTATHIONE CONJUGATES FROM HRMS QTOF DATA


Yun W. Alelyunas, Paul D. Rainville and Mark D. Wrona Pharmaceutical Life Sciences, Waters Corporation, Milford, MA The relationship between reactive metabolites and adverse toxic effects has made reactive metabolite screening a necessary and valuable component of the drug discovery process. The observation of adducts often trigger more complicated studies to either assess the risks associated with observed GSH conjugation levels, or to entirely tune these properties out through lead optimization efforts. While ultimately, radio-labeled experiments represent a gold standard approach, refinements in HRMS and triple quadrupole screening techniques have enabled the use of ‘cold’ studies as an increasingly common DMPK practice. Several popular LC-MS approaches for detecting glutathione conjugates exist, including background subtraction, mass defect filtering, detecting signature GSH neutral loss fragment such as 129, or ESI- precursor scanning for the 272 diagnostic product ion. Current methodologies have shown great success in detecting glutathione conjugates, but software that combines this information, generically mines, deconvolutes the spectral data and tracks this information across samples is still lacking. Relating observations across a variety of samples and incubation conditions allows for more confidence in detection of reactive metabolites versus single control/analyte comparisons. In this presentation, we demonstrate GSH adduct detection using Xevo G2-S QTof and UNIFI software for both acquisition and automated processing of HRMS data. The test compounds were incubated using human and rat liver microsomes in the absence or presence of glutathione over a 2 hour time course. After protein precipitation and centrifugation, the supernatant was analyzed on a ACQUITY UPLC-Xevo G2-S QTof platform using a C18 BEH 1.7 u 2.1 100 mm column under generic gradient conditions. The MSE approach of ‘‘all the data, all the time’’ and the sensitivity afforded by QTof mass spectrometer together provide a complete record of precursors and their fragmentation patterns of all samples in the study. Using compounds including clozapine, diclofenac and carbamazapine, both precursors and metabolites from both phase I and phase II biotransformation were characterized. Tracking and plotting all of these components was automated within the software. Automatically grouping all detected charge states is essential since glutathione adducts commonly exist in up to three charge states and often have little to no residual M þ Hþ peak present. The utility of performing multiple fragment or neutral loss searches through post-acquisition data mining of the MSE data to facilitate detection of even very small reactive metabolites early in the time course will also be discussed. Embedded graphing capabilities then allow the user to view concentration changes over time for each metabolite, multiple components and across species. Reviewing all drug-related components in the incubations, and understanding their changes over time and across species provide a more complete picture for positive identification of both phase I and phase II metabolites and their rates of formation and disappearance.

P152. CHARACTERIZATION OF THE ABSORPTION, METABOLISM, AND EXCRETION OF GDC-0068, AN ATP-COMPETITIVE AKT INHIBITOR, FOLLOWING SINGLE ORAL ADMINISTRATION TO CYNOMOLGUS MONKEYS Ryan Takahashi, Shuguang Ma, Cyrus Khojasteh and Bianca M. Liederer Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA GDC-0068 is a potent small molecule ATP-competitive Akt inhibitor that is being developed for the treatment of solid tumors as a single agent and in combination therapies, currently in Phase I testing. In the current study, the absorption, metabolism, and excretion of GDC-0068 were characterized in male and female cynomolgus monkeys following a single oral dose of 5 mg/kg [14C]GDC-0068 (20 mCi/kg). Based on the recoveries in bile (45% of the dose) and urine (7–13% of the dose), at least 52% of the administered radioactive dose was absorbed. From the intact monkeys, an average of 86–92% of the administered radioactivity was recovered with the majority (78–80% of the dose) eliminated in the first 72-hours following dosing. GDC-0068 was extensively metabolized through primarily oxidative pathways prior to elimination with low amounts eliminated as unchanged drug. The metabolic fate for the drug was determined by profiling the metabolites in plasma and excreta samples by HPLC with radiometric detection coupled with a LTQ-Orbitrap mass spectrometer. Metabolite structure elucidation was based on full scan and product ion spectral data that was acquired simultaneous with the sample radioprofiling. In circulation, unchanged GDC-0068 was the major component. Rapid metabolism was reflected by theCmax plasma sample containing metabolites generated by N-dealkylation, oxidation, and isomerization. In addition to the metabolic pathways reflected by circulating metabolites, GDC-0068 underwent additional oxidation and sulfate conjugation before being excreted. The results of these studies provided the quantitative assessment of the pharmacokinetics,

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metabolic fate, and excretion routes for GDC-0068 in monkeys, and allowed comparison of exposures for human circulating metabolites to this toxicological species.

P153. SYSTEMIC EXPOSURE TO THE METABOLITES OF LESOGABERAN IN HUMANS AND ANIMALS- A CASE STUDY OF METABOLITES IN SAFETY TESTING


Ann Aurell Holmberg, Anja Ekdahl and Lars Weidolf Dmpk, AstraZeneca, Mo¨lndal, Sweden During preclinical and early-phase clinical studies of drug candidates, exposure to metabolites should be monitored in order to assess whether safety conclusions drawn from studies in animals can be extrapolated to humans (ICH, 2009; ICH, 2012). Metabolites in humans accounting for more than 10% of exposure to total drug-related material (DRM) are of regulatory concern, and for any such metabolites adequate exposure should be demonstrated in animals before large-scale phase 3 clinical trials are conducted. We have previously identified six metabolites, M1–M6, of the gastroesophageal reflux inhibitor lesogaberan (Ekdahl et al., 2012). Here, we present an assessment of the exposure of humans, rats and beagle dogs to parent compound and these six metabolites after lesogaberan dosing. Plasma samples were taken at various time points after lesogaberan administration in two clinical and three preclinical studies. Concentrations of lesogaberan and its metabolites were measured, and exposures during a single dosing interval were calculated. Systemic total DRM in humans was shown to comprise parent compound and metabolites M1, M2, M4 and M5. Only M4 and M5 were present at levels of regulatory concern (10.6% and 18.9% of exposure to total DRM, respectively, at steady state). For M5, the highest absolute exposure observed in humans at steady state (52 mmol h/L) was exceeded in rat toxicology studies (117 mmol h/L). Regulatory recommendations for adequate exposure in animals were therefore met for this metabolite. In contrast, exposures to M4 in the animal species tested were less than 50% of the highest absolute exposure observed in humans. Thus, further safety testing of this metabolite may be required to support continued lesogaberan clinical development.

References Ekdahl A, Aurell-Holmberg A, Castagnoli N, Jr. Identification of the metabolites of lesogaberan using linear trap quadrupole orbitrap mass spectrometry and hydrophilic interaction liquid chromatography. Xenobiotica2012:doi:10.3109/ 00498254.2012.725486. ICH. Topic M3(R2): non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals. 2009. Available from:http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/ 09/WC500002720.pdf (Accessed 23 May 2013). ICH. M3(R2) Implementation Working Group, M3(R2) guideline: guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals questions & answers (R2). 2012. Available from: http:// www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Multidisciplinary/M3_R2/Q_As/M3_R2_Q_A_R2_Step4. pdf (Accessed 23 May 2013).

P154. PREDICTION OF HUMAN PHARMACOKINETICS OF EPITINIB FROM PRECLINICAL PHARMACOKINETICS BY A PHYSIOLOGICALLY BASED PHARMACOKINETIC MODEL Hongcan Ren, Yi Gu, Jian Wang, Liang Ni, Weijun Zhang, Li Zhang, Xianli Tang, Dongxia Gu, Jingjing Zhao, Xiaoyan Xu, Haibin Yang, Weihan Zhang, Weiguo Su and Yang Sai Hutchison MediPharma Limited, Shanghai, China Epitinib is a potent and selective inhibitor of Epidermal Growth Factor Receptor (EGFR). The objectives of these studies were to predict its human pharmacokinetics using a physiologically based pharmacokinetic (PBPK) approach. Methods: To support the modeling process, preclinical in vitro and in vivo PK of Epitinib was investigated systematically, with the details to be reported elsewhere. Solubility and LopP of Epitinib were determined. Other compound-related parameters are all generated by the ADMET Predictor Module or default value in Gastroplus 8.0. Then, an Advanced Compartment Absorption and Transit (ACAT) model and dissolution model, coupling to a whole-body PBPK model, was developed by using Gastroplus 8.0, assuming liver is main metabolism organ. Preclinical in vitro PK data and Kp values (AUCtissue/AUCplasma) were integrated into the PBPK model for PK prediction by in vitro-in vivoextrapolation approach in three preclinical species (ICR moues, SD rat, and Beagle dog). The predicted PK profile and related PK parameters were compared with actual PK data for in vitro-in vivo correlation analysis, and part validation of the model. Finally, the partially-validated PBPK model was used for the prediction of human PK and further validated by the observed human PK profiles. Results: The pH-dependent solubility of Epitinib is from 0.000046 to 41.6 mg/mL at pH 8 1.2. The actual LogP is 1.31. The preclinical PK information of Epitinib used for the modeling is followed: the permeability Papp (A!B) is 4 106 cm/s (efflux ratio ¼ 4.0); plasma protein binding of Epitinib is 95%, 93%, 90%, 90% in nude mouse, rat, dog, and human (up to 10 mM); the Phase I metabolic t1/2 in liver microsomes is 38.5, 25.5, 10.8 and

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98.4 min in mouse, rat, dog and human respectively; in rat excretion study, parent Epitinib only contributed to 12% of the dose and minimal Phase II conjugates were found, supporting the modeling assumption that hepatic phase I metabolism is the major elimination pathway for Epitinib; the actually determined Kp value in rats is 7.1, 2.2, 29.5, 8.6, 31.4, 4.7, 1.2, 19.6, 17.4, and 1.1 in adipose, muscle, liver, gut, spleen, heart, brain, kidney, skin and marrow; the Kp values for species other than rats are obtained by the GastroPlus prediction with the correction of the corresponding plasma free fractions. The model predicted concentrationtime curves in mouse, rat and dog are well correlated to observed curves. The ratio (model-predicted exposure/actual exposure) is from 0.3 to 1.6 after intravenous or oral administration in those preclinical species. Other predicted parameters including clearance, Vss, exposure and bioavailability, keep accordance with the actual data in preclinical species. So, this model is reliable and could be reasonably extrapolated to human. The predicted exposure is about 0.7 fold compared with actual data after oral administration at 80 mg. Conclusion: An ACAT/dissolution model coupling to a whole-body PBPK model, characterizing the absorption and disposition of Epitinib, is successfully established. The in vitro-in vivo extrapolation approach demonstrates that this model showed good predictability in animals and also predicted the Epitinib PK in human successfully.


P155. OPTIMIZATION AND QUALIFICATION OF ULTRACENTRIFUGATION FOR PROTEIN BINDING DETERMINATION Helen L. Shen1, Wenbin Liu2, Julia Wang2, Brad Yuska2, Bruce Aungst2 and Zamas Lam2 1 Dmpk, QPS, LLC, Newark, DE, 2Dmpk, QPS, Newark, DE Equilibrium dialysis (ED) has been the gold standard for protein binding determination. However, this method has limitations particularly for compounds with very high non-specific binding, plasma instability, or inability to diffuse through the membrane. Ultracentrifugation (UC) may be an alternative for some compounds. The objective of this study is to optimize UC conditions to determine plasma protein binding. UC was performed using a Beckman preparative ultracentrifuge (Model: L8-70 M) with a Beckman L Series rotor (Model: F50L-24 1.5) and 37 C. The effects of centrifugation time and force were investigated by measuring protein concentration in the supernatant and by visual inspection of the separation of the supernatant phase. In vitro plasma protein binding results using UC were compared with other protein binding determination methods using highly-protein bound reference compounds and plasma from various species. Frozen pooled blank human plasma from at least three males and three females was obtained from commercial sources. Ultracentrifugation 280,000 g or 200,000 g were investigated in this study. The measured protein concentration in the UC supernatant (6 hours at 280,000 g) was approximately 0.8% of the initial plasma concentration, whereas approximately 0.4% was found in the dialysate from ED. Visually clear phase separation was produced and at least 5 aliquots (100 mL each) could be removed from each UC plasma sample supernatant for analysis. Warfarin, docetaxel, and other proprietary compounds, including several peptides were used in this study. Warfarin plasma protein binding results were consistent when determined using UC, ED, RED (rapid equilibrium dialysis), and UF (ultrafiltration). The mean percent bound was approximately 98% in rat, monkey, and human plasma, and approximately 94% in mouse and dog plasma. Docetaxel was approximately 88% protein bound in human plasma using UC, ED, or UF. Another small molecule compound was approximately 99% bound in human plasma using UC, ED, or UF methods. The extent of plasma protein binding could not be determined for the peptides investigated using RED method as the peptides were unable to move across the dialysis membrane even in buffer solutions. However, using UC the extent of plasma protein binding could be determined for all compounds, with the percent free from 0.9% to 10%. These results demonstrate the potential advantage of UC method. Ultracentrifugation can be useful for assessing plasma protein binding of compounds with high molecular weight or high non-specific binding. Further investigation is needed for UC method at higher speed and shorter time, which may be an alternative for compounds with marginal stability.

P156. NEURO-PHARMACOKINETICS BASED PREDICTION OF P-GLYCOPROTEIN LIABILITY IN EARLY DRUG DISCOVERY Nageswara Rao Muddana, Vijay Benade, Pankaj Paliwal, Akhil Babu Muppidi, Ramkumar Subramanian, Muddukrishna Chillakur, Vishwottam Kandikere, Gopinadh Bhyrapuneni and Ramakrishna Nirogi Discovery Research, Suven Life Sciences Ltd, Hyderabad, India Recent preclinical studies indicate that several drugs may be substrates of the multidrug resistance transporter P- glycoprotein (P-gp) at the blood-brain barrier (BBB). Drug efflux by P-gp at the BBB represents a major obstacle in the delivery of pharmacological compounds targeted to brain and prediction of this nature of the compound is essential in the early preclinical discovery. Generally P-gp liability of the compounds is assessed by in-vitro assays. Although, these are high throughput assays but the in-vivo translatability is limited. Thus, the application of these methods is limited to rank order the compounds. In the present investigation, unbound concentration approach was used to predict the P-gp liability of test compounds. According to this approach, unbound concentrations of test compounds will be in equilibrium between three compartments, i.e. plasma, brain and CSF. Further this approach was confirmed using brain microdialysis. For this investigation, four structurally diverse compounds were selected, wherein 2 compounds were predicted to have no liability to the efflux transporter and 2 compounds were substrate

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to the P-gp transporter. Plasma, brain and CSF neuropharmacokinetics was carried for these compounds in male Wistar rats and unbound concentrations (fu) in plasma and brain were calculated by correcting for protein binding. Compound-1 and 2 had fu values in equilibrium between plasma, brain and CSF. However, Compound-3 had higher fu in plasma than in brain (5X) and CSF (9X), respectively. Unbound concentrations for compound-4 were 2 fold higher in plasma than in brain and CSF. From neuropharmacokinetic results, it was predicted to have high PgP affinity for compound-3 and moderate affinity for compound-4. In order to confirm this, brain microdialysis was carried out to measure unbound concentrations in brain with and known PgP inhibitor (verapamil). For compound 1 and 2 there was no change in unbound concentrations was observed in presence and absence of verapamil. However, unbound concentrations of compound-3 in brain were increased 14 fold in presence of verapamil. Similarly, unbound concentrations of compound-4 increased in presence of verapamil. Results from this investigation indicate that neuropharmacokinetics can be reliably used for initial screening of transporter liabilities in early preclinical discovery.

P157. FINE ISOTOPIC PATTERN RECOGNITION FOR SMALL MOLECULE IDENTIFICATION


Caroline Ding and Tim Stratton Thermo Fisher Scientific, San Jose, CA Isotopic patterns arising from natural isotopic abundances have been utilized in mass spectrometric measurements since the beginning of mass spectrometry. With recent advancements in mass spectrometry, we can now routinely achieve very high resolution (4100 K) measurements. This increased resolution allows observation of the fine isotopic pattern, the separation of extremely close isobars in typically unresolved isotopic pattern members. This can be a powerful tool to confirm and aid in small molecule identification. The software tool to aid in accurate mass and fine isotopic pattern recognition is just as important as the instrument capability itself. Here we demonstrate the use of very high resolution to extract metabolites of a sulfur containing drug on a Q ExactiveÔ benchtop HR/AM instrument. Samples of omeprazole and metabolites in human urine were collected at 0–3 hr, 3–5 hr and 5–7 hr post dosing. Sample preparation by SPE was followed by analysis by UHPLC-HRAM MS using a Q Exative instrument coupled to an AccelaÔ UHPLC system. Accurate mass data was acquired using alternating full scan MS (70,000 resolution FWHM atm/z 200) and all-ion-fragmentation (AIF) scans from which accurate mass NL triggered precursor ion selected MS2 scans on potential metabolites were obtained. UHPLC separation was achieved with a gradient of ACN and Water with 0.1% on a Hypersil Gold aQ C-18 column (2 100 mm, 1.9 um). Data was analyzed using Compound Discoverer software to identify peaks displaying a one or two sulfur fine isotopic pattern. Analysis of Omeprazole metabolism samples on the Q Exactive served as a benchmarking study to prove the concept of using fine isotopic pattern recognition for small molecule identification. In this example, the A2 isotope was resolved to show both the one 34S two 13C components in the full MS scan. A fine isotopic search of the urine samples was perfomed using a fine isotopic search algorithm. Briefly, the search used the expected mass shift from any A0 to both the A2 of the heavy sulfur and the A2 of the two heavy carbons combined with the expected ratio contribution of both signals to search the full scan data. A small tolerance for the mass (in ppm) and the ratio (as a percentage of A0) were allowed. This search was performed looking for both single sulfur (omeprazole and phase I metabolites) and on two sulfur (sulfate meteabolites) components. The traces were then used to detect or confirm components that matched the pattern. The approach successfully identified multiple major and minor phase I metabolites of omeprazole including multiple oxidations, sulfone reduction, demethylation/dealkylation, and glucuronidations. The two sulfur trace successfully identified both sulfate conjugates of demethylated and oxidative metabolites of omeprazole. In addition, endogenous sulfur containing components were also identified in the searchs (urothione). Fine isotopic pattern recognition can be used as a new orthogonal way for finding and confirming new metabolites. It can also be applied in any small molecule workflows for detection and confirmation of compounds of interest.

P158. EVALUATION OF PHARMACOKINETIC INTERACTION BETWEEN FIMASARTAN AND HYDROCHLOROTHIAZIDE IN RATS Soo Heui Paik1, Won Sik Seo1, Hyuk Joon Choi1, Jae Soo Lim1, Joo Han Lee1, Yong Ha Chi1, Je Hak Kim1 and Beom Soo Shin2 1 Boryung Research Institute, Boryung Pharm. Co. Ltd, Ansan, South Korea, 2College of Pharmacy, Catholic University of Daegu, Daegu, South Korea Objectives: Fimasartan, 2-butyl-5-dimethylaminothiocarbonylmethyl-6-methyl-3-[[20 -(1H tetrazol-5-yl)biphenyl-4-yl]methyl] pyrimidin-4(3H)-one (BR-A-657), is a new angiotensin II receptor blocker exhibiting potent antihypertensive activity, and being developed as a combination with hydrochlorothiazide, a commonly used diuretic. We evaluated the potential pharmacokinetic drug-drug interaction between fimasartan and hydrochlorothiazide in Rats. Methods: We developed an analytical method for the determination of fimasartan and hydrochlorothiazide in rat plasma using liquid chromatography– tandem mass. Three groups of male SD rats were orally administered with fimasartan 3 mg/kg, hydrochlorothiazide 0.63 mg/ kg, or both drugs at the same time. Time-concentrations profiles of fimasartan and hydrochlorothiazide were determined and pharmacokinetic parameters were calculated by non-compartmental analysis.Results: The assay was validated to demonstrate

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the specificity, linearity, recovery, lower limit of quantification (LLOQ), accuracy, precision and stability. The LLOQ was 0.2 ng/mL and 1 ng/mL, and linear over a concentration range from 0.2–500 ng/mL and 1–1000 ng/mL with correlation coefficients 40.999 for fimasartan and hydrochlorothiazide, respectively. The intra- and inter-day assay accuracies were 93.6– 108.0 and 90.8–101.4% for fimasartan and 90.6–109.8 and 97.7–104.4% for hydrochlorothiazide, respectively. The intra- and inter-day precision were 2.4–4.4 and 3.0–13.4% for fimasartan and 1.0–11.9 and 6.0–9.6% for hydrochlorothiazide, respectively. Both drugs were rapidly absorbed following oral administration, and eliminated showing multi-compartmental properties. For fimasartan, mean peak plasma concentrations (Cmax) were 241.6 ng/ml (fimasartan alone) and 209.5 ng/ml (combined with hydrochlorothiazide). Times to reach Cmax (Tmax), were 0.1 h and 0.2 h respectively and the total areas under the curve (AUC0–inf) were 742.3 ng.h/ml and 675.1 ng.h/ml, respectively. For hydrochlorothiazide, Cmax were 50.2 ng/ml (hydrochlorothiazide alone) and 45 ng/ml (combined with fimasartan). Tmax were 2.8 h and 2.0 h respectively and AUC0– infwere 221.5 ng.h/ml and 189.5 ng.h/ml, respectively. Differences between treatment alone and co-treatment were not significant for both fimasartan and hydrochlorothiazide. Conclusion: The validated assay was adequate for investigating the exposure to fimasartan and hydrochlorothiazide in rat plasma. The experimental data demonstrated that pharmacokinetic interaction between fimasartan and hydrochlorothiazide may be clinically insignificant. Based on these results, other preclinical and clinical studies to understand pharmacokinetic and/or pharmacodynamic interaction between two drugs are in progress.

P159. TIME-DEPENDENT BALANCE BETWEEN INDUCING AND INHIBITING EFFECTS OF P-GLYCOPROTEIN AND CYTOCHROME P450 3A Keizo Fukushima1, Shinji Kobuchi2, Yukako Ito3, Kanji Takada3 and Sugioka Nobuyuki1 1 Clinical Pharmacokinetics, Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan, 2Kyoto Pharmaceutical University, Kyoto Pharmaceutical University, Kyoto, Japan, 3Pharmacokinetics, Kyoto Pharmaceutical University, Kyoto, Japan Background: Some inhibitors of P-glycoprotein (Pgp) and cytochrome P450 (CYP) 3A also act as an inducer by repeated treatment, while the inhibition effects would overcome the immunoreactive inductions whenever the inhibitor is administered; subsequently, the induction effects may gradually become superior to the inhibition effects along with the elimination of inhibitor until next administration. However, there is little information about the power balance between inhibition effects and induction effects; the aim of present study was to test the hypothesis that the power balance between inhibition effects and induced activities of Pgp and CYP3A depends on the elapsed time after last administration of initiator (Time-I). Methods: Ritonavir (RTV) was selected as a model compound of initiator, which has both inhibition and induction effects. Rhodamine 123 (Rho) and midazolam (MDZ) was used as a substrate of Pgp and CYP3A, respectively. The induction was performed by oral repeated administration of RTV for 7 days (20 mg/kg/day) and the expressions of Pgp and CYP3A in small intestine and liver after 7-day treatment of RTV were investigated by Western blotting. Rho and MDZ were administered orally or intravenously at four different Time-I (0, 3, 9 and 24 hr) to rats pretreated with RTV for 1 or 7 days. Results and Discussion: Western blot analysis showed the induction effects of 7-day RTV pretreatment on small intestine and liver: hepatic CYP3A, 1.70-; hepatic Pgp, 1.58-; intestinal CYP3A, 1.76-; intestinal Pgp, 3.35-fold inductions compared to that without RTV pretreatment. After oral administration of Rho and MDZ to rats pretreated with RTV for 7 days, the areas under the plasma concentration-time curve of Rho and MDZ were significantly altered depending on Time-I: 1.27-, 0.79-, 0.95-, and 0.11-fold increases over that of the control for Rho at Time-I ¼ 0, 3, 9, and 24 hr, and 3.12-, 1.50-, 1.27-, and 0.17-fold increases over that of the control for MDZ at Time-I ¼ 0, 3, 9, and 24 hr, respectively. The bioavailabilities of Rho and MDZ were remarkably affected also by Time-I: 2.06-, 1.14-, 1.14-, and 0.13-fold increases over that of the control for Rho at Time-I ¼ 0, 3, 9, and 24 hr, and 2.85-, 0.73-, 0.78-, and 0.15-fold increases over that of the control for MDZ at Time-I ¼ 0, 3, 9, and 24 hr, respectively. These results supported our hypothesis and revealed that the elapsed time after last administration of initiator is one of the most important determinants for the balance between inducing and inhibiting effects.

P160. USE OF NATURAL AND SYNTHETIC CANNABINOIDS TO ESTABLISH CANNABINOID RECEPTORS AS NOVEL TARGETS FOR THE TREATMENT OF BREAST CANCER Centdrika Dates1, FeAna Francis Devaraj1, Aleksandra K. Greer1, Stacie M. Bratton1, Sebastian Pyrek1, Tariq Fahmi2, Fabricio Medina-Bolivar3, Paul L. Prather2 and Anna Radominska-Pandya1 1 Dept. of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences (UAMS), Little Rock, AR, 2Dept. of Pharmacology and Toxicology, University of Arkansas for Medical Sciences (UAMS), Little Rock, AR,3Biological Sciences, Arkansas State University, Jonesboro, AR Breast cancer continues to be one of the leading causes of cancer death among women. In spite of significant medical advancements, major limitations still exist: drug resistance, toxicity to normal tissue, adverse side effects, and increased risk of other cancers. Natural, noninvasive, and less toxic treatments are crucial for breast cancer patients. Here, the cytotoxic effects of


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the well-known compounds Tamoxifen (Tam) and its hydroxylated and/or chlorinated derivatives, gambogic acid (GA), and prenylated stilbenoids (transArachidins; tAs), which we have recently identified as ligands for cannabinoid receptors (CBRs), have been studied in two human breast cancer cell lines, MCF-7 and MDA-231. GA was the most cytotoxic of these compounds as demonstrated by WST-1 cell viability assays (IC50 values of 2.33 mM and 0.867 mM in MCF-7 and MDA-231 cells, respectively) and microscopy. Surprisingly, Tam and 4OHTam were found to be equally cytotoxic in ERnegative MDA-231 cells as in ER-positive MCF-7 cells (IC50 values from 32.8–34.7 mM). Studies to determine the mechanism responsible for the anticancer effects observed in MDA-231 cells revealed that these compounds bind to and act at cannabinoid receptors (CBRs) with high affinity (low mM Kis). Competitive binding assays show that each of these compounds binds to cannabinoid receptors 1 and 2 (CB1 and CB2) in the high nanomolar to low micromolar range. 4OHTam had the highest affinity for both CB1 and CB2 with Ki values of 1.4 mM and 0.9 mM, and G-protein activity assays indicated that these compounds act as partial agonists (GA), antagonists (tAs), and inverse agonists (Tam and 4OHTam). Therefore, we hypothesize that the cytotoxicity observed in these cells may be attributed in part to the binding of these drugs to CB1 and/or CB2 causing activation or suppression of downstream genes regulating cell proliferation. The identification of Tam and its derivatives as inverse agonists for CB1 and CB2 could explain the previously reported activity of these compounds in ER-negative cancer cells. Our goal is to fully characterize the cytotoxic effects of these compounds, assess the varied levels of expression or splice variants of CBRs, and establish whether these effects are mediated by these receptors in breast cancer. If our hypothesis is correct, CBRs could constitute a novel molecular target to which effective, non-toxic, natural and synthetic cannabinoids might be developed for treatment of breast cancer. [DoD-W81XWH1110795 and UAMS Translation Research Institute (CTSA Grant Award #UL1TR000039) ABCRP funds to AR-P; AR-Center for Plant Powered Production-P3 to FMB].

P161. CRIZOTINIB MODULATES ACTIVITIES AND EXPRESSIONS OF CYPS 2B6, 2C9 AND 3A4/5 IN HUMAN HEPATOCYTES MaryAnn Yang, Lining Cai, Qin Shi and Kan He Biotranex, Monmouth Junction, NJ Crizotinib, a potent inhibitor of ALK and cMet, has been recently approved for the treatment of ALK positive non-small cell lung cancer. The objective of this study was to determine the potential of crizotinib to modulate the activities of CYPs 2B6, 2C9 and 3A4 in plated human hepatocytes. Treatment of the plated human hepatocytes with phenobarbital and rifampicin for 72 hr caused a 2.3 and 1.9-fold increase in bupropion hydroxylase (CYP2B6), 1.2 and 2.1-fold increase in diclofenac 40 -hydroxylase (CYP2C9), 1.7 and 2.2-fold increase in midazolam 1-hydroxylase (CYP3A4/5), respectively. The increases of the mRNA levels of the respective CYP enzymes were consistent these activity results. Under the same incubation condition, treatment with crizotinib at 1 and 10 mM decreased CYP3A4/5 activity by 77% and 99%, but increased CYP3A4 mRNA by 2.3 and 9.5-fold, and increased CYP3A5 mRNA by 1.5 and 7.1-fold, respectively. Treatment with crizotinib at 1 mM increased CYP2B6 and 2C9 activity by 33% and 72%, respectively. However, treatment with crizotinib at 10 mM decreased CYP2B6 and 2C9 activity by 75% and 92%, respectively. It is noteworthy that significant hepatocyte toxicity was observed with crizotinib at 10 mM, which should account for the decrease of the enzyme activities measured for this treatment group. In summary, crizotinib caused induction of CYP3A4/5 as measured by mRNA, but inhibition or inactivation of CYP3A4/5 as measured by the enzyme activity in human hepatocytes. Crizotinib might also induce CYP2B6 and CYP2C9.

P162. MECHANISM-BASED INACTIVATION OF CYTOCHROME P450 2D6 BY BERBERINE ISOLATED FROM COPTIS JAPONICA Min-Jung Kim Department of Pharmacology and PharmacoGenomics Research Center, Inje Univ Col of Med, Busanjin-gu, South Korea Coptis japonica has been widely used for the treatment of various diseases such as gastrointestinal diarrhea in China and Korea. Seventy percent ethanolic extracts of Coptis japonica and its major ingredient berberine were evaluated for their inhibition of cytochrome P450 (CYP) isoforms in human liver microsomes and recombinant CYP2D6. Both the extracts of Coptis japonica and berberine strongly inhibited CYP2D6-mediated dextromethorphan O-demethylase activity. The IC50 value of berberine was 5.6-fold reduced after pre-incubation with microsomes in the presence of NADPH, suggesting that berberine is a mechanism-based inactivator. CYP2D6 was selectively inactivated by berberine. The inhibition of dextromethorphan O-demethylase activity by berberine was not restored to control level by extensive dialysis. In addition, the inactivation was protected by the addition of fluoxetine or quinidine, competitive inhibitors of CYP2D6. The KI value of berberine to CYP2D6 was 4.1 mM with a maximal rate constant for inactivation (kinact) value of 0.044 min1 and a partition ratio of 26.1. These results indicate that berberine is a potent mechanism-based inhibitor of CYP2D6. Therefore, in vivo interactions between berberine and CYP2D6 substrates are necessary to determine whether the inhibition of CYP2D6 by berberine is clinically relevant.

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P163. COMPARISON OF THE IC50-SHIFT AND % INHIBITION ASSAY TO EVALUATE TIME-DEPENDENT INHIBITION


Simon G. Wong and Mey Y. Lee Pharmacokinetics and Drug Metabolism, Amgen, South San Francisco, CA Due the risk associated with clinical drug-drug interactions, assessing the potential for time-dependent inhibition (TDI) of CYP enzymes is an integral component of early ADME drug discovery. However, definitive risk assessment for a clinical DDI is not amenable to higher throughput evaluation, because determination of the key input kinetic parameters KI and kinact requires measurement of inactivation rates involving multiple concentrations and time points. Accordingly, alternative formats for assessing TDI potential have been developed that do not require the generation of kinetic parameters and are less resource intensive. Two commonly employed approaches are the (a) IC50-shift assay, which determines the IC50 after a 30-minute preincubation (IC50t¼30 min) and (b) the two-step dilution assay, which determines the % inhibition at a single concentration (10 mM) after a 30-minute pre-incubation. The present study compared the ability of these two assay formats to assess the CYP3A4 TDI potential for a series of structurally related compounds with respect to 1) kinact:KI ratio and 2) the predicted change in AUC (AUCr) of a victim substrate (midazolam). The % inhibition assay demonstrated an excellent correlation (R-square ¼ 0.82) with the kinact:KI ratio, indicating that this assay format is an appropriate indicator for TDI potential. In terms of predicting in vivo DDI, the % inhibition assay classified compounds with low risk (ie AUCr52) as having less than 50% inhibition, and those with high risk (AUCr46) having greater than 80% inhibition. In contrast, IC50t¼30min had a weak correlation with kinact:KI ratio (R-square ¼ 0.46) and poor ability to predict in vivo DDI, with a greater than 100-fold range in IC50t¼30min (0.2 uM to 32 uM) for compounds with a low in vivo TDI potential (AUCr52). In conclusion, although the IC50t¼30min was of limited use in terms of predicting TDI potential and AUCr, the % inhibition assay demonstrated a strong correlation with kinact:KI ratio and successfully categorized compounds based on the predicted magnitude of an in vivo DDI.

P164. EFFECTS OF METRONIDAZOLE ON THE HEPATIC CYP ACTIVITY AND EXPRESSION Toshiyuki Kudo1, Masami Yatsu1, Rina Taguchi1, Izumi Akimoto1, Yoshiyuki Yatsuno1, Yuichi Takeuchi1, Junichi Iida2 and Kiyomi Ito1 1 Dept of Clin Pharmacokinet, Musashino Univ, Tokyo, Japan, 2Dept of Pharmacy, Saiseikai Yokohamashi Nanbu Hospital, Kanagawa, Japan Metronidazole (MTZ) is an imidazole antibiotic frequently used to treat anaerobic, protozoal and Helicobacter pylori infections. Although MTZ has long been used in clinical practice, few studies have investigated the drug interactions involving MTZ, with some controversial findings. For example, patients with cerebral hemorrhage and high prothrombin time values have been reported with concomitant use of MTZ and warfarin, but the mechanism of this interaction has yet to be clarified in detail. The present study investigated whether MTZ affects the activities and expressions of CYP isozymes by using human liver microsomes and HepaRG cells, a human hepatoma cell line. The effect of MTZ on CYP2C9-mediated metabolism was evaluated using S-warfarin as a probe substrate. 5 mM S-warfarin was incubated at 37 C for 30 min with pooled human liver microsomes and NADPH in the presence and absence of MTZ (10–6,000 mM) and the concentrations of metabolites (6-hydroxywarfarin and 7-hydroxywarfarin) were determined by LC/MS. As a result, both hydroxylation pathways of S-warfarin were completely inhibited by 10 mM sulfaphenazole (positive control), while almost unaffected by MTZ at the concentrations close to the plasma unbound concentration after oral dosage of MTZ. Furthermore, the inhibitory effect of MTZ was not enhanced by preincubation of MTZ and human liver microsomes in the presence of NADPH. In order to investigate the effect of MTZ on the expression of various CYP isozymes, HepaRG cells were cultured with or without MTZ (4-2,500 mM) for 2 days and the mRNA expressions of CYP isozymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) were measured by real-time RT-PCR. More than 80% viability was confirmed by MTT assay with the HepaRG cells cultured in the same condition. As a result, the mRNA expressions of all the CYP isozymes investigated were decreased by MTZ treatment in a concentration dependent manner. These results suggest that the reported interaction of MTZ and S-warfarin was not due to the inhibition of CYP2C9-mediated metabolism but could be attributed to the reduction of CYP2C9 expression. The effects of MTZ on CYP expression in human hepatocytes are currently under investigation.

P165. NAMPT INHIBITOR GNE-618 DDI RISK ASSESSMENT: PUTTING IN VITRO DATA INTO PERSPECTIVE Sophie Mukadam1 and Bianca M. Liederer2 1 Dmpk, Genentech, Inc., South San Francisco, CA, 2Genentech Inc., South San Francisco, CA


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Drug-drug interactions (DDI) occur when one drug changes the metabolism of a co-administered drug. This can result in an increase or decrease in clearance leading to changes in plasma concentrations which may result in decreased efficacy or increased adverse drug reactions and even death. Many DDI are manifest via reversible or irreversible inhibition of cytochrome P450 enzymes (CYPs). Evaluating the in vitro propensity of a compound to inhibit CYPs in vitro allows for identification of potential liabilities and SAR. However, in vitro inhibitory potency alone does not reflect the potential risk of DDI manifesting in vivo. Achieving an assessment of clinical significance of DDI is an important aspect of drug discovery and development. Many tools exist to predict clinical DDI using in vitro data, ranging from simple ‘‘rule of thumb’’ assessments such as 1 þ [I]/Ki to complex PBPK simulation packages such as SimcypÕ . Different approaches are appropriate at different stages of drug discovery and development, depending on data availability. Here we describe the DDI risk assessment for the NAMPT inhibitor GNE-618, a compound with potent in vitro CYP2C9 and CYP2C19 reversible inhibition and moderate CYP3A4 time-dependent inhibition (TDI). Using the basic static model to evaluate the potential DDI risk due to reversible inhibition of CYP2C9, a large in vivo interaction was predicted for all scenarios evaluated. This model, however, is known to over-predict DDI risk. Dynamic mechanistic predictions were done using the SimcypÕ software platform to attempt to gain a more accurate prediction of the clinical DDI liability. Taking into account both reversible and TDI, sensitive probe substrates for both CYP3A4 and CYP2C9 showed an approximately 3-fold change AUC when co-administered with GNE-618 at steady state in virtual clinical trials. There was, however, no predicted clinical risk due to CYP2C19 reversible inhibition. Clinical contraindicated medication analysis for potential patient populations was also evaluated; the moderate DDI risk predicted with midazolam (fmCYP3A4 of 0.94) likely has even lower predicted DDI potential for other, less extensively CYP3A4-metabolized substrates. Alternative treatment options (allowing bypass of metabolic pathways dominated by inhibited CYPs) also provide another avenue to avoid potential DDI risk in the clinic.

P166. DRUG INTERACTION STUDIES DURING DRUG DEVELOPMENT:CURRENT STATUS AND REGULATORY PERSPECTIVES IN JAPAN Masanobu Sato, Akihiro Ishiguro, Daisuke Iwata, Tatsuo Kishi, Mai Hirano, Motohiro Hoshino and Naomi Nagai Pharmaceuticals and Medical Devices Agency (PMDA), Tokyo, Japan The drug interaction data packages in the recent new drug applications (NDAs) for approved new molecular entities (NMEs) and regulatory perspectives in Japan are presented. Multiple-drug therapies are often prescribed in current medical practice, making it more difficult for healthcare professionals to adequately manage drug-drug and drug-food interactions. The U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) published a corresponding new regulatory draft guidance in 2012 and a guideline in 2013, respectively. In Japan, intensive works toward the revision of the regulatory document ‘‘Methods of Drug Interaction Studies’’ issued in 2001 by the Ministry of Health, Labour and Welfare (MHLW) has been conducted by the MHLW Drug Interaction Working Group and three Sub-Working Groups (Transporters, Drug Metabolizing Enzymes, and Modeling & Labeling) since December 2012. The new document is drafted based on the progress of clinical pharmacology as well as consideration of global harmonization. The MHLW is asking for comments on the draft version of the new Japanese guidance. In order to refine and finalize this draft document, it is useful to summarize recent situations and discussion points regarding drug interaction studies during new drug development and review processes in Japan. The Pharmaceuticals and Medical Devices Agency (PMDA) examined drug interaction packages in the NDAs for approved NMEs as well as recent consultation cases for drug interaction issues in Japan. The quality and quantity of the data packages for drug interaction studies have been improved, resulting in more informative labeling recommendations on the management of drugdrug and drug-food interactions. Considering the outcome of our examination of drug interaction studies, the following contents should be included in the new regulatory guidance: (1) general concepts and qualified approaches for transporter-mediated drug interaction studies and induction studies, (2) overviews of non-CYP enzymes, therapeutic proteins, and mixed mechanismmediated drug interactions, (3) quantitative decision-making strategies to develop both in vitro to in vivo studies and in vivo studies to labeling implications, and (4) detailed clinical drug interaction study design. 1) The U.S. FDA, Drug Interaction Studies-Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations (Draft Guidance), February 2012. 2) The EMA, Guideline on the Investigation of Drug Interactions. January 2013. 3) The MHLW, The Method of Drug Interaction Studies, Notification No. 813, June 2001.

P167. NEW JAPANESE DRAFT GUIDANCE ON DRUG INTERACTION STUDIES AND LABELING RECOMMENDATIONS Naomi Nagai1, Akihiro Hisaka2, Hiroshi Suzuki2, Toshiyuki Kume2, Kazuya Maeda2, Kiyomi Ito2, Ken-ichi Inui2, Yukio Kato2, Shogo Ozawa2, Hiroshi Watanabe3, Shin-ichi Miura4, Tatsuo Kishi1, Masanobu Sato1, Akihiro Ishiguro1, Toshinari Mitsuoka5, Keiko Maekawa6, Yoshiro Saito6 and Yasuo Ohno6

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1

The MHLW Drug Interaction Working Group and Pharmaceuticals and Medical Devices Agency (PMDA), Tokyo, Japan, The MHLW Drug Interaction Working Group and The Japanese Society for the Study of Xenobiotics (JSSX), Tokyo, Japan, 3The MHLW Drug Interaction Working Group and The Japanese Society of Clinical Pharmacology and Therapeutics (JSCPT), Tokyo, Japan, 4The MHLW Drug Interaction Working Group and Japan Pharmaceutical Manufacturers Association (JPMA), Tokyo, Japan, 5The MHLW Drug Interaction Working Group and the Ministry of Health, Labour and Welfare, Tokyo, Japan, 6The MHLW Drug Interaction Working Group and National Institute of Health Sciences (NIHS), Tokyo, Japan


2

The basic policy and contents of the new Japanese draft guidance on drug interaction studies and labeling recommendations issued by the Ministry of Health, Labour and Welfare (MHLW) are presented, focusing on updated information and new strategies. In light of the current technology and approaches and theoretical and in-depth scientific perspectives as well as global harmonization with the U.S. and EU regulatory documents1,2, the Japanese regulatory document ‘‘Methods of Drug Interaction Studies’’3 issued in 2001, should be revised. The MHLW Drug Interaction Working Group (DI-WG) and three Sub-Working Groups (Transporters, Drug Metabolizing Enzymes, and Modeling & Labeling) organized by scientists from academia, industry and regulatory bodies have been intensively discussing and working collaboratively since December 2012 to complete the draft version of the new guidance on drug interaction studies. The MHLW is asking for comments on the draft document. The new guidance will be finalized and issued in 2014. Compared to the ‘‘Methods of Drug Interaction Studies,’’ the revised document is more clinically oriented, with the latest scientific consensus and global harmonization as follows: (1) information on all pharmacokinetic (PK)-mediated drug interaction studies including CYP, non-CYP, transporter, therapeutic protein and mixed mechanism-mediated drug interactions, with currently acceptable and practically actionable decision trees as well as tables of sensitive substrates, in vitro and in vivo inhibitors and inducers; (2) detailed explanations and rich references for more sciencebased approaches such as applications of modeling and simulations for decision-making processes, and qualifications of in vitro transporter experiments; and (3) new instructions on labeling. The goal of this regulatory guidance on drug interactions is to effectively plan necessary drug interaction studies during the drug development process and to appropriately utilize drug interaction information for the purpose of safe and effective use of drugs after marketing. Extensive communication and discussions are needed among academic and industry scientists and consideration of the U.S. and EU regulations is required to refine and finalize this new guidance document.

References 1. The U.S. FDA, Drug Interaction Studies-Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations(Draft Guidance), February 2012. 2. The EMA, Guideline on the Investigation of Drug Interactions. January 2013. 3. The MHLW, The Method of Drug Interaction Studies, Notification No.813, June 2001.

P168. ANALYSIS OF METABOLISM AND TRANSPORT DRUG INTERACTION DATA IN 2012 NEW DRUG APPLICATIONS Aditi Mulgaonkar1, Isabelle Ragueneau-Majlessi1, Sophie Chung1, Lei Zhang2 and Ping Zhao3 1 Department of Pharmaceutics, University of Washington, Seattle, WA, 2Office of Clinical Pharmacology, Office of Translational Sciences, CDER, U.S. Food and Drug Administration, Silver Spring, MD, 3Office of Clinical Pharmacology, U.S. Food and Drug Administration, Silver Spring, MD Background: The aim of the work was to perform a systematic analysis of DMPK, transport and drug interaction data available in New Drug Applications (NDAs) of drugs approved in 2012, and highlight significant findings. Methods: The University of Washington Drug Interaction Databaseß was used to extract in vitro metabolism and transport data, as well as results of clinical drug interaction studies. Results: Among 33 small molecule new molecular entities (NMEs) approved in 2012, 25 (76%) had drug metabolism or transport data available and were fully analyzed (the other 8 were topical or diagnostic agents, enzymes, surfactant). Ten of the 25 drugs evaluated (40%) were cancer therapies, including 6 kinase inhibitors, making oncology the most represented therapeutic area. In vitro metabolism data were consistent with the current practice and regulatory recommendations, confirming the systematic and mechanistic approach of metabolism-based DDI evaluation. Fourteen of the 25 NMEs (56%) were metabolized by CYP3A, and 7 inhibited at least one enzyme in vivo. Of the 24 NMEs tested for in vitro induction, 9 were found to be CYP inducers in vitro, with only 4 being established in vivo inducers. Twenty-four of the 25 NMEs had in vitro transporter data available, either as substrates or as perpetrators, P-glycoprotein (P-gp) being evaluated in all cases. Eleven NMEs (44%) were found to be substrates of P-gp and 11 inhibited P-gp in vitro. Three NMEs were tested in vivo with the P-gp probe substrate digoxin: mirabegron was found to be a weak P-gp inhibitor, while apixaban had no effect on digoxin exposure. Cobicistat (a component of StribildÔ) had no effect on digoxin AUC but increased its Cmax by 40%. For other transporters recommended in the 2012 draft FDA DDI guidance, BCRP, OATP1B1, and OATP1B3 were the most studied (n ¼ 11, 10, and 8, respectively), while information on OAT1, OAT3, and OCT2 was found in 6, 7, and 7 NDAs, respectively.

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Overall, 15 of the 25 drugs (60%) had at least one in vivo DDI study with a change in exposure of clinical significance (AUC increase 100% (2 fold) or AUC decrease 50% for the affected drugs), either as victims or as perpetrators. Five of them had at least one DDI study with an AUC ratio over 5 fold. These largest DDIs were observed with NMEs that were sensitive substrates of CYP3A (or CYP3A and P-gp). Observed AUC ratios after co-administration of the strong CYP3A/P-gp inhibitor ketoconazole were 8.5-, 8.6-, 12.8-, and 27.3-fold for ivacaftor, bosutinib, avanafil, and lomitapide, respectively, while ritonavir increased the AUC of elvitegravir (a component of StribildÔ) by 19.9 fold. Conclusion: New drugs approved in 2012 were thoroughly evaluated for transporter-based DDIs, reflecting recent regulatory recommendations. Fifteen NMEs had clinically significant DDIs (over 2 fold change in exposure). The largest interaction was observed with the lipid/cholesterol lowering agent lomitapide, a substrate of CYP3A and P-gp, when co-administered with the CYP3A/P-gp inhibitor ketoconazole (27.3 fold change in lomitapide AUC).

P169. ASSESSMENT OF POTENTIAL CYTOCHROME P450S DRUG-DRUG INTERACTIONS USING STATIC AND DYNAMIC MODELS FOR ML-1


Lawrence H. Cohen, Stephen W.J. Wang, Robert J. Griffin and Cindy Q. Xia Drug Metabolism and Pharmacokinetics, Millennium Pharmaceuticals, Cambridge, MA ML-1 demonstrated anti-tumor activity in nonclinical in vivo xenograft models of breast, skin, brain and kidney cancers. In vitro studies with human liver microsomes (HLM) indicate that ML-1 is a reversible inhibitor of major cytochrome P450 (CYP) enzyme isoforms 1A2, 2B6, 2C8, 2C9, 2C19 and 3A4/5 with IC50 values of 133 mM, 42.9 mM, 35.3 mM, 42.5 mM, 20.3 mM and 68.7 mM, respectively. Based on allometric scaling, the projected human efficacious dose for ML-1 was estimated to be 1120 mg/ day with a corresponding Cmax value of 28.8 mM. Using the traditional static prediction model whereby an estimated [I]/Ki ratio greater than 0.1 is considered positive for potential in vivo drug-drug interactions (DDIs), ML-1 is thus categorized as a potential inhibitor of all CYPs aforementioned and a follow-up evaluation in vivo is recommended. In an effort to further assess the potential magnitude of DDIs in the clinic, a dynamic physiologically-based pharmacokinetic (PBPK) model was constructed using SimCYPÕ (Simcyp Ltd) in a bottom-up approach with in vitro and preclinical in vivo absorption, distribution, metabolism and excretion (ADME) data of ML-1. Using this model, ML-1 as a potential inhibitor of CYP1A2, 2B6, 2C8, 2C9, 2C19 and 3A4/5 was individually assessed with specific CYP substrates phenacetin, buproprion, rosiglitazone, diclofenac, omeprazole and midazolam, respectively. Based on this dynamic model, probe substrate exposure (area-under-curve [AUC]) ratio in the presence or absence of ML-1, greater than 1.25 is considered positive for potential in vivo DDI. Simulated AUC ratios of CYP1A2, 2B6, 2C8, 2C9, 2C19 and 3A4/5 were 1.01, 1.00, 1.12, 1.07, 1.04 and 1.24, respectively. Taken together, while the basic static model predicted possible in vivo DDIs for ML-1, dynamic PBPK model simulations show a low potential for DDI with AUCR 1.25.

P170. PACLITAXEL ENHANCES CELLULAR UPTAKE OF GEMCITABINE THROUGH UP-REGULATION OF NUCLEOSIDE TRANSPORTERS CNT1 AND CNT3 Zeen Tong, Usha Yerramilli, Yong Chen, Gondi Kumar and Sekhar Surapaneni Nonclinical Development, Celgene Corporation, Summit, NJ The combination of gemcitabine (dFdC) and nab-paclitaxel (albumin-bound paclitaxel nanoparticles) is being developed for the treatment of advanced pancreatic cancer. Gemcitabine requires active cellular uptake by human nucleoside transporters (hNTs), followed by intracellular activation to its monophosphate (dFdCMP) by deoxycytidine kinase (dCK). Subsequent phosphorylation reactions yield active triphosphate (dFdCTP). Gemcitabine can also be metabolized to dFdU by deamination mediated by cytidine deaminase (CDA). It has been reported in literature that paclitaxel increased dFdCTP accumulation in mononuclear cells and tumor cells, while pharmacokinetics of dFdC was not altered. Published mechanistic studies about the interactions between paclitaxel and dFdC have been focusing on dCK and CDA. Impact of paclitaxel on nucleoside transporters responsible for dFdC uptake has not been thoroughly studied. The present study investigated effects of paclitaxel on dFdC uptake and expression of human concentrative (CNT1, CNT2, CNT3) and equilibrative (ENT1, ENT2, ENT3 and ENT4) nucleoside transporters in MIA PaCa-2 and PANC-1 cell lines. MIA PaCa-2 and PANC-1 cells were treated with vehicle control or paclitaxel at 37 C for 48 hours. Paclitaxel concentrations (10 and 100 nM) used in the treatment were selected based on the cytotoxicity of palitaxel. To study the uptake of dFdC, cells were washed with incubation medium after paclitaxel treatment and incubated at 37 C for 4 hours with 20 mM [3H]dFdC. Medium was collected and cells were lysed for analysis by HPLC with online radioactivity detection to quantify dFdC and its metabolites. To study the effect of paclitaxel on expression of hNTs, cells were collected and lysed after paclitaxel treatment, and total RNA isolated. Messenger RNA (mRNA) content for each hNT was analyzed using TaqManÕ based quantitative Real Time Polymerase Chain Reaction (qRT PCR). After palitaxel treatment, concentration-dependent increases in intracellular concentrations of dFdC, dFdCMP, dFdCTP, dFdU and total radioactivity were observed in both cell lines. Approximately 2-fold increases in radioactivity were observed with 100 nM paclitaxel treatment. The increase in dFdC-related components indicates enhanced uptake of dFdC by palitaxel treatment. mRNA levels of CNT1 and CNT3 in Mia PaCa-2 cells and CNT1, CNT2 and CNT3 in PANC1 cells increased approximately 2- to 8-fold with the treatment of paclitaxel at 100 nM. CNT2 showed little activity toward dFdC in a previous study and was not detectable in MIA PaCa cells.

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Slight increases in ENT1 mRNA in MIA PACa cells and slight decreases in ENT4 mRNA in both cell lines were observed after paclitaxel treatment at 100 nM. mRNA levels of ENT2, ENT3, dCK and CDA were unchanged. Results from this study indicate that paclitaxel may increase dFdC uptake via up-regulation of CNT1 and CNT3.

P171. APPLICATION OF UPCYTEÕ HUMAN HEPATOCYTES TO CYP INHIBITION AND INDUCTION SCREENING


Sarada Devi Ramachandran, Nicola J. Hewitt, Bernhard Mu¨nst and Joris Braspenning Medicyte GmbH, Heidelberg, Germany upcyteÕ human hepatocytes are ‘‘up-regulated’’ primary human hepatocytes (PHHs) which have been driven into proliferation, whilst retaining important adult cell markers and functions e.g. responsive to CYP inducers1. Phase 2 activities were similar to those in freshly isolated PHHs but CYP activities were equivalent to levels after 5 days of culture. We have improved the upcyteÕ technology and this resulted in upcyteÕ Hepatocytes with higher basal CYP activities and a greater responsiveness to inducers. The applicability of these cells to drug interaction screening was investigated using known CYP inhibitors and inducers. For inhibition studies, upcyteÕ Hepatocytes were pre-cultured for 6 days before trypsinization and seeding in 48-well plates. After 24 h, CYP1A2 wells were induced for 3 days with 100 mM omeprazole. All other CYPs did not require preinduction with omeprazole. The inhibitors, a-naphthoflavone (0.01–10 mM), miconazole (0.1–100 mM) and ketoconazole (0.01– 10 mM) were pre-incubated for 5 min prior to the addition of phenacetin, tolbutamide and testosterone addition, respectively. Ticlopidine (a time-dependent CYP2B6 inhibitor, 0.1–20 mM) was incubated for 30 min before the addition of bupropion. For induction studies, upcyteÕ Hepatocytes were seeded into 48-well plates and pre-cultured for 3 days prior to the addition of a range of concentrations of inducers with different CYP3A4 induction potencies (rifampicin, phenobarbital, phenytoin, carbamazepine, troglitazone, pioglitazone, nifedipine and quinidine). The ‘‘Relative Induction Score’’ (RIS) prediction model was used to convert the concentration-response data to predict the in vivo induction potential. In addition, the effect of the inducers on CYP2B6 activity was also assessed. Both endpoints were measured in two independent experiments. The control CYP1A2, CYP2B6, CYP2C9 and CYP3A4 activities were 245 6, 56 4, 11 1 and 74 4 pmol/min/mg, respectively. CYP1A2, CYP2C9 and CYP3A4 activities were inhibited in a concentration-dependent fashion in upcyteÕ Hepatocytes from donor 422A-03, with IC50 values of 0.6, 1.6 and 0.2 mM, respectively. All concentrations of ticlopidine inhibited CYP2B6. Rifampicin, phenobarbital, phenytoin were all potent inducers of CY3A4 (14.8-, 7.1- and 11.8-fold induction, respectively) as well as CYP2B6 (5.5-, 6.8- and 6.3-fold induction, respectively). The RIS prediction model correctly identified the CYP3A4 potency of the compounds e.g. troglitazone was predicted to cause a decrease in the AUC of midazolam but not pioglitazone. The RIS curves were comparable in both biological replicates. These results support the use of upcyteÕ Hepatocytes for CYP inhibition and induction screening. upcyteÕ Hepatocytes are standardised such that the results are reproducible across experiments; this combined with the sheer quantities that can be generated from one vial of PHHs makes them a promising and unique alternative to PHHs for drug interaction screening.

Reference 1. Burkard A, Da¨hn C, Heinz S, Zutavern A, Sonntag-Buck V, Maltman D, Przyborski S, Hewitt NJ, Braspenning J., 2012, Xenobiotica 42(10):939–56.

P172. INHIBITION MECHANISM STUDY FOR HWANG-RYUN-HAE-DOK-TANG ON CYTOCHROME P450 IN HUMAN LIVER MICROSOMES Sang Yoon Lee1, Himchan Jang1, Ji-Yoon Lee1, Jin Yeul Ma2, Soo Jin Oh3 and Sang Kyum Kim1 1 College of Pharmacy, Chungnam National University, Daejeon, South Korea, 2KM-Based Herbal Drug Research Group, Korea Institute of Oriental medicine, Daejeon, South Korea, 3Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, South Korea Hwang-Ryun-Hae-Dok-Tang (HR, Japanese name: Oren-gedoku-to, Chinese name: Huang-Lian-Jie-Du-Tang) is one of the most frequently used traditional Chinese medicines prepared from Coptidis Rhizoma, Scutellariae Radix, Phellodendri Cortex and Gardeniae Fructus. It has been used to treat inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases in the clinical practice of Traditional Chinese Medicine. Although HR has been used widely in oriental countries, there is no information available regarding the effects of HR on hepatic CYP expression and activity. The purpose of the current study was to evaluate and predict the herb-drug interaction potential of HR mediated by CYP inhibition. In addition, the effects of fermentation on the CYP-mediated herb-drug interaction potential of HR were determined using pooled HLM. Using an in vitro CYP inhibition assay, HR-con displayed the inhibition of CYP1A2, CYP2C19, CYP2D6 and CYP3A4 activity determined using testosterone. The CYP1A2 inhibition by HR-con is best fit to a competitive inhibition model with 13.4 1.8 mg/mL of Ki value and the CYP2D6 inhibition may be quasi-irreversible through formation of metabolic intermediate complex(s). The fermentation of HR was not attenuated the HR-induced CYP inhibition, except for Lactobacillus acidophilus (HR-217). The fermentation of

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HR using Lactobacillus acidophilus significantly attenuated the CYP2D6 inhibition by HR-con. Future studies should focus on the identification of CYP regulatory component(s) from HR and the study of its metabolism for estimating herb-drug interactions of HR in vivo.

P173. CHEMOSYNTHETIC LIVERS: EVALUATING PLAUSIBLE DRUG-DRUG INTERACTIONS RELATING TO METABOLITE SUPPRESSION OR ATTENUATION


Mukund S. Chorghade1, Margarita Charnis1, Chiara M. Chapman2, Anjali M. Rahatgaonkar3 and Ingolfur Agustsson1 1 Empiriko Corporation, Newton, MA, 2Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 3 Department of Chemistry, Institute of Science, Nagpur, India Polypharmacy, when a multiple number of drugs are being co-administered, is becoming more common, especially in the elderly population and among the chronically ill. Polypharmacy is a major risk factor for adverse drug reactions (ADRs) and drug-drug interactions (DDIs). One form of DDI is when the metabolites of one drug interfere with the metabolism of another drug, causing irreversible changes to the metabolic pathways of one or both drugs. This manifests itself as suppression or attenuation of the metabolic profile of one drug in the presence of the other drug, relative to the metabolic profile of single drugs. We report herein preliminary experiments using our Biomimiks technology to study and predict the modulation of metabolite formation of common drugs for diabetes (e.g., metformin), hypercholesterolemia (e.g. atorvastatin) and hypertension (e.g., nifedipine) in the presence of each other. In addition, we will assess the linkage between DDI and potential ADRs. Biomimiks is in vitro and ex vivo technology that mimics the in vivo metabolism mediated by cytochromes P-450 to generate and predict oxidative metabolic profiles of drugs. Biomimiks has the potential to be applied at the individual patient level using chemistry-based diagnostics to determine comparative effectiveness and safety – providing a framework for achieving personalized patient treatment.

P174. LONG-LASTING INHIBITION OF INTESTINAL ABSORPTIVE TRANSPORTER OATP2B1 BY FRUIT JUICE Yukiko Murata, Takanori Mori, Akira Kubota, Keisuke Sekiya, Hiroshi Arakawa, Hisakazu Komori, Takeo Nakanishi and Ikumi Tamai Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan It is well known that fruit juice (FJ) affects oral bioavailability of drugs and we have postulated that organic anion transporting polypeptide 2B1 (OATP2B1) is involved in decreased absorption by being inhibited by FJ components1). In addition we observed that apple juice (AJ) shows long-lasting inhibitory effect on OATP2B1 by in vitro studies using Xenopus oocyte in addition to competitive inhibition2), while its mechanism remains unclear. On the other hand Ko¨ck et al indicated that activation of PKC internalizes OATP2B1, resulting in decreased activity3). Accordingly, in this study, we examined whether long-lasting effect of AJ on OATP2B1 is caused by decreased expression of OATP2B1 in plasma membrane by internalization of the protein or not using in vitro cultured cells. Transport activity of OATP2B1 was evaluated in HEK293 cells stably expressing human OATP2B1 (HEK/2B1) using [3H]estrone-3-sulfate (E3S) as the substrate. Expression and localization of OATP2B1 protein was examined by immunostaining. Cellular localization of fusion protein of OATP2B1 and EGFP was analyzed by confocal microscopy. When HEK/2B1 was exposed for 1 hr with FJ prior to uptake measurement, E3S uptake was significantly decreased by AJ and moderately by orange juice but not by grapefruit juice. Extent of decrease of uptake was dependent on exposure time and concentration of AJ. After 1 hr exposure with AJ, the cells were maintained without AJ and recovery of OATP2B1 activity was evaluated. The decreased activity by 1 hr exposure with AJ lasted longer than 4 hrs and its activity was recovered within 8 to 12 hrs. When protein expression and localization of OATP2B1 was examined immunocytochemically and western blot, staining of OATP2B1 at the plasma membrane became unclear and seemed to be located more strongly intarcellularly by 1 hr exposure with AJ, which is similar to the effect of PKC activator PMA. In addition, plasma membrane expression of OATP2B1 protein became clearer by 16 hr culture after washout of AJ, suggesting recovery of protein expression at plasma membrane. Such a change of cellular localization and activity of OATP2B1 by AJ became less significant in the presence of PKC inhibitor BIM-I. Furthermore, the strong expression of fusion protein of OATP2B1 with EGFP at plasma membrane was also changed to intracellularly after exposure with AJ. In conclusion, these results suggest that long-lasting inhibitory effect of AJ on apparent activity of OATP2B1 is explained by internalization of OATP2B1 protein from the plasma membrane and activation of PKC by AJ may be involved as the mechanism.

References 1. Tamai I. Adv. Drug Delivery Rev., 64: 508–514 (2012). 2. Shirasaka et al., Drug Metab. Dispos. 41: 615–621 (2013). 3. Ko¨ck K. et al., J. Biol. Chem., 285: 11336–11347 (2010).

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P175. DRUG-DRUG INTERACTION SIMULATIONS OF TIME-DEPENDENT CYP2C19 INHIBITION ON OMEPRAZOLE AND PROGESTERONE FROM IN VITRO METABOLISM DATA USING SIMCYP SIMULATOR


Jinrong Liu, Ning Liu, Robert A. Carr, Kennan C. Marsh, Sonia de Morais, Volker Fischer Drug Metabolism, Pharmakokinetics and Bioanalysis, AbbVie, North Chicago, IL In vivo data suggested that compound 1 was significantly metabolized across species. Preliminary results suggested that it is a potential CYP2C19 time-dependent inhibitor (TDI). Purposes: 1) characterize CYPs responsible for the metabolism of compound 1 in vitro and determine its potency as a CYP2C19 TDI; 2) simulate the clinical relevance of in vitro CYP2C19 inhibition for omeprazole, a prototypical CYP2C19 substrate; 3) predict DDI with its potential co-medication progesterone. Methods: Recombinant CYPs were used to identify the CYPs and to determine corresponding Km/Vmax. Time- and concentration-dependent decreases in activity, as defined by maximum rate of inactivation (kinact) and inhibitor concentration that gives 50% maximal inactivation (KI) of CYP2C19, was quantified using S-mephenytoin as a marker for CYP2C19 activity in pooled human liver microsomes. In vitro data were used for the DDI simulation using SimCYP software. Study design for simulations mirrored the clinical protocol. Results: CYP2D6 and CYP2C19 were identified as major enzymes with Kmof 4.4 and 2.8 mM and Vmax of 4.5 and 1.4 pmol/min/pmol CYP, respectively. Compound 1 displayed CYP2C19 inactivation with a KI of 1 mM and kinact of 4.8 hr1. The predicted median or mean AUC ratio of omeprazole in the presence or absence of Compound 1 was 2.6 and 3.0, in good agreement with the clinical observation (3.7 and 4.0, respectively). In addition, the simulated mean AUC ratio of progesterone was 1.1 when co-dosed with compound 1. Conclusions: Compound 1 is a CYP2D6 and CYP2C19 substrate, and is a moderate in vivo CYP2C19 inhibitor. SimCYP successfully simulated the extent of clinical interaction with the CYP2C19 substrate omeprazole, and predicted the lack of interaction between compound 1 and progesterone. The in vitro approach coupled with DDI simulations can be used proactively to obviate the need for clinical DDI studies in drug development.

P176. XENOBIOTIC DETOXIFICATION FROM AN ENVIRONMENTAL PERSPECTIVE: THE ROLES OF MICROBIAL ARYLAMINE N-ACETYLTRANSFERASES Sotiria Boukouvala1, Britton Davies2, Barbara Kotseli1, Evanthia Kontomina1, Vasiliki Garefalaki1, Giannoulis Fakis1, Edith Sim3, Ka´roly Ma´rialigeti4 and Anthony E. Glenn2 1 Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece, 2Toxicology & Mycotoxin Research Unit, US Department of Agriculture, Agricultural Research Service, Athens, GA, 3Faculty of Science, Engineering and Computing, Kingston University, Kingston-upon-Thames, United Kingdom, 4Faculty of Science, Department of Microbiology, Eo¨tvo¨s Lorańd University of Budapest, Budapest, Hungary Arylamine N-acetyltransferases (NATs) are enzymes catalyzing the detoxification of many environmental xenobiotics. The NAT isoenzymes have been characterised in higher eukaryotes and a limited number of pathogenic bacteria, but our recent genomic surveys [1] have indicated the presence of NAT genes in a range of phylogenetically diverse prokaryotic and eukaryotic microorganisms, including free-living extremophilic and biodegradative bacteria, as well as plant-symbiotic bacteria and endophytic fungi of agronomical relevance. In ascomycetes, NATs are exclusively found in the subphylum Pezizomycotina, including various plant-pathogenic species. We have undertaken cloning and expression of 13 NAT genes from five mycotoxigenic ascomycetes (the corn pathogen Fusarium verticillioides str. M3125, the wheat pathogen F. graminearum str. NRRL31084, the tomato pathogen F. oxysporum f.sp. lycopersici str. NRRL34936, the grain contaminant A. flavus str. NRRL 3357 and the model ascomycete A. nidulans str. FGSC A4), followed by characterization of the enzymatic properties of recombinant proteins against a panel of established NAT substrates. The results indicate that different homologues have diverse detoxification capabilities, suggesting that they could serve diverse functions within fungal cells. In endophytic Fusaria associated with cereals, of particular interest are the NAT1 homologues which mediate resistance to 2-benzoxazolinone (BOA), a potent anti-microbial compound released by the host. The NAT1 locus of F. verticillioides, is strongly up-regulated upon exposure of the fungus to BOA, as demonstrated by quantitative real-time PCR and microarray analysis. Up-regulation is also evident at the enzymatic activity level, with NAT specific activity increasing 15-fold in cell extracts prepared from cultures amended with BOA. Species with higher NAT1-mediated detoxification capabilities grew more effectively on BOAsupplemented solid medium, while phylogenetic analysis of PCR-amplified NAT1 homologues from 41 plant pathogenic Fusariaidentified a distinct clade for F. verticillioides and other maize pathogens. Apart from their crop-compromising potential as pathogens, Fusarium and Aspergillus are also producers of potent mycotoxins (fumonisins and aflatoxins, respectively) that may cause carcinogenicity or teratogenicity when consumed by human or livestock. Manipulating detoxification pathways that enable mycotoxigenic fungi, like F. verticillioides, to survive the chemical defences of their hosts may be a strategy relevant to global efforts to tackle the health risks and multi-million dollar losses associated with mycotoxin contamination of crops. In the broader context of exploiting microbial xenobiotic metabolism for the purposes of environmental management and detoxification, we are further expanding our investigation of microbial NAT enzymes to free-living bacteria isolated from

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diverse environments, including extreme habitats and heavily polluted areas. Using our previous genomic surveys to guide our selection, we are investigating the NAT genes of diverse bacteria from an extensive in-house collection of isolates, including non-pathogenic, biodegradative actinobacteria and bacteria (e.g. Rodococcus, Streptomyces, Novosphingobium, Delftia), plantassociated bacteria (e.g. Rhizobium, Burkholderia, Xanthomonas) and free-living Bacilli.

References [1] Glenn, A.E., Karagianni, E.P., Ulndreaj, A., Boukouvala, S. (2010). Comparative genomic and phylogenetic investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family. FEBS Lett 584:3158–64.


P177. PROTECTION AGAINST SODIUM ARSENITE –INDUCED TOXICITY IN RATS BY NONI EXTRACT (MORINDA CITRIFOLIA LINN.) Olufunke E. Ola-Davies1, Aina O. Adeogun2 and Oladayo F. Opasina3 1 Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan, Ibadan, Nigeria, 2Department of Zoology, University of Ibadan, Ibadan, Nigeria, 3Department of Veterinary physiology,biochemistry and Pharmacology, University of Ibadan, Ibadan, Nigeria The effect of Noni extract (Morinda citrifolia) on the toxicities induced by sodium arsenite a model clastogen, was investigated in wistar rats. The animals were dosed for 7 and 14 days consecutively with Noni extract as dietary supplement. They were then challenged with a single dose of sodium arsenite (NaAsO2) 2.5 mg/kg body weight on the 7th and 14th day respectively and sacrificed 24hours later. Phytochemical screening and toxicity of Noni was assessed. Degree of clastogenicity was evaluated using the rat bone marrow micronucleus assay and histopathology. The results revealed the presence of alkaloids, cardenolides, saponins and tannins. Noni extract alone did not induce any significant micronucleated polychromatic erythrocytes (PCEs) in the bone marrow after 7 or 14 days administration. Sodium arsenite induced micronuclei in the PCEs of the rat bone marrow by about 18 folds in comparison to the PCEs formed in the negative control rats. However, feeding Noni extract for 7 or 14 days before the administration of sodium arsenite reduced significantly the ability of sodium arsenite to induce micronuclei formation. Furthermore, NaAsO2 alone induced diffuse vacuolation of hepatocytes with foci of hepatocellular necrosis. However, priming with noni extract significantly decreased sodium arsenite induced micronucleated PCE and lesions in the hepatocytes. These findings suggest that Noni extract (Morinda citrifolia) protect against sodium arsenite induced clastogenicity and toxicity.

P178. HEPATIC GENE EXPRESSION PROFILES FROM RATS EXPOSED IN UTERO AND/OR THROUGH LACTATION TO MIXTURES OF ENVIRONMENTAL CONTAMINANTS Cathy Cummings-Lorbetskie, Gong-hua Xiao, Karen Leingartner, Andrea Rowan-Carroll, Mainul Husain, Andrew Williams, Carole Yauk and Daniel Desaulniers Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON, Canada Since they persist in food sources and have been detected in human tissues, environmental contaminants such as polychlorinated biphenyls (PCBs), organochlorine pesticides, and methylmercury, continue to pose a potential health risk. Many studies have examined hepatic gene expression following direct exposure to individual contaminants or to simple mixtures (two to three chemicals). In this study, rats were exposed in utero and/or through lactation to complex chemical mixtures, composed to simulate the contaminant profile detected in the maternal blood of Northern Canadians. Agilent microarrays were used to characterize the hepatic gene expression profiles of male offspring at post natal day (PND) 21 and PND85 following perinatal exposure through dams fed cookies laced with one of three chemical mixtures: 1 – M (PCBs, organochlorine pesticides, and methylmercury) at 0.5 or 1.0 mg/kg/day (0.5 M, and M); 2 – aryl hydrocarbon receptor agonists (Ahr) (non-ortho PCBs, PCdibenzodioxins and PC-dibenzofurans) at 1.8 ng/kg/day; or 3 – a 0.5 M/Ahr mixture. To determine the relative impact of in utero versus lactational exposures, control (C) and M litters were cross fostered at birth creating CC, MM, MC, and CM offspring groups. Exposures to mixtures M, 0.5 M, 0.5 M/Ahr, and Ahr affected expression (false discovery rate [FDR]50.1 and fold change [FC]41.5) in 69, 7, 12, and 1 genes respectively. All mixtures induced Cyp1a1 levels by an FC of 26, 14, 21, and 6 respectively, reflecting the chemical abundance and confirming previous low dose studies. Mixture M induced a significant (p50.005) dose dependent response (C vs 0.5 M vs M) in 6 genes including Cyp1a1, Cyp2b2, Cyp1a2, Ugt1a6, *LOC293989, and *LOC100359554 (*Cyp like genes). Ingenuity pathway analysis found significant enrichment among genes associated with liver toxicity functions such as cholestasis, glutathione depletion, hyperplasia/hyperproliferation, hepatocellular carcinoma, and inflammation. The top upstream regulators (Ingenuity) among M-exposed livers included ligand dependent nuclear receptors, PXR, CAR, and Ahr, and transcription regulator, Nfe2l2. Of 51 genes measured by quantitative real time PCR (qRT-PCR), only one gene, slc24a47, was found differentially expressed following in utero exposure alone (MC) compared to lactational exposure (CM, 23 genes) which displayed similar levels of changes as the MM group (25 genes). In PND21 and PND85 M-exposed rats, 69 and 32 genes were affected respectively, but only a cholesterol transporter gene, Abcg5, was verified (by qRT-PCR) to be

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commonly down regulated. Though few functional gene families were enriched at PND85, M and Ahr exposure significantly altered (FDR50.1 and FC41.5) 32 and 5 genes respectively with aldoketoreductase, Akr1c1, commonly repressed, a finding that was verified through qRT-PCR. This research suggests that (1) lactational and not in utero exposure to contaminant mixtures could modify the hepatic transcriptome at PND21, (2) that these changes did not persist, and (3) that the expression of different genes were altered during adulthood, but with uncertain biological consequences.

P179. DEVELOPMENTAL EFFECTS OF MALATHION EXPOSURE ON LOCOMOTOR AND EXPLORATORY ACTIVITIES IN WISTAR RAT


Pacoˆme Kouadio N’Go1, Fatima-Zahra Azzaoui2, Ahmed Ahami2, Mohamed Najimi3 and Fatiha Chigr4 1 Department of Biology, Unit of Clinic and Cognitive Neuroscience and Health, Faculty of Sciences, Kenitra, Morocco, 2Unit of Clinic and Cognitive Neuroscience and Health, Faculty of Sciences, Kenitra, Morocco, 3Department of Biology, Laboratory of Animal Physiology, Faculty of Sciences and Technology, Beni Mellal, Morocco, 4Laboratory of Animal Physiology, Faculty of Sciences and Technology, Beni Mellal, Morocco Developmental exposure to organophosphate insecticides is well known to induce neurobehavioral impairments, at late period. The present study aims to investigate the effects of chronic exposure to Malathion, from in utero to young adult stage, on locomotor skills and exploratory activity among wistar rat. Four groups of female rats, bred with one non-pesticide exposed male, are used. On gestational day 6, three groups receive daily, by intragastric gavage, 3 different doses of Malathion dissolved in corn oil (100, 200 and 300 mg/kg body weight). The control group receives the corn oil only. On postnatal day 21, weaned offsprings are submitted to the similar treatment until adult age. Neurobehavioral abilities are evaluated using the Open-Field test (OF). Results show that Malathion at 300 mg/kg is toxic to pregnant dams, and pups are stillborns. In males, Malathion level at 100 and 200 mg/kg induced significant impairment of spontaneous locomotor and exploratory activity expressed by the great decrease of number of squares crossed and number of rearing in OF test. In contrast, no discernible changes are observed in Malathion female’s treated-group at both doses. Neurochemistry assay shows that cerebellum and neocortex acetylcholinesterase (AChE) inhibition activity is associated to neurobehavioral deficits in males, but not sufficiently in females. Overall, neurobehavioral outcomes of current study reveal that developmental exposure to Malathion induces sex-selective effects with greater changes in females.

P180. EVIDENCE OF ELEVATED PHTHALATE ESTER LEVELS IN FIN AND SHELL FISH FROM EPE AND LAGOS LAGOON, NIGERIA D. E. Omogbemi1, Rachael A. Adegbola2, G. O. Adewuyi3, Adenike O. Ogunshe4 and Aina O. Adeogun1 1 Department of Zoology, University of Ibadan, Ibadan, Nigeria, 2Chemistry Department, The Polytechnic, Ibadan, Ibadan, Nigeria, 3Department of Chemistry, University of Ibadan, Ibadan, Nigeria, 4Department of Microbiology, The Polytechnic, Ibadan, Ibadan, Nigeria Lagoon systems are places of great biological importance where fishing is the main economic activity, but intensive agriculture, industrialization and tourism have degraded their sensitive environmental structure as waste materials from these sources either directly empty into them or are indirectly discharged through the rivers that drain into lagoons. Epe and Lagos lagoons are part of the major lagoon systems in Nigeria and the occurrence of four classes of phthalate esters (Dibutyl phthalate (DMP), Diethyl phthalate (DEP), Diethylhexyl phthalate (DEHP) and Dimethyl phthalate (DMP)) were evaluated from May-July 2011 to quantify their concentrations in water, sediment and finfish (Tilapia guineeensis, Pseudotolithus senegalensis, Synodontis filamentosus) muscle, liver, kidney and gill and in whole specimen of the shellfish Macrobrachium macrobrachium using High Performance Liquid Chromatography (HPLC). The results indicated that concentrations of these phthalates ranged as follows: DEHP, 230–7600 mg/g; DEP, 10–4100 mg/g; DMP, 51 – 2410mg/g and DBP 69 – 6030mg/g. DEHP had the highest concentration and the concentration of these classes of phthalate esters were DEHP4DBP4DEP4DMP for Epe and Lagos Lagoons respectively in water, sediment and fish. The muscle of Tilapia guineensisfrom Lagos lagoon had the highest mean concentration of 1990ug/g and the liver had the highest phthalate pollution index (PPI) of 352.2ug/g. Although the trend of accumulation in fish were not organ specific, significantly(p50.05) higher concentrations were recorded in samples from Lagos lagoon compared to Epe lagoon- an indication that lagos lagoon is under heavier pollutional load than Epe lagoon.The concentration of these phthalate esters in the two lagoon biota were also above the permissible limit recommended by the United States Environmental Protection Agency (USEPA) for water and fisheries management and this may pose a risk to the development and reproductive potential of wild fish species due to the ability of phthalates to act as Endocrine Disrupting Chemicals.

P181. FUNCTIONAL IMPAIRMENT OF MUSCLE GLYCOGEN PHOSPHORYLASE BY MERCURY Ximing Xu1, Cećile Matthieu1, Julien Dairou2, Jean-Marie Dupret2, Onnick Agbulut1 and Fernando Rodrigues-Lima2 1 Univ Paris Diderot, Paris, France, 2Sciences du Vivant, Universite Paris Diderot, Paris, France

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Muscle glycogen phosphorylase (GP) plays an important role in muscle energy and functions. This is evidenced by the existence of a genetic myopathy (McArdle’s disease) due to mutations that impair GP activity. Clinical features of this disorder encompass exercise intolerance, muscle fatigue, and cramps. Exposure to mercury has been shown to exert toxic effects in skeletal muscle giving rise to different symptoms such as muscle weakness. However, the molecular mechanisms underlying these toxic effects are poorly understood. We show that the glycogenolytic activity of muscle GP is impaired by mercury. Mercury (in particular in its inorganic form Hg2þ) was able to irreversibly inhibit purified GP and endogenous enzyme in muscle myotubes and in mouse skeletal muscle extracts. The inhibition of GP by mercury was found to rely on the formation of thiol-Hg bonds involving cysteine residues of the enzyme. Our data suggest that the irreversible inhibition of GP may contribute to mercury-dependent muscle toxicity.

P182. FIRST EVIDENCE OF ENDOCRINE DISRUPTION IN TILAPIAS FROM OGUN RIVER, NIGERIA


Oju R. Ibor, Azubuike V. Chukwuka and Aina O. Adeogun Department of Zoology, University of Ibadan, Ibadan, Nigeria A total of 69 Tilapias (Oreochromis niloticus, Tilapia zillii, Sarotherodon galilaeus, and Tilapia guineensis) were collected across 5 sampling and control (reference) points from Ogun river (receiving effluents from anthropogenic sources) in January 2013 to investigate the existence of endocrine disruption. Significant decreases (p50.05) in the levels of Luteinizing hormone (LH), Follicle stimulating hormone (FSH), Prognenolone, Triiodothyronine (T3), Thyroxine (T4), Progesterone, Testosterone and Cholesterol were recorded in fish from polluted points compared with the reference point indicating a disruption in the steroid hormone signaling pathway. Consequently, estrogen levels significantly increased (p50.05) in fish from polluted points compared with the reference point. Normal testicular and ovarian development were observed in fish from reference point, while fish from polluted points showed oocytes with testicular tissues (ovo-testis), empty seminiferous tubules and distorted ovaries. Altered hormonal levels and histological alterations in gonads observed in fish from polluted portions of the river may have resulted from the ability of composites mixtures of chemicals to modulate the activity of endogenous steroids.

P183. SPERMATOZOA MORPHOLOGY AND CHARACTERISTICS OF SPONDIA MOMBIN.L.(ANACARDIACEAE) PROTECTED MALE WISTAR RATS EXPOSED TO SODIUM ARSENITE Olumide S. Ajani1, Olufunke E. Ola-Davies2 and Matthew O. Oyeyemi1 1 Department of Veterinary Surgery and Reproduction, UNIVERSITY OF IBADAN, Ibadan, Nigeria, 2Department of Veterinary Physiology, Biochemistry & Pharmacology, UNIVERSITY OF IBADAN, Ibadan, Nigeria Spermatozoa morphology and characteristics of Spondia mombin L. (Anacardiaceae) protected male Albino (Wistar strain) rats exposed to Sodium arsenite was carried out. The leaves bark and fruit juices of the plant Spondia mombin have been widely used for both medicinal and non-medicinal purposes. Thirty five clinically healthy male albino rats (225–228 g) were grouped into 7 (A to G) in which each group contains 5 rats. The treatments were as follows- Group A was treated with 0.1 ml Dimethysulphoxide (DMSO), B (0.1 ml of Distilled water), C (Sodium arsenite (SA) 2.5 mg/kg body weight), D (Ethylacetate fraction (FB), E (Ethylacetate fraction for 7 days and Sodium asenite at 7th day), F (Methanolic fraction for 7days and Sodium arsenite the 7th day) and G (Methanolic fraction for 7 days) It was observed that group G and E had the lowest percentage motility 25.00 15.00% and 38.00 13.90% respectively and significantly lower (p50.05) than groups A and B 89.00 2.45% and 85.00 3.16% respectively which were negative controls. Percentage motility of Groups A to D were similar but higher (p50.05) than groups E to G. The morphological characteristics ranged between 10.88 and 12.74% in all the groups. In Artificial Insemination, it is important that the percentage motility be not less than 60%. In this study, motility of the sperm cells in groups A–D and F were above 60%. This indicated that the percentage motility of sperm cells in these groups was not affected by the treatment. However, the group E treated with Ethylacetate fraction of Spondias mombin prior to Sodium arsenite exposure and group G treated with Methanolic fraction alone showed a reduction in percentage motility and livability which may precipitate infertility. In conclusion, the Spondias mombin fractions did not affect sperm cells structurally but treatment with Ethylacetate fraction and Methanolic fraction caused significant reduction (p50.05) in percentage motility and livability thus may precipitate infertility.

P184. A NOVEL CANCER SUICIDE GENE THERAPY COMBINING THE IMPROVEMENT OF CYCLOPHOSPHAMIDE TUMOR CYTOTOXICITY AND THE DEVELOPMENT OF AN ANTI-TUMOR IMMUNE RESPONSE Walid Touati1, Johanne Seguin2, Thi Tran3, Eric Tartour3, Claude Baillou4, Ge´raldine Lescaille4, François M. Lemoine4, Philippe Beaune1 and Isabelle de Waziers5 1 Inserm, U775, INSERM U775, Paris, France, 2INSERM U1022, Paris, France, 3INSERM U970, Paris, France, 4INSERM U959, Paris, France, 5INSERM U775, Paris, France


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Gene-directed enzyme prodrug therapy (GDEPT) systems have been developed for killing cancer cells by introducing a gene coding for a protein that can convert a harmless prodrug into a cytotoxic one. We built a highly efficient therapeutic suicide gene which metabolizes the prodrug cyclophosphamide (CPA) into cytotoxic metabolites. This gene was formed by the fusion of two human genes: a triple mutant of CYP2B6 (CYP2B6TM) modified by site-directed mutagenesis and the soluble part of NADPH cytochrome P450 reductase (RED). Moreover the linker between both genes was optimized to express a high RED activity to improve electron transfer required for CYP2B6TM catalytic activity. Expression of this fusion gene (CYP2B6TM-RED), by a recombinant lentivirus (LV), allowed switching of a resistant human pulmonary cell line (A549) into sensitive cell line toward CPA. After 48H of CPA treatment, cytotoxic tests showed that A549 cells expressing CYP2B6TM-RED had an IC50 of about 0.5 mM approximately 13-fold weaker than that of cells expressing CYP2B6 wild type-RED. Our strategy was tested in animal models using immunocompetent C57Bl/6 mice and a mouse epithelial pulmonary cell line (TC1). In vitro, this CPA resistant cell line became sensitive to CPA after expression of CYP2B6TM-RED gene with an IC50 comparable to that observed in transduced A549 cells. TC1 cells previously infected by LV-CYP2B6TM-RED or not infected were injected sub-cutaneously into C57Bl/6 mice. CPA was intraperitonealy administered each week at a dose of 140 mg/Kg when the tumor volume reached about 500 mm3. Mice with TC1-CYP2B6TM-RED tumor presented an important decrease of tumor volume immediately after the first injection of CPA and only four injections of CPA were necessary to observe a complete disappearance of the tumors. Similar results were observed when the tumor cells were composed of 25% of TC1 expressing CYP2B6TM-RED and 75% of non-infected cells demonstrating in vivo the contribution of the direct bystander effect to the efficiency of our strategy. Morever cured mice were rechallenged with uninfected TC1 cells. In some cases a complete eradication of the tumor was observed 7 days after cell inoculation which correlated to a cellular immune response against tumor cells that would lead to a long-term protection against potential metastasis To confirm the role of the immune system in the efficiency of our strategy, similar experiments using nonimmunocompetent mice are now under investigation. Moreover, we are administering our fusion gene either by intratumoral lentiviral injection or by infection of mesenchymal stem cells used as delivery strategy, in preparation for clinical human studies.

P185. EVALUATION OF ‘‘R3’’ AND RELATIVE INDUCTION SCORES (RIS) DERIVED FROM ENZYME ACTIVITY AS PREDICTORS OF CYP3A4 INDUCTION RESPONSE IN VIVO J. George Zhang, Thuy Ho, Alanna L, Callendrello, Robert J. Clark, Elizabeth A. Santone, Lisa G, Fox and David M. Stresser Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, MA Recent guidance from the FDA and EMA advocates the use of human hepatocyte as a test system with mRNA as the preferred endpoint in evaluating cytochrome P450 induction potential of drug candidates. The agencies also indicate or recommend the use of basic models [e.g. relative induction score (RIS) and ‘‘R3’’] where data are obtained using cryopreserved hepatocyte lots which have been previously characterized with a sufficient numbers of clinical inducers and non-inducers. Our previous report (1) demonstrated that both RIS and R3 values obtained using CYP3A4 mRNA data could be strong predictors of CYP3A4 induction-mediated drug-drug interactions. In this investigation, we assessed RIS and R3, derived from CYP3A4 enzyme activity data, to predict the induction potential of CYP3A4 in vivo. Hepatocytes from three lots were treated with a set of 20 compounds, including established clinical inducers and non-inducers of CYP3A4 for 2 days, typically with 8 concentration points to enable calculation of EC50 and Emax. After treatment, testosterone 6b-hydroxylase activity was determined by LC-MS/MS. Proposed calibration curves of the observed clinical AUC changes of CYP3A4 substrates versus RIS or R3 values were prepared. The cut-off values for 20% of AUC change indicating a clinical inducer were determined. Our results showed that using the calculated R3 value of 0.9, as suggested by the FDA guidance, effectively categorized strong inducers, but was less effective at identifying mid-range or weak inducers. These results were consistent with our earlier analysis using mRNA as an endpoint. The RIS was reasonably correlated with the clinical AUC changes of inducers and non-inducers. Use of unbound or total Cmax in the calculation of R3 or RIS made no difference in predictive outcomes. Therefore, the use of enzyme activity data alone can be useful in predicting clinical inducers and non-inducers of CYP3A4, when test compounds are not significant inhibitors of enzyme activity. Integrating results of mRNA and enzyme activity may provide a higher level of assurance compared to either endpoint alone.

Reference 1. Zhang et al. The 18th North American Regional ISSX Meeting Program and Abstract (P94). October 14–18, 2012, Dallas, Texas

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P186. TIME-COURSE OF CYTOCHROME P450 (CYP450) INDUCTION IN CULTURED HUMAN HEPATOCYTES: EVALUATION OF ACTIVITY AND MRNA EXPRESSION PROFILES FOR SIX INDUCIBLE CYP450 ENZYMES


David B. Buckley1, Catherine Wiegand1, Pilar L. Prentiss1 and Odette A. Fahmi2 1 XenoTech, LLC, Lenexa, KS, 2Pharmacokinetics and Drug Metabolism, Pfizer, Groton, CT Enzyme induction potential for new drug candidates is typically evaluated in vitro with cultured human hepatocytes with a demonstrated utility for the quantitative prediction of drug interactions involving cytochrome P450 (CYP450) enzymes, particularly CYP3A4. Recently, many researchers have begun to measure CYP450 mRNA expression in vitro for prediction of clinical outcomes for CYP3A4 with relative success. Furthermore, recent regulatory guidance from the FDA and EMA now recommends CYP450 mRNA expression as the primary in vitro endpoint as opposed to CYP450 activity. The purpose of this study was to evaluate the time-course of CYP450 induction in cultured human hepatocytes to determine the adequate treatment period and establish CYP450 mRNA expression or enzymatic activity as reasonable endpoints for each of the six inducible hepatic CYP450 enzymes, namely CYP1A2, 2B6, 2C8, 2C9, 2C19 and 3A4. Three individual lots of cryopreserved human hepatocytes were treated for up to three days (72 hrs) with a vehicle control (0.1% DMSO), multiple concentrations of rifampin (0.05–10 mM) and a single concentration of omeprazole (50 mM) or phenobarbital (750 mM) with medium replacement every 24 hrs, as appropriate. CYP450 enzyme activities were measured in situ at 0, 24, 48 and 72 hrs with a two-step cocktail approach using specific marker substrates for individual CYP450 enzymes. Metabolite formation was quantified by LC-MS/MS. CYP450 mRNA expression was evaluated at 0, 6, 12, 24, 30, 36, 48, 54, 60 and 72 hrs by RT-PCR. As expected, treatment of all three lots of hepatocytes with rifampin caused a time-dependent increase in CYP2B6, all CYP2C enzymes and CYP3A4 activities inasmuch as the highest fold-induction values compared to vehicle controls were typically observed at 72 hrs. Similar results were observed for CYP1A2 activity following treatment with omeprazole. For CYP450 mRNA expression, the highest foldinduction values compared to vehicle controls were oftentimes reached by 48 hrs following initiation of treatment by prototypical inducers and, in some cases, as early as 24–36 hrs. These results suggest that when mRNA is a primary endpoint a 48 hr treatment period may be suitable. In most cases, the maximal fold-induction (Emax) values observed for mRNA expression were equal to, or greater than, the fold-induction values determined by CYP450 activity measurement. However, in the case of CYP2C19, treatment of all three lots of hepatocytes with rifampin up to 72 hrs caused greater than a two-fold induction in enzyme activity compared to vehicle controls (as measured by S-mephenytoin 40 -hydroxylation). Whereas, in many cases, rifampin did not cause more than a two-fold induction of CYP2C19 mRNA compared to vehicle controls. The results presented here provide significant insight into the selection of both appropriate treatment duration and endpoint for in vitro CYP450 induction experiments.

P187. TRANSCRIPT REGULATION OF 42 ADME GENES BY PROTOTYPICAL INDUCERS IN HUMAN HEPATOCYTES Timothy Moeller1, Scott Heyward1, Caitlin Brown1, Cherylle Wilson2 and Brendan Yee2 1 Celsis In Vitro Technologies, Baltimore, MD, 2Affymetrix, Santa Clara, CA Due to recent FDA draft guidance, a fundamental change from measuring enzyme activities to transcripts will have profound implications for studying induction drug-drug interactions (DDI). Discrete enzymatic assays for individual CYP enzymes are replaced by multiplexed assay to measure several genes from a single induction sample. This increase in data from a simplified system allows for basic screening with target markers as well as broader transcript surveys. In this study, we used the QuantiGene Plex Assay to simultaneously measure 42 ADME transcripts spanning Phase I (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, FMO1, FMO3,FMO5, AOX1, XDH, MAOA, MAOB) and Phase II (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15, SULT1A1, 2A1, 2B1) enzymes, transporters (ABCA1, B1, B11, C1, C2, G2, SLC10A1, 22A1, 2A1, 3A1, O1B1, O1B3, O2B1) and eight control genes (ACTB, GAPDH, GUSB, HRPT1, PGK1, POLR2A, PPIB, TBP). We screened 11 donors of human cryoplateable hepatocytes at single concentration of omeprazole, phenobarbital and rifampicin after 48-hour exposure in 96-well format to observe individual variations in induction and suppression potentials. Inducers did modulate certain control gene expression levels. Selective use of control genes was necessary to normalize the data. For omeprazole, induction greater than 2-fold was observed for CYP1A1, 1A2, 2B6, 3A4, UGT1A1 and 1A4. Phenobarbital induced CYP2A6, 2B6, 2C8, 2C9, 3A4, 3A5, UGT1A1 and 1A4. Rifampicin induced CYP1A1, 2A6, 2B6, 2C8, 2C9, 3A4, UGT1A1 and 1A4. Suppression greater than 30% was observed in several genes. For two donors, concentration response curves were generated and EC50 values were calculated where applicable. Omeprazole EC50 value for CYP1A2 was 2 mM, phenobarbital for CYP2B6 was 1.3 mM., and rifampicin for CYP3A4 was 260 nM and CYP2C9 170 nM. Dose dependent suppression was measured for several genes. The data shows the power of generating profiles of transcript levels from a single concentration or a concentration response curve of an inducer in a multiplex format. Primary markers such as CYP1A2, 2B6, 2C9 and 3A4 can be used to fulfill regulatory

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requirements with a potential of adding secondary markers such as phase II enzymes or transporters to probe gene regulation from test articles.

P188. TRACING CYP INDUCTION: A NOVEL APPROACH TO MEET FDA RECOMMENDATIONS ON DRUG-DRUG INTERACTIONS


Hinnerk Boriss1 and Jari Rautio2 1 Sovicell GmbH, Leipzig, Germany, 2Plexpress Oy, Helsinki, Finland Induction of cytochrome P450 activity in the clinic can result in therapeutic failure as a result of activation of drug metabolizing enzymes. Activity based assays for CYP inductions are well established in the drug discovery routine. However, these standard CYP activity screens are less sensitive in detecting CYP inductions and become obscured when induction and activity inhibition of CYP3A4 co-occur. Hence, both FDA and EMEA recommend CYP mRNA expression profiling as the preferred endpoint to reduce the false negative rate of standard activity screens. Expression level profiling of CYP genes by PCR methods is laborious, costly and prone to inter-experiment variation. TRAC is a novel technology for studying gene expression levels of CYP and other drug metabolism related genes. By directly measuring transcripts without RNA extraction, cDNA conversion and amplification, the method is more accurate, much faster and less costly than standard mRNA profiling techniques. Multiplexing facilitates screening of the most relevant transcripts of phase I and II enzymes simultaneously e.g. with hepatotoxicity markers in one experiment and easy automation provides scalability of the method.

P189. PREDICTION OF CLINICAL CYP3A4 INDUCTION USING POOLED HUMAN HEPATOCYTES ON CELL ARRAY 3D-CULTURE PLATE FOR DRUG-METABOLIZING ENZYME INDUCTION STUDIES IN DRUG DISCOVERY Jiro Kuze Discovery Drug Metabolism & Pharmacokinetics, Taiho pharmaceutical co., ltd, Tsukuba, Japan The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug (DDIs) interactions, To predict and reduce a DDI risk of a drug candidate caused by CYP3A4 induction in preclinical study is one of the challenges facing pharmacokinetic researchers. To account for inter-individual variability in drug-metabolizing enzyme activities and induction potential, the FDA guidance suggests the induction studies be carried out using freshly isolated or cryopreserved human hepatocytes prepared from at least three individual donor livers. We compared the induction potentials between individual and pooled cryopreserved human hepatocytes (CryostaX?) using microfabricated cell array plate. In addition, we developed an approach with in vitro assays of the pooled human hepatocytes for predicting clinical CYP3A4 induction by using a novel value (Emax’), determined from the ratio of Emax value of an inducer to that of rifampicin as a positive control by metabolic activity or mRNA. Cryopreserved human hepatocytes obtained from multiple donors and the pooled ones were cultured (1 10^5 cells/well) on 48-well cell array (Cell-Able?) preconditioned with 3T3 cells as feeder cells. Activities and comparative mRNA of 5 cytochromes P450 (CYP) isoforms (1A2, 2B6, 2C9, 2C8, 3A4/5) were estimated after 2 or 3-day treatment using LC-MS/MS or QuantiGene Plex, respectively. Induction potencies of the CYPs with prototypical some inducers to CYP3A4 were also evaluated. The obtained Emax’ values were related to the free plasma concentration at steady-state of each inducer (Css,u). All CYP activities and responsiveness to the inducers were maintained well on the cell array plate and the pooled cells showed the average activities of individual data. In addition, a good relationship between Emax’ values and clinically observed induction response was found in the in vitro system using pooled human hepatocytes. Our 3D-cell array plate maintained stable druginduction potentials using pooled hepatocytes. Thus, a novel Emax’ approach to the prediction of clinical potency of CYP3A4 inducers was established by using pooled cryopreserved human hepatocytes on 3D-cell array plate. These results suggested that the 3D human hepatocytes spheroid culture system is a useful tool for screening the induction potency of new drug candidates in drug discovery.

P190. ESOMEPRAZOLE MODIFIES HEME IN HUMAN LIVER MICROSOMES: PRELIMINARY HIGHRESOLUTION MASS SPECTROMETRIC EVIDENCE FOR THE MECHANISM OF INACTIVATION OF CYTOCHROME P450 Brian W. Ogilvie1, Joanna E. Barbara2 and Andrew Parkinson3 1 XenoTech, LLC, Lenexa, KS, 2Analytical Sciences, XenoTech, LLC, Lenexa, KS, 3XPD Consulting, Shawnee, KS The proton-pump inhibitor omeprazole decreases the efficacy of clopidogrel, an anti-platelet drug that depends (at least in part) on conversion to its active metabolite by CYP2C19. We previously identified omeprazole, esomeprazole, omeprazole sulfone


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and 5-O-desmethyl omeprazole, but not R-omeprazole, as irreversible (or quasi-irreversible) metabolism-dependent inhibitors of CYP2C19 [1–3]. For esomeprazole, omeprazole sulfone and 5-O-desmethyl omeprazole, these studies established the following ‘‘Silverman’’ criteria for mechanism-based inhibition: 1) inactivation of CYP2C19 occurs in a concentration-, time- and metabolism-dependent manner, 2) the inactivation is largely irreversible (by ultracentrifugation and washing), 3) the inactivation is saturable, 4) there is substantial protection by the CYP2C19 substrate, pantoprazole (200 mM), and 5) there is minimal protection from GSH, superoxide dismutase or catalase. Additional studies were conducted to provide further evidence for mechanism-based inactivation of CYP2C19 by esomeprazole and its inhibitory metabolites, and to begin to elucidate the mechanism of this inactivation. Esomeprazole (100 mM), solvent or 1-aminobenzotriazole (2 mM – positive control) were incubated in 3-mL incubation mixtures (pH 7.4) containing high purity water, potassium phosphate buffer (50 mM), MgCl2 (3 mM), EDTA (1 mM), NADP (1 mM), glucose-6-phosphate (5 mM), glucose-6-phosphate dehydrogenase (1 Unit/mL), and HLM (1 mg protein/mL). Reactions were terminated by 1 N HCl and extracted with two volumes of methylene chloride. The organic fraction was evaporated to dryness and the residue reconstituted with acetonitrile. Heme and potential adducts were monitored by ultra-performance LC-UV/HRMS with a Waters Synapt G2 quadrupole time of flight mass spectrometer equipped with a Waters Acquity LC system. For chromatographic separation, formic acid-modified water (A) and acetonitrile (B) were applied to an Agilent Zorbax 300 SB-C18 LC column (2.1 150 mm; 5 mm) with a gradient ranging from 2–98% mobile phase B. The mass spectrometer was operated in positive, resolution MSE mode. Under these conditions nearly complete loss of intact heme occurred in incubations with 1-ABT and approximately half was lost with esomeprazole. No apparent heme-associated ions (other than intact heme) were found in the control sample, whereas a component associated with a protonated molecule of m/z 723.2240 was found in the esomeprazole sample, with an isotopic distribution similar to that of the intact heme. An ion of this m/z would correspond (within 2.8 ppm) to the mass of intact heme adducted with the 5-methoxy-benzene portion of esomeprazole (theoretical mass of 107.0467 amu). The results of this study provide preliminary evidence for modification of the heme of P450 by esomeprazole in HLM in the presence of NADPH.

References 1. Ogilvie BW, Yerino P, Kazmi F, Buckley DB, Rostami-Hodjegan A, Paris BL, Toren P and Parkinson A (2011). Drug Metab Dispos 39:2020–2033. 2. Ogilvie BW, Toren P, Kazmi F, and Parkinson A. (2011). Drug Metab Rev, 43 (Suppl 2, Abstract 145). 3. Ogilvie BW and Parkinson A. (2012). Drug Metab Rev, 44 (Suppl 1, Abstract 40).

P191. POTENT INHIBITION OF HUMAN SULT1E1 BY TRICLOSAN Katie Jo Rohn-Glowacki1, Margaret O. James2 and Charles N. Falany3 1 Pharmacology & Toxicology, University of Alabama at Birmingham, Birmingham, AL, 2Medicinal Chemistry, University of Florida, Gainesville, FL, 3Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL Human cytosolic sulfotransferases (SULTs) are Phase II conjugating enzymes that facilitate the transfer of a sulfonate group from 30 -phosphoadenosine 50 -phosphosulfate (PAPS) to the hydroxyl or amine group of the substrates. SULT substrates include endogenous, exogenous, and xenobiotic compounds that are sulfated to expedite excretion or bioactivation of the compounds. SULT1E1, known as estrogen SULT, is important in estrogenic hormone homeostasis in humans. Estrogen, specifically 17bestradiol (E2), is the primary substrate of SULT1E1 with a reported Km of E2 of 5 0.8 nM (1). SULT1E1 is expressed in many hormone sensitive tissues in the body such as prostate, liver, kidney, and placenta. Triclosan is an antibacterial agent which is found in many household items such as soap and toothpaste and is currently being investigated by the FDA for safety. Triclosan has been shown to inhibit estrogen sulfation and hormone homeostasis in mammalian systems. Our study focuses on the kinetic properties of SULT1E1 with triclosan including sulfation, inhibition, and binding. Triclosan is a substrate for SULT1E1, however, it is not a great substrate with a Km ¼ 95.3mM and no detectable activity at concentrations less than 1mM. With a calculated Km of almost 100mM we would expect that if the binding of triclosan is the rate limiting step, that the disassociation constant (Kd) of triclosan to be around 70–80mM. However, when binding studies were performed a calculated Kd was determined to be approximately 0.3mM to SULT1E1 alone and approximately 0.05mM with the PAP (30 50 -diphosphoadenosine) bound enzyme. The difference in Kd with and without PAP bound raises the possibility that PAP(S) binding elicits structural changes in the enzyme that affect substrate selectivity and binding. Molecular modeling and docking studies of triclosan to the published SUTL1E1 crystal structure with PAP bound indicate that triclosan binds with high affinity in a non-catalytic orientation in the active site. The modeling of triclosan to SULT1E1 without PAP bound was not performed since, to date, no validated open homology model for SULT1E1 has been generated. Together the modeling and kinetic data suggest that triclosan may be a potent inhibitor of SULT1E1. Therefore, we tested the ability of triclosan to inhibit E2 sulfation by SULT1E1 in vitro and the calculated Ki was 20–55 nM. We conclude that triclosan is a potent inhibitor at low nanomolar concentrations while it is not a substrate until micromolar concentrations. This inhibition of SULT1E1 by triclosan in humans may potentially be dangerous to hormone homeostasis with exposure to triclosan.

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References 1. Zhang H, Varlamova O, Vargas FM, Falany CN, Leyh TS, Varmalova O. Sulfuryl transfer: the catalytic mechanism of human estrogen sulfotransferase. J Biol Chem. 1998;273(18):10888–92. PubMed PMID: 9556564.

P192. ENZYME-SPECIFIC INHIBITION OF HUMAN GLUTATHIONE TRANSFERASES BY NAPHTHALENE ANALOGUES OF 1-CHLORO-2,4-DINITROBENZENE


Hilary Groom1, Moses Lee2 and P. David Josephy1 1 Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, 2Department of Chemistry, Hope College, Holland, MI Glutathione transferase (GST) superfamily enzymes initiate the detoxication of many xenobiotics and byproducts of intermediary metabolism. GSTs catalyze the conjugation of glutathione with reactive functional groups of xenobiotics, including (among many classes of electrophiles) halonitroaromatics and chemotherapeutic alkylating agents. We hypothesized that greater enzyme specificity of GST substrates and inhibitors could be achieved by expanding the ring size of commonly-used substrates for GST activity assays, such as 1-chloro-2,4-dinitrobenzene (CDNB). We have studied the interactions of naphthalene analogues of CDNB with recombinant human GST enzymes (Alpha, Mu, and Pi classes) expressed in E. coli. Spectrophotometric enzyme assays were conducted. Several nitronaphthalene derivatives were found to be GST inhibitors. 1- Chloro-2,4-dinitronaphthalene (CDNN) reacted readily with glutathione (GSH) in strong alkali to give the corresponding adduct; nevertheless, GST catalysis of the reaction between CDNN and GSH was undetectable. CDNN inhibited the CDNB conjugation activity of GST enzymes A1-1, M1-1, and P1-1, with IC50values 3.6, 35.6, and 91.2 micromolar, respectively. The halogen substituent on the naphthalene ring is not required for inhibitory activity: 2,4-dinitro1-naphthol (Martius yellow) also inhibited GST activity, with IC50 values 1.7, 4.1, and 89.7 micromolar, respectively. Study of the interactions of nitronaphthalene derivatives with hGST enzymes can provide insight into the toxic properties of these compounds and may identify novel lead compounds for development of clinical GST inhibitors. (Supported by the Natural Sciences and Engineering Research Council of Canada.)

P193. INDUCTION POTENCY AND INHIBITORY EFFECT OF A NOVEL DEOXYURIDINE TRIPHOSPHATASE INHIBITOR, TAS-114, ON DRUG-METABOLIZING ENZYMES IN HUMANS: IN VITRO AND IN VIVO STUDIES Kunihiro Yoshisue1, Eiji Matsushima1, Kaku Saito2 and Ken-ichiro Yoshida1 1 Pharmacokinetics Research Laboratory, Taiho Pharmaceutical Co. Ltd., Tokushima, Japan, Department, Taiho Pharmaceutical Co. Ltd., Tokyo, Japan

2

Clinical Development I

Deoxyuridine triphosphatase (dUTPase) expression prevents the misincorporation of 5-fluorouracil (5-FU) and uracil into DNA, and induces resistance to FU-based chemotherapy. Therefore, the use of a dUTPase inhibitor in combination with a thymidylate synthase inhibitor such as fluoropyrimidine would afford a novel approach for cancer treatment. TAS-114 is a novel smallmolecule inhibitor of dUTPase and is now being developed in combination with a 5-FU derivative drug. In this study, we investigated the inhibitory effects of TAS-114 on the activities of cytochrome P450 (CYP) isoforms and dihydropyrimidine dehydrogenase (DPD), a 5-FU-degrading enzyme, and its potency for inducing CYP1A2 and CYP3A4 activities by using a human liver fraction or hepatocytes. Furthermore, we evaluated CYP3A4 induction and DPD inhibition by TAS-114 in healthy volunteers by monitoring urinary excretion of cortisol and 6b-hydroxycortisol, and plasma concentration of uracil, respectively. In vitro metabolism studies suggested a major involvement of CYP3A4 in TAS-114 metabolism, and induction of CYP3A4 activity by TAS-114. The possibility that plasma concentration of unchanged TAS-114 may decrease with repeated dosing (which could lead to autoinduction of enzymes) was considered. Regarding the inhibitory effect of TAS-114 on CYP and DPD activities, although the half-maximal inhibitory concentration (IC50) values for all CYP isoforms were more than 50 mM, TAS114 competitively inhibited DPD activity and the inhibition constant (Ki) value was approximately 2.1 mM. The plasma concentration of TAS-114 and the urinary excretion of cortisol and 6b-hydroxycortisol in healthy volunteers suggested that the extent of autoinduction was minimal even after repeated administration of TAS-114 at doses of 60 mg and 150 mg bid. Furthermore, plasma uracil concentrations significantly increased after the administration of a single 150 mg dose of TAS-114. These results suggested that although alteration of exposure to TAS-114 during its repeated administration at doses up to 150 mg was not caused by CYP3A4 autoinduction, the increased exposure to 5-FU was a result of DPD inhibition after TAS-114 administration at a dose of more than 150 mg. Further, in this presentation, we will discuss the relationship between in vitro properties of TAS-114 that influence CYP induction and DPD inhibition and in vivo responses.

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P194. EVALUATION OF TIME-DEPENDENT INHIBITION OF CYP3A4 IN PLATED AND SUSPENDEND HUMAN HEPATOCYTES BY MODEL INHIBITORS


J. George Zhang, Thuy Ho, Alanna L, Callendrello, Elizabeth A, Santone, Deqing Xiao and David M. Stresser Corning Gentest Contract Research Services, Corning Life Sciences, Woburn, MA Time-dependent inhibition/inactivation (TDI) is a major concern during drug discovery and development as it can cause drugdrug interactions (DDI). Human liver microsomes (HLM) are a convenient test system and are traditionally used to determine TDI potential. However, depending on the test compounds, this system may result in over or under prediction of DDI potential, possibly by failing to account for conditions found in vivo. To bridge this gap, human hepatocytes have been increasingly used for evaluation of cytochrome P450 TDI on the premise that the model provides a complete and relevant system. Using both plated (HP) and suspended (HS) hepatocytes, the TDI potential for four model CYP3A4 inhibitors - ketoconazole, verapamil, diltiazem and troleandomycin were evaluated and compared. Hepatocytes in suspension or plated cultures in a 96-well plate format were pre-incubated with the test compounds for time periods ranging from 15 to 240 min at different concentrations, prior to a four-fold dilution or a washout step and incubation with midazolam, a CYP3A4 probe substrate. The metabolite 10 -hydroxymidazolam was then quantified by LC-MS/MS to determine the enzyme activity remaining after each condition, followed by calculation of kinetic parameters KI and Kinact. Preincubation with verapamil, diltiazem and troleandomycin resulted in TDI in both methods (e.g. for diltiazem, KI ¼ 0.38 mM, Kinact ¼ 0.18 h1 in HS, and KI ¼ 0.32 mM, Kinact ¼ 0.25 h1). Ketoconazole caused a strong inhibition of CYP3A4 but was not time-dependent in either method, as expected. The data demonstrate that both plated and suspended hepatocytes methods can be used for the assessment of TDI potential. The merits of each method will be discussed.

P195. EVALUATION OF SANDWICH CULTURED HUMAN HEPATOCYTES (SCHH) AS AN IN VITRO MODEL TO ASSESS TIME DEPENDENT INHIBITION (TDI) OF CYTOCHROME P450 3A4 Aneesh Arvind Argikar1, Dung Nguyen2 and Konstantine W. Skordos3 1 Pharmaceutical Sciences, Temple University, Philadelphia, PA, 2Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, King of Prussia, PA, 3Dept of DMPK, GlaxoSmithKline, King of Prussia, PA Human liver microsomes (HLM) are the preferred in vitro model to screen for risks and determine inactivation kinetic parameters. These parameters are essential for quantitative drug interaction risk assessment. However, HLM data sometimes provide an over-estimate of the drug-drug interaction (DDI) risk compared to actual clinical observations Sandwich cultured hepatocytes (SCHH) may provide a more relevant, holistic system to study time dependent inhibition and assess DDI. Cryopreserved human hepatocytes were seeded at a cell density of 0.5 106 cells/mL in collagen coated plates. Matrigel was added after 4 hours of plating and the hepatocytes were incubated overnight at 370C, 5% CO2. Pre-incubation was performed at increasing inhibitor concentrations and time points (n ¼ 2). Midazolam was used as a probe substrate at 20 mM for 20 min. The formation of 10 -hydroxymidazolam was analyzed using UHPLC-MS/MS. The rate of inactivation (kobs) values were calculated from the natural log of the percentage of enzyme activity remaining at the end of pre-incubation with inactivators plotted against pre-incubation time. KI and kinact values were estimated from non-linear regression of kobs plotted against inhibitor concentrations by using Graphpad v.6.0. The estimated KI and kinact values were used for DDI predictions using static model. Potential mechanism based inactivation by 8 commercial compounds was studied in SCHH and in two donor lots. The KI values across the two lots were troleandomycin (6.57 mM, 1.85 mM, mibefradil (0.047 mM, 0.068 mM), verapamil (20.8 mM, 23.8 mM), diltiazem (2.12 mM, 1.44 mM) and raloxifene (50, 53). The kinact values were troleandomycin (0.082 min–1,0.052 min–1), mibefradil (0.0109 min-1,0.025 min-1), verapamil (0.060 min-1, 0.036 min-1), diltiazem (0.023 min-1, 0.015 min-1) and raloxifene (0.0804 min-1,0.0517 min-1). The predicted DDI for inactivators were troleandomycin (9.2, 10.9), mibefradil (6.6, 8.6), verapamil (2.2, 2), diltiazem (4, 3.9), and raloxifene (1.2, 1.1). Tamoxifen, dihydralazine did not show clinically relevant DDI. The assay was able to distinguish between inactivators that do or do not show clinically relevant DDI. The kinact values for troleandomycin, verapamil and diltiazem were within two fold of those reported for HLM (1). The predicted KI values were significantly higher than those reported in HLM (mibefradil was an exception). (1,2,3,4,5,6). Predicted DDI values for inactivators like troleandomycin, mibefradil, verapamil were within two fold of the clinically reported DDI values (7,8,9) and were within 2–3 fold of the DDI values reported in HLM. In general, the predicted DDI values from SCHH under predicted the DDI values in HLM reported in literature.

References 1. Xu L et.al.DMD 2009;37(12):2330–9. 2. Albaugh et.al. DMD 2012;40(7):1336–44. 3. Zhao XJ et.al. Xenobiotica 2002;32(10):863–78

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4. 5. 6. 7. 8. 9.

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Chen Q et.al Chem Res Toxicol 2002;15(7):907–14 Zhao P et.al. DMD 2005;33(6):853–61 Masubuchi Chem Res Toxicol 1998;12(10):1028–32 Kharasch et.al. Clin Pharmacol Ther 2004;76(5):452–66 Veronese et.al. J.Clin. Pharmacol 2003;43(10):1091–100 Backman et.al. Br J Clin Pharmac 1994;37(3):221–225

P196. RELATIONSHIP OF PASSIVE PERMEABILITY AND CYTOCHROME P450 (CYP) 3A4 INHIBITION


Ling Li, Julia Zhou, Amr Nouraldeen and Alan GE Wilson Department of DMPK/Toxicology and Pathology, Lexicon Pharmaceuticals Inc, The Woodlands, TX The industry standard practice to evaluate CYP inhibition is to assess the inhibition using human liver microsomes. Because CYPs are located in the endoplasmic reticulum (ER) in the cells, the inhibitors need to pass through the cell membrane to reach the target site. Therefore, the inhibitor permeability could affect the inhibition potency. In this study, we have examined the relationship between permeability and CYP3A4 inhibition, and compared the inhibition from human liver microsomes (HLM) and human hepatocytes (HH) for 30 compounds with a wide range of physicochemical properties (ALogP 0.75–3.5, and PSA 110–170). These compounds demonstrated CYP3A4 inhibition in HLM with IC50 values of less than 5 mM. Passive permeability was determined in MDCK cells. Compounds were divided into two groups with apparent passive permeability (Papp) less than 50 nm/sec and greater than 50 nm/sec, respectively. CYP3A4 activity was determined using midazolam as a probe substrate. CYP3A4 KM for midazolam 10 or 40 - hydroxylation was determined in HLM and HH. IC50 values for the selected compounds were then determined. For the compounds with Papp550 nm/sec, the IC50 values in HH were significantly higher than those in HLM (p50.003). The average-fold error (AFE) between HH and HLM IC50 values was 8 and ranged from 2 to 25. In contrast, for the compounds with Papp450 nm/sec, IC50 values were comparable with AFE of 1.2, and there was no significant difference between two groups (p ¼ 0.85). This study demonstrated that permeability did not have a major impact on the inhibitory effect for the compounds with Papp450 nm/sec, which suggests that the intracellular concentrations could be similar to extracellular concentrations. In contrast, for compounds with Papp550 nm/sec, the inhibitory potency could be significantly affected by permeability. If this is the case, the inhibition determined in HLM may overly predict the potency inside the cell. Therefore DDI prediction based on CYP inhibition in the GI tract should take into consideration of the compound permeability.

P197. DEVELOPMENT AND VALIDATION OF AN IN VITRO, SEVEN-IN-ONE CYTOCHROME P450 ASSAY FOR EVALUATION OF BOTH DIRECT AND TIME-DEPENDENT INHIBITION Dominik Dahlinger, Sabrina Du¨chting, Konrad Sydow, Sebastian Frechen and Uwe Fuhr Department of Pharmacology, Clinical Pharmacology Unit, University of Cologne, Cologne, Germany Background: Direct and time-dependent inhibition of cytochrome P450 enzymes (CYPs) are major causes of drug-drug interactions (DDIs). In particular time-dependent inhibition (TDI) characterized by a delayed and often (quasi-)irreversible loss of enzyme function was identified to provoke several of the most clinically severe DDIs [1, 2]. Although several N-in-one (aka cocktail) methods have been documented [3], to our knowledge, no method has been published which simultaneously assesses TDI of the major human CYP enzymes [4] in a single, multiple probe substrate cocktail incubation. Objective: The objective of this study was to develop an enhanced throughput method to simultaneously assess the direct as well as time-dependent inhibitory potential of xenobiotics on seven CYPs using a seven-in-one approach. Methods: An in vitro cocktail of specific, clinically relevant probes was incubated with pooled human liver microsomes for 10 minutes. The substrates were used in concentrations close to their respective Km: 220 mM caffeine for CYP1A2, 10 mM bupropion for CYP2B6, 5 mM rosiglitazone for CYP2C8, 67 mM tolbutamide for CYP2C9, 17 mM omeprazole for CYP2C19, 2 mM dextromethorphan for CYP2D6 and 3 mM midazolam for CYP3A4. In all experiments the organic solvent concentration was set at a standard of 1.0% acetonitrile. After the 10 minute incubation period, labetalol was added as an internal standard. The formation of the major metabolites was analyzed using a single, fully validated[5] , 7 minute, step-gradient, reverse-phase liquid chromatography/tandem mass spectrometry method. The direct inhibitory potential of 7 direct and 7 time-dependent reference CYP inhibitors was studied and evaluated using nonlinear regression. Subsequently, a single concentration equivalent to the IC25, determined in the previous direct inhibition experiment, was used to examine TDI of the test compounds. After a 30 minute preincubation with NADPH, a TDI was identified by a decrease in enzyme activity. Results: The inhibitory effect of each probe substrate on the activity of the other CYPs was less than 30%. The seven-in-one assay was validated against the results obtained from single substrate incubations by determining IC50 values for 7 well-characterized direct and time-dependent reference inhibitors: fluvoxamine/furafylline for CYP1A2; voriconazole/ticlopidine for CYP2B6; montelukast/gemfibrozil for CYP2C8; sulfaphenazole/tienilic acid for CYP2C9, fluconazole/fluoxetine for CYP2C19, quindine/paroxetine for CYP2D6 and ketoconazole/verapamile for CYP3A4. The resulting IC50 values (standard error) for both methods were similar with an average error of 1.24-fold (e.g. for 3A4 IC50 (cocktail) vs. IC50(single substrate): ketoconazole 0.36 0.08 mM vs. 0.58 0.11 mM; verapamile 28.1 7.8 mM vs.

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30.1 10.1 mM) and corresponded with published values. After preincubation, all prototypical time-dependent inhibitors were identified using the described IC25 inactivation experiment, whereas the direct inhibitors resulted in no significant decrease of activity. Conclusion: The presented seven-in-one assay allows for the detection and discrimination between direct and time dependent CYP inhibition. This cocktail study is a valuable tool to efficiently screen larger libraries of uncharacterized drugs for their inhibitory potential and will facilitate the determination of optimal parameters for more detailed inhibition-mechanism and inactivation kinetics experiments.


References 1. Grimm, S.W., et al., The Conduct of in Vitro Studies to Address Time-Dependent Inhibition of Drug-Metabolizing Enzymes. Drug Metabolism and Disposition, 2009. 37(7): p. 1355–1370. 2. Obach, R.S., R.L. Walsky, and K. Venkatakrishnan, Mechanism-based inactivation of human cytochrome P450 enzymes and the prediction of drug-drug interactions. Drug Metabolism and Disposition, 2007. 35(2): p. 246–255. 3. Otten, J.N., et al., An in vitro, high throughput, seven CYP cocktail inhibition assay for the evaluation of new chemical entities using LC-MS/MS. Drug Metab Lett. 5(1): p. 17–24. 4. FDA, Guidance for Industry, Drug Interaction Studies. Available from URL http://www.fda.gov/cder/guidance/index.htm. 2012. 5. FDA, Guidance for Industry, Bioanalytical Method Validation. Available from URL: http://www.fda.gov/cder/guidance/ index.htm. 2001.

P198. AUTOMATED UPLC-MS/MS ASSAYS FOR REGULATORY-COMPLIANT ASSESSMENT OF REVERSIBLE AND METABOLISM DEPENDENT INHIBITION OF HUMAN CYP ENZYMES David Wilkinson, Anthony Glazier, Sarah Andrews, Emma Alexander, Thomas Heeley, Iain Tipping and David Lankester Metabolism, Covance Laboratories Ltd, Harrogate, United Kingdom Cytochromes P450 catalyse the biotransformation of numerous pharmaceutical, environmental and endogenous compounds across a structurally diverse range. Due to the broad binding capacity of the cytochromes P450, xenobiotic interactions at the enzymatic level are of considerable pharmacokinetic and toxicokinetic importance. Selective substrates are utilised as markers for activities associated with individual cytochrome P450 (CYP) isoforms in vitro for assessing interaction potential in vivo. In this presentation, we describe the use of the Hamilton Microlab STAR liquid handling platform for the complete automation of human liver microsome-based inhibition assays for all of the major human CYP enzymes, with analyte quantification by UPLCMS/MS. A bespoke deck configuration incorporating ancillary devices for heating, cooling and centrifugation, coupled with fast, sensitive UPLC-MS/MS detection has enabled us to develop rapid, precise, accurate and reproducible assays for the purpose of characterising test substance-mediated inhibition potential. The performance of these assays and their utility in studies which comply with the 2012 FDA and EMA guidelines for the evaluation of drug interaction potential is discussed. In addition, data generated from 4400 assays will be summarised with respect to the effects of common organic solvents and extended preincubation times (used in metabolism dependent inhibition assays) on basal enzyme activities of the major human CYP isoforms.

P199. HUMAN HEPATOCYTE BASED CYP3A4 INHIBITION IC50-SHIFT ASSAY – PREDICTION OF CLINICAL DRUG-DRUG INTERACTIONS Jacob A. Olsen SCP Disposition, Safety & Animal Research, Frankfurt, Germany, Sanofi, Frankfurt am Main, Germany Assessment of a NCE CYP450 inhibition potential is an important parameter for preclinical and clinical development and is conventionally evaluated first in vitro using a human liver microsomal (HLM) based test system.1 In vitro HLM CYP inhibition data successfully predict clinical drug-drug interactions. Despite the success of HLM based in vitro systems other possible more predictive in vitro system has lately been reported in the literature. In particular CYP inhibition data from in vitro systems based on cryopreserved human hepatocytes have been claimed to be more predictive than data obtained with HLM.2,3 Human hepatocytes are a more liver like in vitrosystem and possess the full spectrum of metabolic activities in contrast to human liver microsomes. An in vitro CYP450 inhibition test system based on pooled cryopreserved human hepatocytes was established in order to investigate if this test-system results in better quantitative prediction of observed clinical drug-drug interactions for CYP3A4. The selected cryopreserved human hepatocyte pool of 10 donors possessed the expected metabolic activity towards all of the major CYP450 enzymes and the cell viability was found to be above490%. The most optimal assay conditions for the two CYP3A4 substrates midazolam and testosterone were determined; a hepatocyte cell density of 0.3 106 cells/ml was determined to fulfill the condition of initial velocity. The Km values of midazolam and testosterone were determined and the approximate Km value of 5 mM and 100 mM were utilized in the assay. Under these conditions an IC50-shift assay with midazolam and testosterone was validated with 4 known CYP3A4 inhibitors; ketoconazole, erythromycin, troleandomycin and azamulin. For the 3 known

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mechanism-based CYP3A4 inhibitors a significant increase in CYP3A4 inhibition was observed as expected upon preincubation. The AUCi/AUC ratio was predicted from the hepatocyte data and from HLM data using a mechanistic static model approach and compared with the clinical observed data.1 In conclusion, prediction of the AUCi/AUC ratios for the 3 evaluated inhibitors from the hepatocyte inhibition data was not improved compared to HLM data based predictions.

References 1) Guidance for Industry, Drug Interaction Studies, FDA, 2012. 2) Zhao, P. Expert Opin. Drug Metab. Toxicol. 2008, 4(2),151–164. 3) Chen, Y. et al Drug Metab. Dispos. 2011, 39(11), 2085–2092.


P200. A COMPARISON BETWEEN A NEW BRONCHODILATOR CONTAINING A THIOPHENE RING AND TIENILIC ACID, IN AN IN VITRO STUDY OF MECHANISM-BASED INACTIVATION OF CYP450 Michela Salvadori, Giancarlo Aquino, Benedetta Riccardi and Paola Puccini preclinical-PKBM, Chiesi Farmaceutici, Parma, Italy The in vitro assessment of mechanism-based inactivation (MBI) of cytochrome P450 enzymes is requested by regulatory agencies to evaluate the potential drug-drug interaction and the safety of a new chemical entity. The formation of chemically reactive metabolites able to inhibit the oxidation reactions catalized by P450 could induce serious changes in drug metabolism up to toxicity (1). In the present work, CHF 5407, an antimuscarinic compound containing a thiophene ring was investigated to evaluate a possible MBI of CYP450. The diuretic agent tienilic acid, a P450 2C9 inactivator, was used as positive control. The thiophene ring of the tienilic acid undergo a selective P450 2C9-mediated bioactivation, leading to the formation of a reactive intermediate that covalently modify the enzyme, resulting in immunoallergic hepatitis (2)(3). In the present study, MBI and potential formation of reactive metabolites were assessed by the following in vitro methods: (a) Inhibition study of CYP450selective marker activity by the tested compound. In this assay a reversible or a time-dependent mechanism of inhibition can be evaluated: the residual enzymatic activity was determined after a preincubation of 30 minutes of the drug compound with or without b-NADPH and human liver microsomes (4); (b) Investigation of P450-mediated toxicity in an human hepatocarcinoma cell line (HepG2), wild-type and transfected with the CYP450 isoforms of interest. The cellular toxicity was evaluated with a viability assay based on the cleavage of the tetrazolium salt (WST) to formazan, by cellular mitochondrial dehydrogenase of living cells; (c) Detection of glutathione adducts, as stable products that follow the conjugation of the reactive metabolites and glutathione, in rat and human liver microsomes and S9 fractions using MRM scan. The data obtained suggested that the tested compound does not behave like mechanism-based inactivator of CYP450. Moreover, tienilic acid produced a reactive metabolite capable to bind glutathione not only in the presence of liver microsomes (as expected), but also in their absence, suggesting that tienilic acid has a structural reactivity per se such as to easily react with nucleophilic compounds like glutathione.

References (1) (2) (3) (4)

S. W. Grimm et al., Drug. Metab. Dispos. 2009, 37: 1355–1370. J. P. O’Donnell et al., Drug. Metab. Dispos. 2003, 31: 1369–1377. C. Y. Chan et al., Toxicol. Lett. 2011, 206: 314–324. C. L. Crespi et al.Poster P192, 9th International ISSX Meeting, Istanbul.

P201. SUBSTRATE-SPECIFIC INACTIVATION OF CYP3A BY THE HIV PROTEASE INHIBITORS RITONAVIR, SAQUINAVIR AND AMPRENAVIR Faraz Kazmi1, Phyllis Yerino1, Ken Goodell1, Brian Kirby2, Brian W. Ogilvie1, David B. Buckley1 and Jashvant Unadkat3 1 XenoTech, LLC, Lenexa, KS, 2Clinical Pharmacology, Gilead Sciences, Foster City, CA, 3Department of Pharmaceutics, University of Washington, Seattle, WA HIV protease inhibitors (PIs), such as ritonavir, saquinavir, and amprenavir, produce profound and clinically significant drugdrug interactions (DDIs) by time-dependent inactivation of CYP3A enzymes. Therefore, it is surprising that these PIs often do not produce a clinically significant DDI with some CYP3A substrates when one is expected. For example, multiple dose ritonavir significantly increases midazolam AUC but has no effect on alprazolam AUC. Since CYP3A4 has multiple binding sites, we hypothesized that the PIs inactivate CYP3A enzymes in a substrate-dependent manner. Therefore, in the present study, we evaluated the in vitro CYP3A inactivation kinetics of ritonavir, saquinavir or amprenavir with several model CYP3A probe substrates, namely alprazolam, testosterone, nifedipine, alfentanil, or midazolam. Inactivation of CYP3A enzymes in human liver microsomes (HLM) was quantified by determining the maximum inactivation rate constant (kinact) and the inactivation

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constant (KI). Inactivation incubations were performed with NADPH-fortified pooled human liver microsomes (HLM) at 0.3 mg/ mL for 1–5 min with ritonavir (0.03–1 mM), 5–30 min for saquinavir (0.3–30 mM), and 2.5–20 min for amprenavir (0.3–10 mM), followed by a 10-fold dilution (to 0.03 mg/mL HLM) and a 5 min probe substrate incubation at the Km for each substrate. CYP3A activity was measured by LC/MS analysis of alprazolam a-hydroxylation, testosterone 6b-hydroxylation, nifedipine oxidation, alfentanil N-dealkylation, midazolam 10 -hydroxylation and midazolam 4-hydroxylation, respectively. For ritonavir, the inactivation potencies (kinact/KI) were within 3-fold across all substrates (300–960 mM1 min1). Saquinavir kinact/KI values were also similar (2 mM1 min1) across substrates. Amprenavir kinact/KI values were similar between midazolam (10 -hydroxylation, 49 mM1 min1; 4-hydroxylation, 42.5 mM1 min1), alprazolam (29 mM1 min1) and alfentanil (57 mM1 min1); however, an approximate 5- and 10-fold difference was observed with testosterone (242 mM1 min1) and nifedipine (497 mM1 min1), respectively. Assuming that testosterone and nifedipine are selective CYP3A probe substrates in HLM, these data suggest that some HIV protease inhibitors, such as amprenavir, may inactivate CYP3A in a substrate specific manner. These findings may provide a rationale for previously observed paradoxical clinical DDI with HIV PIs. In addition, these findings have implications for choice of probe substrates for accurate IVIVE of DDI where CYP3A inactivation is involved. Additional studies with recombinant enzymes are needed to confirm these findings.

P202. COMPARISON OF INHIBITION OF CYP1A2, 2C9 AND 3A4 USING HUMAN LIVER MICROSOMES AND HEPATOCYTES Timothy A. Moeller1, Tracy Worzella2, Dongping Ma2, James J. Cali2 and Bradley R. Larson3 1 Celsis In Vitro Technologies, Baltimore, MD, 2Promega Corporation, Madison, WI, 3BioTek Instruments, Winooski, VT Determination of inhibition of drug metabolizing enzymes is a key component to assessing the risk of drug-drug interactions (DDI). Inhibition may lead to greater bioavailability and lower clearance of concomitant drugs which will alter efficacy and duration that may lead to toxicities and dysfunction. Due to this importance, drug candidates are screened for potential inhibition during drug discovery and development. Typically, this is performed using human liver microsomes (HLM), a simple system of membrane bound enzymes such as cytochrome P450s (CYPs) which are responsible for the majority of phase I drug metabolism in humans. However, growing evidence indicates CYP inhibition may be influenced by factors that are absent from microsomes. Passive membrane impermeability, transporters and cytosolic enzymes may affect the intracellular concentration of drug and thereby alter its effective inhibition potential. The drug, too, may be sequestered in intracellular compartments such as lysosomes which will decrease the availability of the drug to elicit inhibition of an enzyme. To better predict in vivo inhibition potential, whole cell incubations with hepatocytes are recommended to incorporate such influences that are lacking in microsomal preparations. Here, we designed an automated high-throughput screen (HTS) system utilizing both HLM and human hepatocytes to be performed in 384-well format with luminescent probes for CYP1A2, 2C9 and 3A4. Z’-factor for both HLM and hepatocytes utilizing the three substrates were all greater than 0.7, indicating an excellent assay system. Twelve inhibitors (four specific inhibitors per CYP) were profiled against the three CYPs in an 11-point concentration response curve. IC50 values were determined and compared between HLM and hepatocyte incubations. Alpha-napthoflavone provided approximately 9-fold higher IC50 in hepatocytes versus HLM, while less than 3-fold difference for fluvoxamine, furafylline and propranolol. The inhibitors for CYP2C9 (diclofenac, fluconazole, fluoxetine and sulfaphenazole) provided similar IC50 between the two systems. For CYP3A4, ketoconazole IC50 in HLM was 4-fold lower than in hepatocytes. Ritonivir and verapamil IC50 were higher in HLM than hepatocytes by 8- and 11-fold, respectively. Troleandomycin provided similar IC50 between the two systems. The design of parallel experiments using HLM and hepatocytes allows for comprehensive profiling of inhibition potentials. Differences between the two systems may be elucidated to determine mechanism of action which may aid in designing a more efficient and informative in vivo study.

P203. VARIATION IN THE IN VITRO INHIBITION OF CYP3A4, CYP2D6 AND CYP1A2 BY DIFFERENT COMMERCIAL RHODIOLA ROSEA PRODUCTS Ole Kristian F. Thu1, Bent H. Hellum1 and Odd Georg Nilsen2 1 Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway, 2Department of medicine, Norwegian University of Science and Technology, Trondheim, Norway Purpose: Rhodiola rosea is a popular natural remedy produced by several different companies with a worldwide distribution. Rhodiola rosea has earlier been shown to be a strong inhibitor of CYP3A4 in vitro [1] and the presence of CYP3A4 both in the small intestine and liver opens for possibilities of metabolic interactions also in vivo. The aim of this study was to evaluate how different commercial products of Rhodiola rosea inhibit CYP3A4, CYP2D6 and CYP1A2 (CYP) activities in vitro. Methods: Six commercial Rhodiola rosea products were investigated; ‘‘Nuodikang’’ (Tibet, granulate), ‘‘Super Rhodiola’’ (USA, granulate), ‘‘Artic Root’’ (Sweden, tablet), ‘‘Rosenrot Vitalas’’ (Sweden, tablet), ‘‘Rosenrot Forte’’ (Norway, tablet) and ‘‘Rosenrot tincture’’ (Norway, tincture). Extraction was performed with 50% ethanol. CYP3A4, CYP2D6 and CYP1A2 activities were

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measured by incubations for 10, 25 and 20 minutes at 37 C with human CYP isolated from baculovirus-infected cell system with testosterone, dextromethorphan and phenacetin as substrates, respectively. Incubation was stopped with ice-cold methanol (CYP3A4) or acetonitrile (CYP2D6 and CYP1A2). The formation of the substrate metabolites 6-OH-testosterone (3A4), dextrorphan (2D6) and acetaminophen (1A2) were determined by a validated high performance liquid chromatography methodology. IC50 concentrations were calculated using non-linear regression. Ketoconazole, quinidine and b-naphtoflavone were used as positive inhibition controls, respectively. Results: The IC50 inhibition constants of the commercial products ranged from 7.2-186 mg/ml. CYP3A4 was significantly more inhibited than CYP1A2 and CYP2D6 with IC50 concentrations (mg/ ml SD): 7.2 0.7 (Rosenrot Tincture), 11.6 1.1 (Artic Root), 13.1 0.4 (Rosenrot Vitalas), 20.4 1 (Super Rhodiola), 29.5 1.4 (Goldenroot) and 106.6 3 (Rosenrot Forte). The ‘‘Rosenrot tincture’’ was the strongest and ‘‘Rosenrot Forte’’ the weakest inhibitor of all the investigated enzymes. Conclusions: The investigated commercial products had a significant difference in their CYP inhibition potential. Based on the daily recommended dosages the ‘‘Rosenrot tincture’’, ‘‘Artic Root’’ and ‘‘Rosenrot Vitalas’’ will reach their IC50 concentrations of CYP3A4 in the small intestine. The ‘‘Rosenrot tincture’’ might reach the IC50 concentration of CYP3A4 and CYP1A2 in the liver after repeated dosing, all dependent on the bioavailability of the active components of Rhodiola rosea. No interaction effect is expected in vivo on CY2D6.


References 1. Hellum, B.H., et al., Potent in vitro inhibition of CYP3A4 and P-glycoprotein by Rhodiola rosea. Planta Medica, 2010. 76(4): p. 331–8.

P204. GOSSYPOL EXHIBITS POTENT INHIBITORY EFFECTS TO HUMAN CARBOXYLESTERASES Zhao-Ming Liu1, Guang-Bo Ge1, Jing Ning1, Chao Zou1, Jie Hou2 and Ling Yang1 1 Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Dalian Medical University, Dalian, China Gossypol, a naturally polyphenolic compound isolated from cottonseed, was used as a male antifertility agent. Recently, it was also found to exhibit inhibitory activity against a wide type of human carcinoma cell lines including human T-47D breast cancer cells, human SW-13 adrenocortical carcinoma, endometrial (RL95-2) and ovarian (SKOV-3) cell lines. Carboxylesterases (CES), a member of serine hydrolase superfamily widely distributed in the human body, played an important role in the bioactivation and hydrolytic metabolism of various ester or amide drugs, were the major determinants of the pharmacological and pharmacokinetic behaviors of many therapeutic agents containing ester or amide. The present study aimed to investigate the inhibitory effects of gossypol towards the hydrolytic activities of three recombinant human carboxylesterase isozymes including CES1b, CES1c and CES2, in vitro. Methyl 4-nitrobenzoate (MNB) and fluorescein diacetate (FD) were used as specific substrates for CES1 (CES1b & CES1c), and CES2, respectively. The results demonstrated that gossypol can inhibit the CES1 (CES1b & CES1c) mediated hydrolysis of methyl 4-nitrobenzoate, and the CES2 mediated hydrolysis of fluorescein diacetate (FD) in a concentration-dependent manner, with the IC50 values of 8.4 mM, 8.1 mM, and 8.2 mM, respectively. The inhibition types and the inhibition kinetic parameters (Ki) of gossypol towards these human CES were further investigated, using various concentrations of CES substrates in the presence of different concentrations of gossypol. The Dixon and Lineweaver-Burk plots indicated that the inhibition types of gossypol towards three human esterases (CES1b, CES1c, and CES2) were noncompetitive inhibition, with the Ki values of 4.4 mM, 7.1 mM, and 12.0 mM, respectively. In summary, gossypol showed moderate to potent inhibitory effects to human CES, and the pharmacokinetic behaviors of CES substrates could be changed by co-administration of gossypol. Given that the large contributions of human CES to the bioactivation or metabolic clearance of most therapeutic drugs containing ester or amide bonds, the potential drug-drug interactions should be paid more caution.

P205. USE OF THE CYP3A4 SELECTIVE INHIBITOR CYP3CIDE IN CYP3A5 GENOTYPED CRYOPRESERVED HUMAN HEPATOCYTES TO EXPLORE THE INDIVIDUAL CONTRIBUTION OF CYP3A4 AND CYP3A5 IN DRUG METABOLISM Scott Heyward, Rachel NV Schultz and Stephen Dennell Celsis In Vitro Technologies, Baltimore, MD The human cytochrome P450(CYP) 3A family is of critical importance to drug discovery and development due to its involvement in the metabolism of the majority of drugs on the market. Of the four isozymes within the human CYP3A family, CYP3A4 is the most abundant and well studied CYP, however the polymorphic member CYP3A5 can contribute significantly to the metabolism of many drugs, such as midazolam. The expression levels of CYP3A5 varies greatly in the population but has been reported to contribute between 2 and 60% of the CYP3A activity.1 Understanding the contribution of each CYP in individual donors is important for gauging the impact of CYP3A5 expressers on safety and efficacy of CYP3A substrates.

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CYP3cide (PF-4981517; 1-methyl-3-[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]1H-pyrazolo[3,4-d]pyrimidine) has been shown to be a potent, efficient, and specific time-dependent inactivator of human CYP3A4 in recombinant CYPs (rCYP) and liver microsomes (HLM).2 We tested the ability of this compound to function in intact cryopreserved human hepatocytes and allow the exploration of compound behavior in the absence of CYP3A4 activity. Human cryopreserved hepatocytes were genotyped using a realtime single nucleotide polymorphism assay (SNP) assay for CYP3A5*3 which encodes an early stop codon generating a non-functional protein. Inhibition of midazolam metabolism with CYP3cide in human hepatocytes gave an average IC50 of 0.2uM similar to that seen in HLM and recombinant CYP3A4.2 The IC50 was similar across genotype groups in hepatocytes. Time dependent inhibition experiments were performed measuring metabolism of midazolam to determine inactivation kinetic parameters. We utilized an 8-point dilution series over 6 timepoints to determine Kinact and Ki. Human hepatocye lots with CYP3A5 *3/*3, *1/*3 and *1/*1 genotypes were screened using optimized inhibition conditions to determine the inhibition of midazolam metabolism. Remaining activity of 1-OH-midazolam formation in the CYP3A5 *3/*3, *1/*3 and *1/*1 groups ranged from 37–94%, 53–63% and 14–25% respectively. CYP3cide allows for the investigation of the impact of CYP3A5 expression on drug metabolism, in intact genotyped cryopreserved human hepatocytes.


References 1. Williams JA, Cook J and Hurst SI.(2003) A Significant Drug-Metabolizing Role For CYP3A5?. Drug Metab Dispos 31:1526–1530 2. Walsky RL, Obach RS, Hyland R, Kang P, Zhou S, West M, Geoghegan KF, Helal CJ, Walker GS, Goosen TC, and Zientek MA. (2012) Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs. Drug Metab Dispos 40:1686–1697

P206. SKIN METABOLISM OF PRO OR PRE HAPTENS: ITS ROLE IN ALLERGIC CONTACT DERMATITIS Joan Eilstein, Guillaume Lereaux, Alexia Garrigues-Mazert and Daniel Duche´ Predictive Methods and Models Development, L’Oreal Research and Innovation - Advanced Research, Aulnay sous Bois, France Although known as a barrier function to its environment, Skin is also an extra hepatic metabolizing organ. It can be involved in the metabolism of chemicals which could have penetrated it. Consequently, skin metabolism impacts the bioavalibility of chemicals and their ability to trigger skin injury such as allergic contact dermatitis (ACD). ACD results from the interaction between haptens (chemicals with low molecular weight and an electrophilic site) and endogenous proteins to form antigenic complexes. 2,4-Dinitrochlorobenzene (DNCB), p-phenylenediamine (PPD) and Cinnamic alcohol (CIN-OH) are very well known hapten, pre-hapten and pro-hapten, respectively. In this work, we studied the cutaneous metabolic fate of these compounds using the reconstructed human skin model EpiskinÔ from SkinEthicÔ Laboratories. Results showed that DNCB, PPD and CIN-OH are biotransformed by the skin model through glutathione S-transferase, N-acetyltransferase and alcohol/aldehyde deshydrogenase activities, respectively. Apparent enzymatic parameters such as affinity (Km), maximal velocity (Vmax) and in vitro metabolic clearance (estimated by the Vmax/Km ratio), were calculated from metabolites assay in dose-effect studies. For GST activities its glutathione cofactor (GSH) was also quantified. We observed that: DNCB is detoxified giving the glutathione-adduct DNP-SG (apparent Vmax/Km ratio: 1.0 0.2 mL.min1.mg prot1). Also, the skin is able to biosynthesized GSH until 32 mM of DNCB after what the DNP-SG formation is impacted by a lack of cofactor. PPD is detoxified to monoacetylated PPD (apparent Vmax/Km ratio: 0.6 0.02 mL.min1.mg prot1) which is also quickly biotranformed into diacetylated PPD. Monoacetylated PPD being more affine for the NAT than the PPD impacts the detoxification of PPD itself. ALDH activity (0.8 0.1 mL.min1.mg prot1) was higher than that of ADH (0.2 0.1 mL.min1.mg prot1). Regarding these results, ACD process would be triggered more likely when the local concentration of aldehyde exceeds the capacity of ALDH isozymes to metabolize it. In conclusion, while a generally accepted idea that a compound is bioactivated by the skin to become toxic, findings show that, for the metabolic pathways here studied, skin reveals a more detoxifying behavior than a bioactivating one. However, the toxicity (ACD) can appear when the local concentration of compounds exceeds the capacity of the isozymes to metabolize it, especially when the skin is deficient in the gene expression of a key isoform (e.g. ALDH) or when a cofactor is running out. In the case of ACD, the kinetic parameters between the detoxification velocity of pro, pre or haptens, mediated by cutaneous metabolizing enzymes and the reaction velocity to turn pro or pre-haptens into haptens chemically and the reaction velocity between haptens and proteins have to be considered to assess the hazard.

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P207. METABOILSM AND ELIMINATION OF GYRAR COMPOUND, AZ1419; CONTRIBUTION OF GUT BACTERIA


Karthick Vishwanathan1, Jian Guo2, Linda Otterson3, Michael Huband3 and Scott Grimm2 1 Infection iMED DMPK, AstraZeneca Pharmaceuticals, Waltham, MA, 2Infection DMPK, AstraZeneca Pharmaceuticals, Waltham, MA, 3Infection Bioscience, AstraZeneca Pharmaceuticals, Waltham, MA Understanding drug metabolism and elimination pathways is important for human dose predictions and in vitro in vivo correlation assessments. Drug-related components are usually eliminated via renal excretion into urine or biliary excretion into feces although other mechanisms can come into play and are therefore important to explore. Fecal elimination after an IV dose can come from the liver via bile or from the gut after circulation in blood. To understand metabolism and elimination route, a novel Gyrase inhibitor compound, (no AZ#) was administered IV to bile duct-cannulated rats. The distribution of the compound was also evaluated after administration to male Long Evans rat in a Quantitative whole body autoradiography study. Unusual metabolites were observed in feces and differences in patterns of elimination were observed. Feces collected after an IV dose contained several components which were neither unchanged drug nor metabolites normally formed by liver enzymes or seen in other matrices (bile, urine and/or blood). A study in mouse showed the same phenomenon observed in rats. One potential source of these oxidative products is metabolism in the gut, but these products were not observed when the compound was studied with intestinal microsomes. Further evaluation identified that these products were formed by gut microflora and not by liver or gut metabolism. In addition, the distribution and elimination of radioactivity observed from a QWBA study coincides very well with the metabolism and elimination seen in the mass balance study. Differentiating between bacterial and hepatic metabolism help us better rationalize the in vitro-in vivo clearance correlations and an improved mechanistic understanding of the metabolism and elimination of this compound, therefore increase the confidence in the human PK predictions.

P208. FMO1-MEDIATED COPE ELIMINATION IN THE METABOLISM OF E7016 IN HUMAN KIDNEY S9 Y. Amy Siu1, George A. Moniz2, J. Eric Carlson3, Eric T. Williams1, James Cutter2, Y. Nancy Wong1, and W. George Lai1 1 DMPK-Andover, Biopharmaceutical Assessments, Eisai Inc, Andover, MA, 2Process Research-Andover, Eisai Inc, Andover, MA, 3NEXSY Library Strategy, Synthesis, and Purification Eisai Inc, Andover, MA The role of renal flavin-containing monooxygenase 1 (FMO1) in the metabolism of E7016 was investigated. E7016, an inhibitor of poly (ADP-ribose) polymerase, is being developed for anticancer therapy. A previous study showed a unique metabolic pathway that involves a two-step oxidation process: dehydrogenation of the 4-hydroxypiperidine to a 4piperidinone followed by a FMO1-mediated ring-opened metabolite via a Michael Acceptor intermediate [1]. We hypothesize this unique FMO1-mediated metabolic pathway follows a mechanism resembling Cope elimination, the N-oxide of a tertiary amine leads to the formation of a hydroxylamine and an alkene. In the case of E7016, the intermediate of 4piperidinone could be metabolized to an N-oxide by FMO1 followed by Cope elimination to the Michael Acceptor that could further be trapped with either water or glutathione. In humans, FMO1 is mostly expressed in adult kidney [2]. To verify the proposed mechanism, the water adduct of the Michael Acceptor was generated by incubating E7016 with human kidney S9 fraction or recombinant human FMO1 that was fortified with human kidney cytosol in the presence of NAD(P)þ. Oxidized form of the cofactor was necessary for the conversion of 4-hydroxypiperidine to 4-piperidinone in the first oxidation step, during which the cofactor could be reduced and provided for the FMO1-mediated second oxidation step. The results showed this metabolism was time-dependent as well as cofactor-dependent. The metabolite formation was more favorable using NADþ rather than NADPþ as the cofactor, with about 2-fold greater turnover. The overall metabolic pathway could be inhibited by methimazole, or briefly heating the microsomes at 50 C. The alcohol dehydrogenase inhibitor 4-methypyrazole blocked 25% and 75% of the metabolite formation in rFMO1 and kidney S9 incubations, respectively. The selectivity of FMO1 for this metabolic pathway was verified by comparing the metabolite formation in recombinant enzymes of the major CYP and FMO forms. Additionally, the FMO1-mediated metabolite could be chemically generated by reaction of the 4-piperidinone with hydrogen peroxide, and additional synthesis and structural confirmation of this metabolite by NMR is ongoing.

References [1] Lai WG, Farah N, Moniz GA, and Wong YN, 18th Regional ISSX Meeting Oct 14–18 2012: Abstract P205. [2] Yeung CK, Lang DH, Thummel KE, Rettie AE(2000) Immunoquantitation of FMO1 in human liver, kidney and intestine. Drug Metab Dispos 28:1107–1111.

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P209. REGULATION OF REDOX-DEPENDENT GENE EXPRESSION UNDER OF DRUG RESISTANCE OF CANCER CELLS Elena Kalinina, Nikolay N. Chernov and Maria Novichkova Biochemistry, Peoples’ Friendship University of Russia, Moscow, Russia


The aim of the present study was the study of expression of redox-dependent genes (isoforms of glutaredoxin, thioredoxin, peroxiredoxin) under development of cancer cells resistance to anticancer agent doxorubicin (DOX) possessed pro-oxidant action. Under development of resistance of human erythroleukemia K562 and ovarian carcinoma SKOV-3 cells to DOX coordinative enhanced expression of genes encoding glutathione synthetase(GS), heavy and light subunits of g-glutamylcysteine synthetase (g-GCSH, g-GCSL, g-GCS, respectively) was found in resistant cells in compare with wild cells. In addition, growth of GSH/GSSG ratio as index of cellular redox state, enhanced expression of redox-regulated genes of isoforms of glutaredoxin (GLRX1, GLRX2), thioredoxin (TRX2), peroxiredoxin (PRDX6) as well as elevated level of transcription factor Nrf2 were observed in both types of resistant cells. We suggest that mechanism of development of cancer cells resistance to doxorubicin causes adaptive antioxidant response to its pro-oxidant action. Its can include activation of GSH synthesis de novo owing to the increase of expression of -GCSH, -GCSL and GS genes, redox-dependent elevation of expression of GLRX1, GLRX2, TRX2, PRDX6 which are co-ordinative regulated by redox-dependent transcription factor Nrf2 and elevated level of GSH/GSSG ratio.

P210. ENERGY SENSING COACTIVATOR PGC-1 REGULATES NOVEL MOUSE HEPATIC CYP GENES INCLUDING VITAMIN D METABOLIZING CYP24A1 AND CYP2R1 S.M. Aatsinki, M. Buler, P. Viitala and J. Hakkola Pharmacology and Toxicology, University of Oulu, Oulu, Finland PGC-1a (PPAR-g-coactivator-1a) is a master regulator of gluconeogenesis in liver during times of metabolic challenge such as fasting. PGC-1a regulates also both mitochondrial function and biogenesis in energy-demanding tissues such as heart and skeletal muscle in addition to liver. PGC-1a requires the help of transcription factors to facilitate gene activation of target genes. The liver-enriched HNF-4a, PXR, CAR, PPARs and other nuclear receptor (NR) family members are coactivated by PGC-1a. In fact, PGC-1a has been shown to activate the transcription of several members of cytochrome P450 (CYP) family which are responsible for the xenobiotic and endobiotic metabolism in liver, such as Cyp2a5 and 2A6, CYP7A1, CYP17A1, CYP11A1 with the aid of NRs. Hypothesis: We show that PGC-1a regulates novel set of CYP genes in mouse primary hepatocytes. These include vitamin D metabolizing CYP24a1 and CYP2r1 which we hypothesize to be regulated by PGC-1a in conditions such as fasting and type II diabetes. Methods: Mouse primary hepatocytes were used as basic model to study the over-expression of PGC-1a by adenoviral transduction. In vivo fasting for 12 and 24 hours was performed using DBA/2 mice. The phenomenon was also studied in continuous cell lines. mRNA and protein expression were measured by real-time qPCR and Western blotting. Results: The over-expression of PGC-1a resulted in the induction of Cyp24a1, Cyp17a1, Cyp2s1, Cyp4a12, Cyp3a25 and consequently down-regulation of Cyp2r1 and Cyp39a1. These genes are mainly involved in endogenous metabolism of e.g. retinoids, androgens, eicosanoids and fatty acids in the liver. Interestingly, CYP24a1 and Cyp2r1 are both involved in vitamin D biosynthesis pathway. Cyp2r1 is considered the major hepatic 25-hydroxylase resulting in the activation of biologically active form of vitamin D3. Conversely, Cyp24a1 is the major vitamin D3 inactivating enzyme. In the livers of 12 and 24 hours fasted mice the Cyp2r1 expression was dramatically down-regulated while Cyp24a1 could not be detected. Conclusion: The results imply an overall down-regulation of circulating active vitamin D3 by the energy-sensing coactivator PGC-1a and possibly the metabolic conditions like fasting or type II diabetes.

P211. ROLE OF ER STRESS AND PPARALPHA IN THE REGULATION OF CYP3A4 BY FGF21 IN A MODEL OF NON-ALCOHOLIC FATTY LIVER DISEASE Sarah J. Woolsey1, Christopher Pin1, Melanie Beaton2, Richard B. Kim3 and Rommel G. Tirona3 1 Physiology and Pharmacology, The University of Western Ontario, London, ON, Canada, 2Department of Medicine, Lawson Health Research Institute, London, ON, Canada, 3Division of Clinical Pharmacology, The University of Western Ontario, London, ON, Canada Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease typically associated with obesity, diabetes, dyslipidemia and hypertension. We have previously shown that Fibroblast Growth Factor 21 (FGF21) levels are increased in patients with NAFLD as well as in human hepatoma (Huh7) and mouse models of the disease. Importantly, we have demonstrated that FGF21 downregulates CYP3A4 expression leading to decreased drug metabolism. Here, we investigated the roles of endoplasmic reticulum (ER) Stress and Peroxisome Proliferator Activated Receptor a (PPARa) signaling pathways in the regulation of FGF21 and CYP3A4. The mRNA expression levels of FGF21, CYP3A4, ER stress markers (ATF3/ATF4), and PPARalpha target genes (PDK4) were analyzed in Huh7 cells treated with either fatty acids to induce steatosis, thapsigargin to

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induce ER stress, or fenofibrate to activate PPARalpha signaling. A 50% reduction in CYP3A4 levels, an upregulation of FGF21 above 20 fold, and an upregulation in ATF3/ATF4 expression of at least 3 fold, was observed in fatty acid and thapsigargin but not fenofibrate treated cells. As expected, fenofibrate induced the expression of PDK4 with an observed 6-fold change. These findings indicate that induction of FGF21 expression with fatty acid loading is an ER stress response rather than a result of PPARa activation. We conclude that reduced CYP3A4 expression in an in vitro model of hepatic steatosis results primarily from fatty acid-mediated ER stress leading to induction of FGF21. These findings are expected to provide new insights for rational drug development and optimal pharmacotherapy in NAFLD.

P212. ENHANCED SENSITIVITY OF LDMM-STABILIZED HUMAN HEPATOCYTES TOWARDS CYTOKINE-INDUCED DOWN REGULATION OF GENE EXPRESSION OF P450 ISOFORMS, UPTAKE TRANSPORTERS AND EFFLUX TRANSPORTERS


Qian Yang and Albert P. Li In Vitro ADMET Laboratories LLC, Columbia, MD It is now known that cytokines are potent regulators of gene expression of P450 isoforms, uptake and transporters. It is important to develop a robust experimental approach for the evaluation of cytokine effects to aid the understanding of relationship between inflammation and drug disposition and toxicity, as well as for the assessment of the drug-drug interaction potential of biotherapeutics which are agonists or antagonists of cytokines. Due to species differences, primary human hepatocytes represent the most relevant experimental system for the evaluation of cytokine effects in hepatic gene expression. We have previously reported that a complicating factor towards the investigation of the down-regulatory effects of cytokines on P450 and transporter gene expression in primary cultured hepatocytes is the spontaneous down-regulation of the same genes with time in culture (Yang Q, Utkarsh D, Li N, and Li AP, Current Drug Metabolism 13: 938-946, 2012). We hypothesize that the down regulatory effects of cytokines could be readily quantified in hepatocytes in which spontaneous down regulation is minimized. Based on our discovery that hepatocytes cultured for 7 days in Li’s Differentiation Maintenance Medium (LDMM) consistently have gene expression for most P450 isoforms, uptake transporters and efflux transporters at levels similar to that of the first day of culture, we examined the effects of the prototypical pro-inflammatory cytokine IL-6 in the ‘‘LDMM-stabilized (LS)’’ human hepatocytes. The LS-human hepatocyte cultures were found to be highly responsive to IL-6 induction of the inflammatory gene marker C-reactive protein (CRP), suggesting the expression of IL-6 receptors and the subsequent signaling pathways. Extensive dose-and time-dependent down regulation of the gene expression of CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5, uptake transporters SLCs (10A1, 22A1, 22A7), SLCOs (1B1, 1B3, 2B1), and efflux transporters (ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, and ABCG2) were observed. The effects were substantially higher levels than that reported by others using the routine culture conditions where spontaneous down regulation occurs (e.g. 490% suppression of CYP3A4 gene expression was observed in hepatocytes from all four donors after a 48 hr. treatment period with 1 ng/mL and higher concentrations of IL-6). The high sensitivity of the hepatocytes to IL-6 is attributed to the restored and stabilized expression of the genes studied, thereby allowing a higher dynamic range of response. The results suggest that the LS-human hepatocytes represent a physiologically relevant model for investigation of the down-regulatory effects of xenobiotics and may be applicable for the evaluation of biotherapeutics.

P213. DOWN-REGULATION OF MOUSE HEPATIC CYTOCHROME P450 3A PROTEIN BY 3METHYLCHOLANTHRENE DOES NOT REQUIRE CYTOCHROME P450-DEPENDENT METABOLISM David S. Riddick1, Chunja Lee1 and Xinxin Ding2 1 Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada, 2Wadsworth Center, New York State Department of Health, Albany, NY The aryl hydrocarbon receptor (AHR)-dependent induction of cytochromes P450 (P450) such as CYP1A1 by 3-methylcholanthrene (MC) and related polycyclic aromatic hydrocarbons (PAHs) is well characterized. We reported previously that MC treatment triggers a pronounced down-regulation, particularly at the protein level, of mouse hepatic Cyp3a11, a relative of the key human drug-metabolizing enzyme CYP3A4 [1]. To determine if this effect of MC requires hepatic microsomal P450 activity, we studied liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of NADPH-cytochrome P450 oxidoreductase (POR) [2]. In vehicle-treated animals, basal levels of CYP3A11 mRNA and CYP3A protein were elevated by approximately 9-fold in LCN mice compared to wild-type mice, whereas CYP3A catalytic activity was profoundly compromised in LCN mice. MC treatment caused suppression of CYP3A11 mRNA, CYP3A protein, and CYP3A catalytic activity in wildtype mice, and the MC effects at the mRNA and protein levels were maintained in LCN mice. Hepatic Por genetic status did not influence basal levels of CYP3A13 and 3A25 mRNA, and MC caused down-regulation of CYP3A25 mRNA levels, a response that achieved statistical significance in LCN mice. Flavin-containing monooxygenase-3 (Fmo3) induction by PAHs was suggested previously to occur via an AHR-dependent mechanism involving p53 activation following conversion of the parent compound to DNA-damaging reactive metabolites [3]. However, we found that hepatic FMO3 mRNA levels were dramatically

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increased following MC treatment in both wild-type and LCN mice. MC did not function as a mechanism-based inactivator of CYP3A enzymes in hepatic microsomes prepared from untreated wild-type mice. Under the same experimental conditions, the positive control 1-aminobenzotriazole caused a marked NADPH-dependent loss of total P450 content and CYP3A catalytic activity, consistent with mechanism-based inactivation of P450s through heme adduct formation [4]. These results indicate that MC down-regulates mouse hepatic CYP3A protein via a pre-translational mechanism that does not require hepatic microsomal P450-dependent activity. [Support: CIHR, NIH-NCI]


References [1] Lee C, Hutson JR, Tzau VKF and Riddick DS. Regulation of constitutive mouse hepatic cytochromes P450 and growth hormone signaling components by 3-methylcholanthrene. Drug Metab Dispos 34(9):1530–1538, 2006. [2] Gu J, Weng Y, Zhang QY, Cui HD, Behr M, Wu L, Yang WZ, Zhang L and Ding X. Liver-specific deletion of the NADPHcytochrome P450 reductase gene: impact on plasma cholesterol homeostasis and the function and regulation of microsomal cytochrome P450 and heme oxygenase. J Biol Chem 278(28):25895–25901, 2003. [3] Celius T, Pansoy A, Matthews J, Okey AB, Henderson MC, Krueger SK and Williams DE. Flavin-containing monooxygenase-3: induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver. Toxicol Appl Pharmacol 247(1):60–69, 2010. [4] Ortiz de Montellano PR and Mathews JM. Autocatalytic alkylation of the cytochrome P-450 prosthetic haem group by 1-aminobenzotriazole: isolation of an NN-bridged benzyne-protoporphyrin IX adduct. Biochem J 195(3):761–764, 1981.

P214. GLUCOCORTICOID INDUCTION OF THE ARYL HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR IN RAT LIVER Sarah R. Hunter and David S. Riddick Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada The aryl hydrocarbon receptor nuclear translocator (ARNT) is a shared partner between the hypoxia signaling pathway and the dioxin-responsive aryl hydrocarbon receptor signaling pathway. Glucocorticoid induction of hepatic ARNT mRNA was first observed in rats treated with the glucocorticoid receptor (GR) agonist methylprednisolone [1]. Subsequently, dexamethasone (DEX), a synthetic glucocorticoid known to activate both the GR and the pregnane X receptor (PXR), was shown to increase rat hepatic ARNT mRNA levels [2]. To examine the roles of GR and PXR in this response, we treated male rats with the GRselective agonist triamcinolone acetonide (TA), the PXR-selective agonist pregnenolone-16a-carbonitrile (PCN), and several doses of DEX. At 6 h following TA treatment, there was a 7.5-fold induction of ARNT mRNA and a 4.5-fold increase in levels of an unidentified lower molecular weight protein reacting with ARNT antibody. There was a trend for increased ARNT protein levels at 24 h after TA exposure. PCN treatment had no effect on these ARNT parameters. A low dose of DEX (1 mg/kg), shown to activate GR but not PXR, caused a 10-fold induction of ARNT mRNA at 6 h along with a 6.5-fold increase in levels of the unidentified ARNT antibody-reactive protein. A trend for increased ARNT protein levels at 24 h was observed, but only at higher doses of DEX (10 to 50 mg/kg), shown to activate both GR and PXR. Preliminary results suggest that the increased expression of ARNT mRNA caused by a low dose of DEX (0.5 mg/kg) is attenuated by the GR antagonist mifepristone (RU486). These findings are consistent with a role for the GR as a key mediator of the induction of rat hepatic ARNT expression by glucocorticoids. [Support: CIHR]

References [1] Almon RR, Dubois DC, Jin JY and Jusko WJ. Pharmacogenomic responses of rat liver to methylprednisolone: an approach to mining a rich microarray time series. AAPS J 7(1):E156-E194, 2005. [2] Mullen Grey AK and Riddick DS. Glucocorticoid and adrenalectomy effects on the rat aryl hydrocarbon receptor pathway depend on the dosing regimen and post-surgical time. Chemico-Biol Interact 182 (2–3): 148–158, 2009.

P215. DUAL ROLES OF NUCLEAR RECEPTOR LXRA IN THE CYP3A4 EXPRESSION IN HUMAN HEPATOCYTES AS A POSITIVE AND NEGATIVE REGULATOR Keisuke Watanabe, Yasushi Yamazoe, Kaori Sakurai and Kouichi Yoshinari Division of Drug Metabolism and Molecular Toxicology, Gradueate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan CYP3A4 is a major human drug-metabolizing enzyme, whose expression level shows large inter-individual variations and is associated with several factors such as genetic polymorphism, nutritional, physiological and pathophysiological status, and xenobiotic exposure. Xenobiotic-responsive nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor


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(CAR), are known to be involved in the xenobiotic induction of CYP3A4, but there is little information on how physiological and nutritional status regulates CYP3A4 expression. In this study, we have investigated a possible involvement of liver X receptor a (LXRa), a critical regulator of cholesterol homeostasis, in the hepaticCYP3A4 expression since several reports suggest the involvement of CYP3A enzymes in the cholesterol metabolism in humans and mice. Reporter assays using wild-type and mutated CYP3A4-luciferase reporter plasmids and electrophoretic mobility shift assays revealed that ligand-activated LXRa transactivates CYP3A4 through the known DNA elements critical for the PXR-dependent CYP3A4 transcription, suggesting LXRa as a positive regulator for theCYP3A4 expression and also a crosstalk between PXR and LXRa in the CYP3A4 expression. In fact, LXRa co-activation attenuated the PXR-dependent CYP3A4 transcription in reporter assays. Moreover, co-treatment with the LXRa agonist GW3965 attenuated rifampicin (the strong human PXR agonist) treatment-dependent increase in CYP3A4 mRNA levels in human primary hepatocytes and differentiated HepaRG cells. Similar inhibitory effects of LXRa agonist treatment were observed on the increase in CYP2B6 mRNA levels by CAR or PXR agonist treatment. However, when LXRa expression was knocked-down with siRNA in HepaRG cells, GW3965 treatment showed no influence on the extent of CYP3A4 induction by rifampicin. Finally, we found that treatment with T0901317, the dual agonist of LXRa and PXR, activated PXR-mediated CYP3A4 expression in reporter gene assays more significantly than rifampicin at the same concentration (1 mM), but it was less effective than rifampicin for CYP3A4 induction in HepaRG cells. In conclusion, the present results suggest that sterol-responsive LXRa positively regulates the basal expression of CYP3A4but acts negatively on the PXR- or CAR-dependent CYP3A4 expression in human hepatocytes. Therefore, nutritional, physiological and disease conditions affecting LXRa’s transcriptional activity might be one of the determinants for the basal and xenobiotic-induced expression of CYP3A4 in human livers. Moreover, our results suggest that if a drug (candidate) of interest strongly induces CYP3A4 through PXR or CAR, conferring the ability to activate LXRa on it may prevent strong CYP3A4 induction in vivo in humans.

P216. RNA-SEQ REVEALS TRANSCRIPTIONAL RESPONSE OF CODING RNAS AND LONG NON-CODING RNAS TO RIFAMPICIN TREATMENT IN PRIMARY HUMAN HEPATOCYTES AND HEPARG CELLS Chad Pope1, Lai Peng2 and Xiao-bo Zhong3 1 Pharmaceutical Sciences, University of Connecticut, Storrs, CT, 2Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS, 3Department of Pharmaceutical Sciences, University of Connecticut School of Pharmacy, Storrs, CT Transcriptional response is a basic cellular process in response to environment stimuli. Transcriptional induction and repression are examples of transcriptional responses in cells in response to drug treatment. Previous studies in drug induction and repression mainly focused on the alteration of coding genes, however, recent studies have demonstrated that the majority of the human genome is transcribed in a particular cell type and many long non-coding RNAs (lncRNAs) have important functions in cell differentiation, organ development, and response to intercellular signals. The purpose of this study is to examine transcriptional responses of both coding and lncRNAs in liver cells to drug treatment. Rifampicin is selected as a model drug. The transcriptional responses were determined by RNA-Seq in primary human hepatocytes (PHH) from two individuals and the human liver cell line, HepaRG. The expression level of each gene was determined by Cufflink and presented as fragments per kilobase exon per million reads (FPKM). Approximately 9850, 9800, and 10,300 coding genes were expressed in PHH1, PHH2, and HepaRG cells, respectively. Among them, 182, 57, and 15 are upregulated (fold increase 42) and 68, 19, and 11 are downregulated (fold decrease 42), in PHH1, PHH2, and HepaRG cells, respectively. As expected, many drug metabolizing enzyme genes, such as CYP3A4, CYP2C9, CYP2B6, UGT1A1, and the transporter gene ABCB1 are induced by rifampicin in both PHH and HepaRG cells, however, compared to two duplicate HepaRG samples, two PHH samples showed significant interindividual variations in fold induction and repression after rifampicin treatment. PHH1 has a relatively higher basal level of many P450s than PHH2. Fold induction of these genes in PHH1 is significantly lower than that in PHH2 and HepaRG. Approximately 700, 700, and 1000 lncRNAs were expressed in PHH1, PHH2, and HepaRG cells, respectively. Among them, 56, 47, and 18 are upregulated (fold increase 42) and 85, 40, and 28 are downregulated (fold decrease 42), in PHH1, PHH2, and HepaRG cells, respectively. Again, two PHH samples varied considerably in the number of lncRNA genes expressed before rifampicin treatment and the level of change in expression after rifampicin treatment compared to HepaRG cells. We identified several specific lncRNAs induced by rifampicin in either PHH or HepaRG cells, such as AL162065 on chromosome 7 and AK058009 on chromosome 11. And, we also found some lncRNAs were suppressed by rifampicin, such as X65706 on chromosome 15 and AF147447 on chromosome 16. The relationship of lncRNAs to coding RNAs in response to drug treatment will be a future direction to determine the roles of lncRNAs in regulation of gene expression in response to drug treatment.

P217. INDUCTION OF RAT HEPATIC NADPH-CYTOCHROME P450 OXIDOREDUCTASE BY DEXAMETHASONE: ARE MULTIPLE RECEPTORS INVOLVED? Alex Vonk and David S. Riddick Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada

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NADPH-Cytochrome P450 oxidoreductase (POR) is the key electron transfer partner for all microsomal P450s, important catalysts of endobiotic and xenobiotic metabolism. Electron transfer to microsomal P450s is considered to be a rate-limiting step in the P450 catalytic cycle and, therefore, alterations in POR expression and activity may impact the capacity for P450dependent metabolism. Hypophysectomized and adrenalectomized rats show decreased hepatic POR activity that is rescued by thyroxine and cortisone, respectively [1,2]. Dexamethasone (DEX), a synthetic glucocorticoid known to activate both the glucocorticoid receptor (GR) and the pregnane X receptor (PXR), increases hepatic levels of POR mRNA in rats via mRNA stabilization [3]. In addition, hepatic POR protein levels are increased following DEX administration to both wild-type and Gr-null mice [4]. To study the roles of GR and PXR inPOR regulation, we treated male rats with the GR-selective agonist triamcinolone acetonide (TA), the PXR-selective agonist pregnenolone-16a-carbonitrile (PCN), and several doses of DEX. At 6 h after dosing, PCN caused a 3-fold induction of POR mRNA and a trend for increased POR mRNA levels was seen in response to TA. Neither PCN nor TA alone affected POR protein levels or cytochrome c reduction activity. A low dose of DEX (1 mg/kg), shown to activate GR but not PXR, caused a 3-fold induction of POR mRNA at 6 h, whereas a high dose of DEX (50 mg/kg), shown to activate both GR and PXR, increased POR mRNA levels by nearly 5-fold at 6 h and 4-fold at 24 h. POR protein levels and cytochrome c reduction activity were increased at 24 h after the high dose of DEX. Both GR and PXR may contribute to the rat hepatic POR mRNA induction, but activation of either receptor alone seems insufficient to elevate POR protein and activity. [Support: CIHR]

References [1] Waxman DJ, Morrissey JJ and Leblanc GA. Hypophysectomy differentially alters P-450 protein levels and enzyme activities in rat liver: pituitary control of hepatic NADPH cytochrome P-450 reductase. Mol Pharmacol 35(4): 519–525, 1989. [2] Castro JA, Greene FE, Gigon P, Sasame H and Gillette JR. Effect of adrenalectomy and cortisone administration on components of the liver microsomal mixed function oxygenase system of male rats which catalyzes ethylmorphine metabolism. Biochem Pharmacol 19(8): 2461–2467, 1970. [3] Simmons DL, McQuiddy P and Kasper CB. Induction of the hepatic mixed-function oxidase system by synthetic glucocorticoids: transcriptional and post-transcriptional regulation. J Biol Chem 262(1): 326–332, 1987. [4] Schuetz EG, Schmid W, Shutz G, Brimer C, Yasuda K, Kamataki T, Bornheim L, Myles K and Cole TJ. The glucocorticoid receptor is essential for induction of cytochrome P-4502B by steroids but not for drug or steroid induction of CYP3A or P450 reductase in mouse liver. Drug Metab Dispos 28(3): 268–278, 2000.

P218. GSK3B INHIBITORS REGULATE CYP EXPRESSION IN PRIMARY HUMAN HEPATOCYTES BY MANY WAYS: ACTIVATION OF CTNNB1, AHR AND PXR Sabine Gerbal-Chaloin1, Edith Raulet1, Philippe Briolloti1, Cedric Duret1, Jean-Marc Pascussi2, Patrick Maurel1 and Martine Daujat-Chavanieu1 1 Hepatic Differentiation of Stem Cells and Biotherapy of Liver Diseases, INSERM U1040, Montpellier, France, 2Cancer signaling, INSERM U661, Montpellier, France Background: The liver displays a large variety of functions in the body, sustained by a remarkable property known as functional zonation. Hepatocytes present highly specialized metabolic functions along the liver cord organized from the portal space to the centrilobular vein, defining respectively periportal (PP) and perivenous (PV) hepatocytes. Recently, Wnt/b-catenin pathway has been identified as the major liver ‘‘zonation-keeper’’. In the absence of Wnt ligand stimulation, b-catenin (CTNND1) is phosphorylated by a protein complex including glycogen synthase kinase 3b (GSK3b) and adenomatous polyposis coli (APC), leading to its proteasomal degradation. In the presence of Wnt ligands, b-catenin remains unphosphorylated and accumulates in the cytosol. This accumulation results in a nuclear translocation of b-catenin and a transactivation of its target genes by binding to T cell factor/lymphoid-enhancing factor transcriptional activator (TCF/Lef). Data from the literature demonstrate that CYP2E1, CYP1A2, CAR and AhR are mainly expressed in PV hepatocytes, where the Wnt/b-catenin pathway is mostly activated. In conditional null mice for b-catenin, loss of expression of several CYPs, especially CYP2E1 and CYP1A2, has been observed. Moreover, in this model the basal expression of most drug metabolism-related genes was diminished as well as the response to CAR and AhR agonists. Conversely, several cytochrome P450 isoenzymes are upregulated in liver tumours harbouring constitutive b-catenin activation. The liver conditional loss of APC causes reexpression of PV genes and the suppression of b-catenin-negative PP target genes. Aim: To identify Wnt/b-catenin target genes in primary human hepatocytes (PHH) and restore PV expression of CYPs. Methods: PHH were submitted to GSK3b inhibitors (CHIR99021: 6-((2-((4-(2,4Dichlorophenyl)-5-(4methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino) nicotinonitrile, BIO: 6-bromoindirubin-30 oxime, lithium chloride) or Wnt b-catenin activators (mWnt3a) in combination with siRNA-mediated invalidation of CTNNB1, APC, AhR or PXR. CYP and xenoreceptor expression were then evaluated. In parallel, luciferase reporter activity was assessed to evaluate CTNNB1, AhR and PXR transcriptional activation in PHH or wild type- or CTNNB1-mutated hepatoma cell lines. Results: We first show that the canonical Wnt/b-catenin pathway can be activated in PHH. We then demonstrate that CYP2E1 is a Wnt/b-catenin target gene in PHH. Its expression and metabolic activity are restored after 48H of CHIR99021 treatment.

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However, CYP1A1 and CYP1A2 expression is strongly increased after CHIR99021 and BIO stimulation but in a Wnt/b-catenin independent and an AhR dependant manner. BIO was shown to activate AhR in a nanomolar range while it activates Wnt/bcatenin at a micromolar range. Finally, we show that CYP3A4 expression is increased by CHIR99021 (but not by BIO) in a PXR dependent way. In contrast to rodents, PXR and CAR expression is not affected by modulation of the Wnt/b-catenin pathway activity. AhR expression is increased when APC expression is invalidated. Conclusion: PHH constitute a powerful model to study Wnt/b-catenin pathway activity on drug metabolism enzyme expression and regulation, and its activation could be used to restore CYP expression in primary culture. Moreover, discrepancies with rodents should be further investigated.

P219. EXPRESSION OF PH REGULATORY PROTEINS MAY INFLUENCE DRUG RESISTANCE


Lukas Klameth1, Martin Svoboda2, Theresia Thalhammer2, Ulrike Olszewski1, Klaus Geissler3, Walter Ja¨ger4 and Gerhard Hamilton1 1 Ludwig Boltzmann Cluster Translational Oncology, Vienna, Austria, 2Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria, 35. Medizinische Abteilung und Onkologie, Krankenhaus Hietzing, Vienna, Austria, 4Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria Background and aims: Small Cell Lung cancer (SCLC) is characterized by a poor prognosis, because of late diagnosis and early resistance to standard chemotherapy. Since under hypoxic conditions, tumor cells irreversibly shift their energy metabolism from oxidative phosphorylation towards an increased anaerobic glycolysis, the pH regulatory proteins carbonic anhydrases IX (CA IX) and XII (CA XII), sodium proton exchanger 1 (NHE1) and monocarboxylate transporters 1 (MCT1) and 4 (MCT4) may provide new chemotherapeutic targets. However, targeting these proteins may also influence the efficacy of standard chemotherapeutic drugs. Method and results: The influence of cisplatin, etoposide and topotecan on the expression of these pH regulating proteins in two chemoresistant SCLC cell lines, DMS-153 and NCI-H417, and one chemonaı¨ve SCLC cell line, NCI-H526, was assessed by qPCR. Protein levels of CA IX were analyzed by Western Blot. Regulation of gene expression depended on their degree of chemoresistance. CAIX mRNA downregulation in response to cisplatin treatment was seen in the higher resistant cell lines DMS-153 and NCI-H417 (0.4fold each), while CA XII mRNA expression is upregulated 2.5fold in sensitive NCI-H526 cells. NHE1 gene expression decreases in DMS-153 cells (0.5fold), while sensitive NCI-H526 and more chemoresistant NCI-H417 cells have an upregulated NHE1 gene expression to 2.5 and 1.5fold, respectively, in response to cisplatin treatment. No change in MCT1 gene expression was seen in chemoresistant cell lines in response to cisplatin, while a 0,5fold reduction was observed in the chemonaı¨ve cell line. Etoposide is able to increase MCT 1 expression by 2fold in NCI-H526 cells, but has no effects in DMS153 and NCI-H417 cells. MCT4 gene expression decreases in NCI-H526 cells (0.2fold) but increases in DMS-153 and NCIH417 cells by 2.2 and 1.8fold, respectively. Importantly, the NHE1 inhibitor zoniporide prevented cisplatin cytotoxicity resulting in an increased survival of the tumor cells. Conclusion: Targeting pH regulating proteins in SCLC cell lines may lead to an altered response to standard therapy in SCLC patients: chemotherapy influences the expression of pH regulating proteins and this has to be considered for a possible second-line therapy involving inhibitors directed to CA IX, CA XII, MCT1, MCT4 and/or NHE1.

Acknowledgement This work was funded by the ‘‘Medical Scientific Fund of the Mayor of the City of Vienna‘‘, project number 11016.

P220. 1-BROMOPROPANE INCREASES THE EXPRESSION OF PROINFLAMMATORY CYTOKINES AND SECRETION OF BETA-HEXOSAMINIDASE IN RBL-2H3 CELLS Hwa Jeong Han1, Hyung Gyun Kim1, Tae Cheon Jeong2 and Hye Gwang Jeong1 1 College of Pharmacy, Chungnam National University, Daejeon, South Korea, 2Yeungnam University, College of Pharmacy, South Korea 1-BROMOPROPANE INCREASES THE EXPRESSION OF PROINFLAMMATORY CYTOKINES AND SECRETION OF beta-HEXOSAMINIDASE IN RBL-2H3 CELLS Hwa Jeong Han 1, Hyung Gyun Kim 1, Tae Cheon Jeong 2, Hye Gwang Jeong 1,* 1 Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon, South Korea 2 College of Pharmacy, Yeungnam University, Gyeongsan, South Korea *Corresponding author: [email protected] Mast cells are secretory cells that are essential to specific and innate immunity, allergic responses, and inflammation. Recently, 1-BP was known to inflammatory effect, but the mast cell degranulation and allergic responses is unclear. In this study, we investigated secretion of proinflammatory cytokines and degranulation via Ca increase in RBL-2H3 cells mediated with 1-Bromopropane. 1-BP significantly increased release of beta-hexosaminidase and histamine in mast cells. 1-BP also increased the production and mRNA expression of proinflammatory cytokines such as IL-4, IL-5 and COX-2. In addition, 1-BP increased IL-4, IL-5 promoter and NF-AT-driven luciferase activity. Furthermore, 1-BP increased phosphorylation of MAP kinase (p-38, p-Erk1/2, and

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p-JNK1/2), Akt and Ca2þ/calmodulin- dependent kinase (CaMKII). 1-BP increased influx of extracellular Ca2þ in RBL-2H3 cells. 1-BP induced release of beta-hexosaminidase and mRNA expression of IL-4, IL-5 and COX-2 was inhibited by treatment with a p38 inhibitor (SB203580), an Erk inhibitor (PD98059), a JNK inhibitor (SP600125), and a PKC- inhibitor (GO6976), a Calcium inhibitor (W7). Therefore, these results clearly demonstrated that 1-BP enhances mast cell-derived allergic inflammatory reactions by inducing b-hexosaminidase, histamine release and proinflammatory cytokine expression via extracellular calcium influx. Key words: 1-Bromopropane, beta-hexosaminidase, histamine, degranulation

P221. EXPRESSION OF POORLY CHARACTERIZED MEMBERS OF SDR SUPERFAMILY IN HUMAN TISSUES


Beata Malcekova1, Jakub Hofman1, Lucka Skarydova1, Petra Matouskova1, Eva Novotna1, Hana Stambergova1, Miroslav Safr2 and Vladimir Wsol1 1 Department of biochemical sciences, Charles University in Prague, Faculty of Pharmacy, Hradec Kra´love´, Czech Republic, 2 Institute of Legal medicine, Medical Faculty of Charles University and University Hospital, Hradec Kra´love´, Czech Republic A variety of compounds of endogenous or xenobiotic origin containing carbonyl groups are metabolized into corresponding alcohols by NAD(P)H-dependent reductases. Carbonyl reduction is a significant step inbiotransformation of endogenous (aldehydes, steroids, prostaglandins, retinoids and fatty acid derivatives) and xenobiotic (NNK, alfatoxin B1, quinones, doxorubicin and daunorubicin) compounds (Skarydova L. and Wsol V. 2013). Based on structural and functional data, carbonyl reducing enzymes have been generally classified into two distinct protein superfamilies: the short-chain dehydrogenases/ reductases (SDRs) and aldo-keto reductases (AKRs). SDRs represent one of the largest and oldest groups of enzymes, whose members occur in all life forms including humans. The SDR superfamily currently contains more than 160 000 primary structures available in sequence databases (Persson B. and Kallberg Y. 2013). The SDR enzymes also play an important role in some serious pathological diseases as some types of cancer, obesity, metabolic syndrome and endometriosis (Jansson A. 2009, Morton N.M. 2010). Over 75 SDR genes have been identified within the genome of human beings (Persson B. and Kallberg Y. 2013). The most of the known information refers about the role of cytosolic forms of carbonyl reducing enzymes in the metabolism of xenobiotics (e.g. CBR1, AKR1C1-4). The knowledge about microsomal forms is limited and only one well-known microsomal form involved in biotransformation of xenobiotics - 11b-hydroxysteroid dehydrogenase 1 (11b-HSD1) - has been reported so far (Maser E. et al. 2006). But it is probable that other microsomal members of SDR superfamily participate in biotransformation. This study has investigated expression and tissue localization of mRNA of four human poorly characterized microsomal enzymes included to SDR superfamily - DHRS3, DHRS7, DHRS8 and DHRS12. The total of 16 tissues was collected from humans. The RNA was extracted using Trizol reagent from all frozen tissues and subsequently RNA was reversely transcribed to cDNA. The expression and tissue localization of mRNA was determined using real-time SYBR Green qPCR with specific primers. Tissue localization together with knowledge of enzyme activity of these four carbonyl reducing enzymes will open the way for studies of its potential role in the metabolism of important endogenous and xenobiotic compounds in human. Publication is co-financed by the European Social Fund and the state budget of the Czech Republic. Project no. CZ.1.07/ 2.3.00/30.0022.

References Jansson, A. (2009) J Steroid Biochem Mol Biol, 114, (1–2) 64–67. Maser, E., Wsol, V., Martin, H.J. (2006) Mol Cell Endocrinol, 248, (1–2) 34–37. Morton, N.M. (2010) Mol Cell Endocrinol, 316, (2) 154–164. Persson, B., Kallberg, Y. (2013) Chem Biol Interact, 25, (1–3) 202. Skarydova, L., Wsol, V. (2013) Drug Metab Rev, 44, (2) 173–91.

P222. DISTAL PROMOTER REGIONS OF HUMAN OROSOMUCOID 1 AND 2 GENES DIFFERENTIALLY REGULATE THEIR GENE EXPRESSIONS AND ACUTE PHASE RESPONSES Kimie Sai1, Kouichi Kurose1, Tomoko Koizumi1, Noriko Katori1, Jun-ichi Sawada2, Yasuhiro Matsumura3, Nagahiro Saijo3, Noboru Yamamoto4, Tomohide Tamura4, Haruhiro Okuda1 and Yoshiro Saito1 1 National Institute of Health Sciences, Tokyo, Japan, 2Pharmaceuticals and Medical Devices Agency, Tokyo, Japan, 3National Cancer Center Hospital East, Kashiwa, Japan, 4National Cancer Center Hospital, Tokyo, Japan Backgrounds and Purpose: Human orosomucoid (ORM), or alpha-1-acid glycoprotein, is a major acute-phase plasma protein, and encoded by two different genes ORM1 and ORM2 which have high sequence homology. Plasma concentration of ORM1 is known to be higher than ORM2. Induction of human ORMs is assumed to be regulated by each proximal promoter region where


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putative glucocorticoid responsive element (GRE) and C/EBPb binding sites are located. However, detailed regulation mechanism of each ORM gene is still unknown. To explore ORM regulatory mechanisms which might differentiate each ORM expression and induction, we conducted in vitro luciferase-reporter assay for each ORM 50 -flanking region. Methods: Reporter assay was conducted using HeLa cells by co-transfection of ORM-reporter construct and GRa. For assessing a role of distal promoter region of each ORM gene, the reporter activities of full construct containing up to approx. 1.1 kbp upstream from the transcription start site (FULL) and those of deletion constructs containing up to 777 bp upstream region (DEL1) and up to 188 bp upstream region with GRE and C/EBPb binding sites (DEL2) were examined for basal activities and treatments of inducers [dexamethasone (DEX) and/or IL-1b] under C/EBPb overexpression or non-overexpression conditions. For assessing differential effects of FULL between ORM1 and ORM2, the ratio profile (FULL/DEL2) was also compared among ORMs. Results: No significant differences in reporter activities between DEL1 and DEL2 were observed for both ORM1 and ORM2. Significantly higher reporter activity in FULL than DEL2 (higher ratio of FULL/DEL2) was observed in IL-1b treatment for ORM1. In contrast, for ORM2, the reporter activities in FULL were significantly lower than those of DEL2 in basal level and DEX treatments (alone and co-treatment with IL-1b). Under C/EBPb overexpression condition, significantly higher FULL activities were detected for basal level and IL-1b treatment in ORM1 and those for basal level and DEX and/or IL-1b treatments in ORM2. Enhancing effects of FULL for modulating DEL2 activity were more evident in ORM1 than ORM2 under C/EBPb non-overexpression condition, while those were inverse under C/EBPb overexpression condition. Conclusions: The findings in this study suggest that ORM distal promoter region (approx.780 bp - 1.1 kbp upstream from the transcription start site) might have a differential role for each ORM gene expression and modulation of acute phase responses. Further studies on specifying the responsive elements in the distal promoter regions should be needed.

P223. PXR IS A LIGAND-ACTIVATED SIGNAL SCAFFOLD THAT FACILITATES PP2C DEPHOSPHORYLATING SGK2 TO REGULATE HEPATIC GLUCONEOGENESIS Saki Gotoh and Masahiko Negishi Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences/National Institute of Health, Research Triangle Park, NC Based on recent clinical findings that statins can increase blood glucose levels, the Food and Drug Administration has added a new safety warning to the statin label; statins may increase the risk of developing Type 2 diabetes. Statins activate the key gluconeogenic genes such as G6Pase and PEPCK1 in HepG2 cells and human primary hepatocytes. The nuclear pregnane X receptor (PXR) regulates this activation but requires serum and glucocorticoid-regulated kinase 2 (SGK2). Here we have investigated the molecular mechanism by which PXR, acting as the cell signal scaffold, facilitates dephosphorylation of SGK2 to activate it as a transcription factor. Western blot analysis with an anti-phosho-Thr193 peptide revealed that SGK2 is phosphorylated at this residue in HepG2 cells. Mimicking by SGK2 T193A or T193D mutant, non-phosphorylated SGK2 translocated into the nucleus, bound to the G6Pase promoter (1,613/1,593 and 196/156 bp regions) and determined the PXR-mediated promoter activation. Statin treatment decreased phosphorylation of threonine 193 only in the presence of liganded PXR. The enzyme that dephosphorylated SGK2 was protein phosphatase 2C, as indicated by experimental results using siRNA, PP2C inhibitor and co-immunoprecipitation. Pure PP2C enzyme dephosphorylated SGK2 only when statin-activated PXR was present in in vitroassays. In response to statin treatment, PXR interacted with phosphorylated SGK2 and facilitated PP2C dephosphorylating SGK2 in the cytoplasm. Therefore, our findings have defined a novel function to PXR as a ligandactivated signal scaffold. The identified PXR-SGK2-G6Pase pathway can be a drug-induced regulatory signal that regulates hepatic gluconeogenesis. Further investigations should be helpful for understanding and decreasing the statin-caused side effects as well as developing drug candidates.

P224. ENDOTOXIN UP-REGULATES THE PROINFLAMMATORY CYTOKINES TNF-A AND IL-6 IN FRESHLY-ISOLATED HUMAN KUPFFER CELLS Diana Li Lambert, Kevin C. Lyon, Maciej Czerwinski and David B. Buckley XenoTech, LLC, Lenexa, KS Kupffer cells, macrophages endogenous to the liver, can modulate hepatic inflammation and injury associated with various pathophysiologies and toxicities. Several proinflammatory cytokines released by activated Kupffer cells, such as TNF-a and IL6, are associated with up-regulation of acute-phase response proteins and suppression of CYP enzymes. For new biological entities, particularly modulators of immune system, evaluating the potential for Kupffer cell stimulation is an emerging concept in preclinical development. As testing develops, robust and predictable test systems are necessary, thus the need to evaluate Kupffer cell isolation, culturing and stimulation for in vitro applications. In the present study, Kupffer cells were freshly isolated from non-transplantable human livers (n ¼ 3) with a two-step Percoll gradient centrifugation method and maintained under standard cell culture conditions (37 C, 5% CO2, 95% RH, Dulbecco’s Modified Eagle Medium) for up to 12 days. Cell morphology was evaluated throughout the culture period by light microscopy. On either day 9 (n ¼ 1) or day 11 (n ¼ 2), the

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expression of scavenger receptor CD68 and hemoglobin scavenger receptor CD163 were evaluated by immunofluorescence staining, with anti-CD68 and CD163 antibodies, respectively. Based on co-staining with a nuclear dye (DAPI), approximately 90% of the cells in culture expressed CD68. Furthermore, most of the cells expressing CD68 co-expressed CD163, a second macrophage marker. Kupffer cells, with expression of macrophage markers confirmed at the aforementioned time points, were treated for 24 hr with 5 mg/mL endotoxin (E.coli lipopolysaccharide), an agonist of human Toll-like receptor 4, to evaluate their potential for stimulation in culture. As expected, the Kupffer cells responded to endotoxin treatment with an increase in TNF-a and IL-6 released into the cell culture medium, as evaluated by ELISA, and corresponding changes in their mRNA expression as evaluated by RT-PCR. In general, stimulation of IL-6 production following treatment with endotoxin was more pronounced than that observed for TNF-a. Cultures of freshly-isolated human Kupffer cells maintained phenotype and responsiveness to endotoxin, a prototypical proinflammatory stimulus, for up to 12 days post isolation. The stimulation of cytokine release observed in cultured human Kupffer cells upon treatment with endotoxin is similar to that observed in the clinical setting. Taken together, these results suggest that freshly-isolated human Kupffer cells may be a suitable test system for preclinical evaluation of pro-inflammatory effects of new biological or chemical entities.

P225. INDUCTION OF UBCM4 BY AHR RESULTS IN C-FOS DEGRADATION Alejandro Mejia-Garcia, Emmanuel Gonzalez-Barbosa, Guillermo Elizondo Cell Biology, CINVESTEV-IPN, Mexico, Mexico

Carmen

Martinez-Guzman,

Maria

Cabanãs-Cortes

and

The aryl hydrocarbon receptor (AhR) is a transcription factor that regulates the expression of various genes in a liganddependent manner. It has been determined that the AhR modulates several cell processes such as cell proliferation, cell differentiation and homeostasis of the immune system, among others. Previous studies indicate that the AhR is involved in the regulation of the ubiquitin-proteasome system (UPS). UPS is an essential process, and is responsible for ubiquitination and controlled degradation of nucleus, cytosol and endoplasmic reticulum proteins. Recently, we have showed that TCDD, an AhR ligand, induce UbcM4 expression in mouse liver. UbcM4 is an ubiquitin conjugating enzyme involved in the ubiquitination of several key proteins, including p53, c-myc, and c-Fos, among others. The aim of the present study is determine whether AhR mediates UbcM4 gene expression as well as to evaluate the effect on c-Fos degradation. For this purpose, Hepa-c7 cells that express constitutive levels of the AhR and Hepa-c12 cells that exhibit reduced levels of AhR were treated with b-naphthoflavone, an AhR agonist ligand. Our results show that AhR activation increases, in a time-dependent manner, UbcM4 mRNA and protein levels. Actinomycin-D treatment indicates that this modulation is at transcriptional level. Furthermore, the AhR-dependent induction of UbcM4 results in a decreased level of c-Fos protein. The proteasome pathway mediates the observed c-Fos degradation. All together, the present data suggest that AhR may be part of a new signal mechanism through which UPS-dependent degradation is modulated.

P226. EXPOSURE ASSESSMENT OF PESTICIDES AND XENOBIOTICS IN SPOT URINE COLLECTIONS Tauri Senn1, Melinda Vredevoogd2, Eric Vigoren2, Elaine Faustman2 and Yvonne S. Lin1 1 Department of Pharmaceutics, University of Washington, Seattle, WA, 2Environmental and Occupational Health Sciences, University of Washington, Seattle, WA Accurate characterization of human exposures to xenobiotics is a critical component of epidemiologic studies, risk assessment and public health measures. Human exposures can be assessed in various ways, including self-report, use of historical information, environmental measurements (e.g., dust and air), personal monitoring, and measurements in biologic specimens. The latter may provide a real-time snapshot of an individual’s exposure. Our goal was to develop a rapid, adaptable screening tool for possible human exposures based on global metabolomic procedures. Initially, we focused on pesticides and selected xenobiotics. Spot urine collections were obtained from 12 families, consisting of farmworkers or non-farmworkers, and their children on two days during three seasons (harvest, non-spray, and thinning). We used a simple sample preparation method (acetonitrile protein precipitation) to maximize the diversity of the compounds we would be able to analyze. Liquid chromatography-quadrupole time-of-flight (LC-QTOF) was used to analyze the samples and authentic standards of over 130 pesticides and selected xenobiotics, such as dextromethorphan and its metabolites (dextrorphan, dextrorphan glucuronide), antiinflammatory drugs (naproxen, ibuprofen, acetaminophen, and aspirin), caffeine and its metabolites (theophylline, paraxanthine, and theobromine), and tetracycline. Samples and standards were run on two columns with different chemistries (Agilent SB-Aq column and a TOSOH Gel column) in both positive and negative electrospray ionization modes. We collected accurate mass and retention time information from the samples and standards. Metabolomics data from the samples were interrogated against our standards database. Collision induced dissociation (CID) spectra were used to confirm the presence of a compound. Tributylphosphate and dimethylphosphate, could be detected and confirmed in urine samples using neutral mass, retention time, and similar CID spectra. Tributylphosphate, used as a plasticizer and solvent for pesticides, was found in 66% of all urine

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samples. We were able to detect the presence of various xenobiotics including: acetaminophen (8 individuals), ibuprofen (6 individuals), tetracycline (1 individual), naproxen (3 individuals), and dextrorphan glucuronide (4 individuals). Several other compounds were detected in urine that were similar to authentic standards based on mass and retention time, but the sample CID spectra intensities were too low or differed too greatly from standard CID spectra to confirm the presence of the compound. In addition, due to the longitudinal nature of these samples, we could identify individuals with chronic exposures (e.g., one individual with naproxen in four urine samples collected over two seasons). Using this approach, we have created an adaptable screening method for a panel of pesticides and xenobiotics which will permit exposure assessment determinations in urine samples.

P227. ABSTRACT WITHDRAWN


P228. INVESTIGATION OF HOST-GUT MICROBIOME INTERACTION IN TACRINE-DOSED LISTER-HOODED RATS THROUGH FECAL METABOLIC PROFILING Lian Yee Yip1, Han Chen Ku1, Chiu Cheong Aw2, Woei Shin Chen3, Edward Browne2, Ratha Mahendran4, Yuan Kun Lee5 and Eric Chun Yong Chan1 1 Department of Pharmacy, National University of Singapore, Singapore, Singapore, 2GlaxoSmithKline R&D Centre, Singapore, Singapore, 3GlaxoSmithKline R&D Center, Singapore, Singapore, 4Surgery, National University of Singapore, Singapore, Singapore, 5Microbiology, National University of Singapore, Singapore, Singapore The human gut microbiota is a key player in modulating drug responses as they make up a significant portion of cells in the human body and are highly involved in host drug metabolism and disposition, both directly and indirectly. The indirect effect is facilitated via host-gut microbiome interaction where bidirectional flow and exchange of metabolites between the host and gut microbiota metabolic pathways cause perturbations to drug disposition. While this mechanism has been shown to account for a number of pharmacological and toxicological idiosyncrasies, the host-gut microbiome interaction has not been well studied owing to its complexity. Tacrine (THA) is an acetylcholinesterase inhibitor that demonstrated a large inter-individual variability in its hepatotoxic side effects in humans. In a preliminary in-house study where 28 Lister-hooded rats were given a single oral dose of 20 mg/kg THA, this inter-individual variability in hepatotoxicity (in the form of transaminitis) was reproduced. We aimed to investigate the potential host-gut microbiome interaction in THA-induced transaminitis by screening for toxicity biomarkers via global GC/TOFMS metabonomic profiling of rat fecal samples from these rodents. Principal component analysis (PCA) of the fecal metabolomes revealed a similar pre-dose metabolomes across all rats. Post-dose 8 to 24 h fecal metabolome analysis revealed distinct clustering of the rats exhibiting toxic phenotypes, especially those with most severe transaminitis (ERs, n ¼ 4), from the non-toxic phenotypes (NRs, n ¼ 6) (R2X ¼ 0.542 and Q2(cum) ¼ 0.107). A closer analyses of the ERs revealed a metabolic trajectory that followed the progression and resolution of transaminitis (R2X ¼ 0.472 and Q2(cum) ¼ 0.291, PCA) over the period of 24 h to 96 h. At the period of highest transaminase elevation (8 to 24 h), we found 124 metabolites correlated strongly with the 24 h transaminase levels (jrj40.6, Pearson’s correlation). The criteria of VIP41.4 from partial least squares discriminant analysis, p50.05 from t-test, jrj40.6 from Pearson’s correlation were used to screen toxicity-related biomarkers strongly discriminating the ERs from NRs at 8 to 24 h. From this screening, we uncovered 11 biomarkers, all of which are in higher abundance in the ERs. A majority of these are amino acids, fatty acids and saccharides, whose fecal levels were known to be affected by bacterial fermentation or host-gut microbiome co-metabolism. Alterations of amino acid levels such as isoleucine (10.9-folds, ERs vs NRs), alanine (4.3-folds, ERs vs NRs) and glutamic acid (2.9-folds, ERs vs NRs) as found in this study have been previously demonstrated when gut microbiota was perturbed upon antibiotic treatment1,2. Apart from gastrointestinal absorption, factors like proteolysis by microbial proteases and metabolism by gut microbes with different growth requirements are known to influence amino acid levels. Our finding suggests a perturbation of the gut microbial species in rats exhibiting THA-induced transaminitis and illuminated a significant potential involvement of host-gut microbiome interaction.

References 1. Zheng X et al. The footprints of gut microbial-mammalian co-metabolism. J Proteome Res. 2011;10(12):5512–22. 2. Zhao Y et al. Gut Microbiota Composition Modifies Fecal Metabolic Profiles in Mice. J Proteome Res. 2013. Epub 2013/ 05/02. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed.

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P229. ABSOLUTE QUANTIFICATION OF TRANSPORTER PROTEINS ALONG THE INTESTINAL TRACT OF PIGS


Evita van de Steeg1, Anita M van Adrichem1, Arjen HG Gootzen1, Kitty CM Verhoeckx2, Jan JP Bogaards1, Wouter HJ Vaes1, Jeroen DeGroot1, Miriam Verwei1 and Heleen M. Wortelboer1 1 Kinetics Research for Food & Pharma, TNO, Zeist, Netherlands, 2Risk Analysis for Products in Development, TNO, Zeist, Netherlands The gastrointestinal tract is the primary site for the intake, metabolism and absorption of pharmaceutical and nutritional compounds. To be able to better predict the oral bioavailablity of these compounds in early development, TNO recently developed the InTESTineÔ model, using ex-vivo intestinal tissue to assess the fraction absorbed. As the GI tract of pigs closely resembles that of humans, intestinal tissue from pigs is used. The aim of the study was to determine the absolute expression levels of various transporter proteins along the intestinal tract of pigs. By using four different protein isolation techniques (i.e total protein, crude membrane protein, cytosolic protein and plasma membrane protein isolation) we additionally explored the option to work with small human biopsy samples. Absolute quantification of a wide range of transporter proteins (including MDR1, BCRP, MRP1-3, OATP2B1, OATP4A1, OCT1, OCT3, PEPT1, ASBT and OATP4A1) was performed using an Ultra Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS-MS) method to determine the protein levels of different membrane embedded transporter proteins. Stable isotope labeled peptides were used as a reference for quantification. All data were corrected for the expression of villin, which is an epithelial-specific marker protein. First results demonstrate that the expression of MRP2, MRP3, OCT3 and MCT1 was higher in pig duodenum compared to jejunum and ileum, whereas the expression MDR1, BCRP, and ASBT was equal along the small intestinal tract of pigs. Importantly, expression of the different transporter proteins determined after total protein isolation was comparable to the sum of the expression established after crude membrane and cytosolic protein isolation, indicating no loss of proteins during these isolation steps. Our future perspective is to compare these data with the abundance of transporter proteins in human (adult and pediatric) intestinal samples. Together with absorption data from our InTESTineÔ model we will use these expression data as input for better prediction of oral bioavailability in humans using PBPK modelling.

P230. USE OF HIGH RESOLUTION ACCURATE MASS SPECTROMETRY WITH AN EXOGENOUS MIXTURE OF GSH AND STABLE ISOTOPE-LABELED GSH TO TRAP REACTIVE METABOLITES OF TROGLITAZONE IN HUMAN HEPATOCYTES Igor Mezine, Chris Bode, Bethany Raughley, Sid Bhoopathy, Albert J. Owen and Ismael J. Hidalgo Absorption Systems, Exton, PA CYP-mediated biotransformation of some drugs can lead to the formation of reactive metabolites, the binding of which to intracellular and/or extracellular macromolecules may be associated with idiosyncratic toxicity. Troglitazone (TGZ) is one such drug and is frequently used as a model compound in studies of reactive metabolites, most of which have been conducted with subcellular fractions such as liver microsomes rather than intact cell-based systems. In this study, metabolites of TGZ were generated by incubation with cryopreserved human hepatocytes in suspension and trapped in the presence of an exogenous mixture of unlabeled and stable isotope-labeled (SIL; [1,2-13C,15N]-glycine) glutathione (GSH/SIL-GSH). Samples were analyzed using liquid chromatography-high resolution accurate mass spectrometry (LC-HRAMS) on an LTQ Orbitrap instrument. GSH conjugates (trapped reactive metabolites) were detected based on a characteristic mono-isotopic pattern (peaks separated by 3.0037 mass units). A total of five conjugates, two of which have not previously been described in the literature, were detected and partially characterized based on accurate mass measurements and MS/MS data. The addition of exogenous GSH/SIL-GSH increased the amount of GSH conjugates formed; the magnitude of the effect was conjugate-specific. Furthermore, the mono-isotopic intensity ratios of the conjugates varied from 1:0.15 (apparently for conjugates formed mostly inside the cells, where there was no SIL-GSH) to near the 1:1 ratio applied (for conjugates formed mostly outside the cells). Results obtained with hepatocytes from different donors suggest that not all GSH conjugates are formed in all cases, consistent with the idiosyncratic nature of drug-induced liver toxicity. The results of experiments in which hepatocytes were exposed to exogenous SIL-GSH, then separated from the medium by centrifugation through oil, clearly indicate that most conjugation with exogenous GSH occurs extracellularly. The finding that GSH conjugates are formed extracellularly raises the question of which transporter(s) is/are involved in the efflux of TGZ and its metabolites. It also suggests a facile mechanism by which reactive drug metabolites could be presented on the extracellular surface of liver cells, constituting immunogenic neoantigens as has been proposed by others [1,2].

References [1] Waring JF and Anderson MG, Idiosyncratic toxicity: mechanistic insights gained from analysis of prior compounds. Curr Opin Drug Discov Devel. 2005;8:59–65. [2] Ju C and Uetrecht JP, Mechanism of idiosyncratic drug reactions: reactive metabolite formation, protein binding and the regulation of the immune system. Curr Drug Metab. 2002;3:367–377.

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P231. CULTURED HUMAN HEPATOCYTES AS A MODEL FOR EVALUATION OF POTENTIAL SUBSTRATES OF ALDEHYDE OXIDASE: EFFECTS ON METABOLIC CLEARANCE OF DISCOVERY COMPOUNDS


Mary Grubb and Yue-Zhong Shu Biotransformation, Bristol-Myers Squibb Co, Princeton, NJ Determining the in vitro clearance and metabolic profile of drug discovery compounds is typically accomplished by incubation with human and animal liver microsomes. Oxidation by aldehyde oxidase (AO) has emerged increasingly as a significant metabolic pathway, especially for nitrogen containing aromatic heterocycles. However, AO is not present in liver microsomes and thus this potential route of metabolism would be missed in studies using liver microsomes, resulting in underestimation of metabolic clearance. Additionally, AO has been implicated in the formation of toxic human metabolites, as with SGX5231. Primary cultures of hepatocytes have recently been employed for measuring the half-life of low clearance compounds, since hepatocyte suspensions do not retain activity for longer than a 4 h incubation period2. As some low clearance compounds are also AO substrates, hepatocyte cultures were evaluated for use in determining the clearance and metabolic profile of AO substrates. Cryopreserved human hepatocytes (n ¼ 3 donors) were inoculated into 24-well plates (one plate per donor) at 0.8e6 cells/mL, 0.5 mL/well, and were allowed to attach for 24 h. AO substrates XK469 (low clearance in humans), zaleplon (moderate), SGX523 and BIBX1382 (high) were added in media and incubated for up to 24 h at both 1 and 30 mM. Samples were taken at 0, 1, 2, 4, 8 and 24 h (1 mM), and at 0, 8 and 24 h (30 mM) and analyzed by accurate mass mass spectrometry. Parent compound half-life was obtained from measuring parent disappearance in 1 mM samples, while metabolite identification was achieved from analysis of 30 mM samples. The major metabolites of XK469, zaleplon, SGX523, and BIBX1382 in human heptocytes were 3-oxo-XK469, 5-oxozaleplon and desethylzaleplon, hydroxy-SGX523, 2-quinolinone SGX523 and N-desmethyl SGX523, and BIBU1476 (2-oxo-pyrimido[5,4-d]pyrimidine), respectively. Formation of 5-oxo-zaleplon, 2-quinolinone SGX523 and BIBU1476 were abolished by co-incubation with AO inhibitor raloxifene. Furthermore, as evidenced by the plots of metabolite formation with time, these AO metabolites contributed significantly to the clearance of these compounds in human hepatocytes. Because rodent and dog hepatocytes possess very little or no AO activity, use of human or primate hepatocytes are instrumental in defining the presence of AO metabolites prior to clinical studies. Furthermore, in order to meet FDA’s and other regulatory agency’s requirements, the use of plated human hepatocytes would be highly valuable to assess the need of monitoring and safety testing of drug metabolites.

References 1. Diamond S, Boer J, Maaduskuie TP, Falahatpisheh N, Li Y and Yeleswaram S (2010). Species-specific metabolism of SGX523 by aldehyde oxidase and the toxicological implications. Drug Metab Dispos 38 (8) 1277–85. 2. Schaeffer O, Ohtsuki S, Kawakami H, Inoue T, Liehner S, Saito A et al. Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDP-glucuronosytransferases in cultured primary hepatocytes (2012). Drug Metab Dispos 40(1) 93–103.

P232. CHARACTERIZATION OF THE HEPATIC TRANSPORT AND DISPOSITION OF GEMFIBROZIL AND ITS METABOLITE Emi Kimoto, Manthena Varma and Yurong Lai Pharmacokinetics, Dynamics & Metabolism, Pfizer Inc., Groton, CT Background: Gemfibrozil, a hypolipidemic agent which decreases serum triglycerides and very low density lipoprotein, causes drug-drug interactions (DDIs) with several drugs including repaglinide and statins. Both gemfibrozil and its major metabolite, gemfibrozil 1-O-B-glucuronide (GG), are known to inhibit drug transporters and metabolic enzymes. However, their in vitro inhibition potency and plasma free concentration underpredict the magnitude of DDIs with repaglinide and cerivastatin, particularly at subtherapeutic doses. We hypothesized that gemfibrozil and/or GG may accumulate in hepatocytes to the high free concentrations that are required for complete inhibition of CYP2C8 activity. Methods: The hepatic uptake and disposition of gemfibrozil and its metabolite were assessed using sandwich-cultured human hepatocytes (SCHH) and transfected Human Embryonic Kidney (HEK) 293 cells. Results: Uptake of both gemfibrozil and GG in SCHH were reduced in the presence of rifamycin SV. The uptake clearance was 1.5- and 3-fold higher than passive diffusion for the parent and metabolite, respectively. Hepatocyte accumulation of GG was higher following an incubation with gemfibrozil, as compared to incubation with the GG itself. The glucuronidation was also characterized in SCHH. GG showed to be a substrate for organic anion transporting polypeptide (OATP), although gemfibrozil was not transported. Following an incubation with gemfibrozil, rifamycin SV reduced secretion of the metabolite into media and increased its accumulation in SCHH, whereas the secretion and accumulation of gemfibrozil were not significantly different. Both an increase in hepatic accumulation and reduction in efflux to the medium were observed for the GG in the presence of rifamycin SV, thereby suggesting inhibition of basal efflux for the glucuronide metabolite. Conclusion: Active transport contributed to the hepatic uptake of both gemfibrozil and its metabolite.

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This suggests that the free liver concentrations of gemfibrozil and its 1-O-B-glucuronide could be higher than would be predicted from their plasma concentration-time profiles and thus have additional implications in the prediction of enzyme-mediated DDIs that are based on plasma free concentrations.

P233. BIOACTIVATION AND TOXICITY OF ACETAMINOPHEN IN RAT PRIMARY HEPATOCYTES CULTURED IN MICROPATTERNED CO-CULTURES


Okechukwu Ukairo1, Michael McVay1, Stacy Krzyzewski1, Kelly Rose2, Melvin Andersen2, Salman Khetani1 and Edward LeCluyse2 1 Hepregen Corporation, Medford, MA, 2The Hamner Institutes, Research Triangle Park, NC Drug- induced liver injury is often caused by cytochrome P450- dependent activation of drugs into reactive metabolites. In vitro models, which can mimic in vivo responses and allow the evaluation of initial and adaptive responses to bioactivated compounds over prolonged periods, offer potentially valuable tools for toxicological assessment. We have previously developed a model in which primary hepatocytes (rat, human) are seeded onto ECM-coated domains of optimized dimensions and subsequently cocultivated with murine embryonic fibroblasts. This model retains key biochemical functions of in vivo liver with long term stability. Here, we assess the bioactivation and cytotoxicity of acetaminophen (APAP) as a function of culture age in the 96-well micropatterned co-culture (MPCC) format. APAP is a well-known hepatotoxin and exerts its toxic effects through bioactivation associated, in part, with cytochrome P450 3A (CYP3A). Primary rat hepatocytes cultured in the MPCC platform were exposed to increasing concentrations of APAP (over 5 days) and assessed for changes in hepatic ATP content, glutathione (GSH) levels, albumin secretion and urea synthesis. Similar concentration-dependent cytotoxicity profiles (AC50 ¼ 8.4 2.4 mM for GSH depletion and 14.17 3.5 mM for urea synthesis inhibition) were obtained over the course of the 4-week study. Addition of 200 mM L-buthionine (S, R)-sulfoximine (BSO), an inhibitor of GSH synthesis, or 10 mM dexamethasone (DEX), an inducer of rat CYP3A1/2, to MPCC rat hepatocyte cultures potentiated APAP- induced hepatotoxicity in these cultures irrespective of culture age (over 4 weeks). These findings are consistent with the known in vivo mechanisms of APAP toxicity in rats. In conclusion, rat hepatocyte MPCCs in a 96-well format provided reproducible APAP-induced cell cytotoxicity profiles over a 4 week period and can be used to assess the effects of chronic exposure to bioactivated compounds.

P234. FUNCTIONS AND GENE EXPRESSIONS OF SANDWICH CULTURED-PRIMARY RAT HEPATOCYTES ON OXYGEN-PERMEABLE MEMBRANES UNDER PHYSIOLOGICAL OXYGEN CONCENTRATIONS Wenjin Xiao1, Hitoshi Matsui2, Marie Shinohara1, Kikuo Komori1, Tomoharu Osada2 and Yasuyuki Sakai1 1 Institue of Industrial Science, The University of Tokyo, Tokyo, Japan, 2Mitsubishi Chemical Medicine Corporation, Tokyo, Japan Oxygen supply is particular a critical tissue in hepatocytes culture. The concentration of the signal oxygen in the liver falls corresponding to a drop from 13% (arterial) to 9–11% (mixed periportal) and then to 5–7% (hepatovenous) from the periportal to the perivenous zone [1]. It suggests that culturing primary hepatocytes under physiological oxygen concentrations will help to prolong the cell survival and reduce the rates of decline in hepatic functions. However, the oxygen requirement cannot be matched in conventional TCPS (Tissue culture treated-polystyrene) culture so that in vitro realization of functional zonation in lobules is unfeasible. We therefore aimed to enhance rat hepatocyte functions and durations in culture through improving both oxygen supply and its actual concentrations at the cell level. Oxygen-permeable PDMS (polydimethylsiloxane) membrane can meet oxygen demand in a very easy manner by diffusion through the membrane, in which the actual oxygen concentration at the cell level can be strictly controlled [2]. Rat primary hepatocytes were cultured in sandwich culture method with Matrigel on PDMS membrane under various oxygen concentrations (20%-O2 (þ), 10%-O2 (þ) and 5%-O2 (þ)) for 14 days to investigate their effects on the performances of culture. In parallel, TCPS cultures were carried out as control groups (20%-O2 (), 10%-O2 () and 5%-O2 ()). Cellular level oxygen tensions (pO2) on PDMS membranes were almost at the equilibrium concentrations in incubator: 18– 20%, 9–10% and 4–5% under the cultures of 20%-O2 (þ), 10%-O2 (þ) and 5%-O2 (þ), which were remarkably lower on TCPS surfaces. HIF staining confirmed that hepatocytes were normoxic under 20%-O2 (þ) and 10%-O2 (þ) but hypoxia under other cultures associated with cell death. Albumin secretion increased greatly under 10%-O2 (þ) from day 2 and maintained at the highest level throughout the culture, 3 times higher than that under 20%-O2 (). Moreover, CYP1A1/2 activity also increased greatly from day 8 under 10%-O2 (þ) and 5%-O2 (þ) and maintained at a higher level on day 14 under 10%-O2 (þ) which was 2 times higher than that under 20%-O2 (). Furthermore, bile excretion assay showed the enhanced bile canaliculi formation under 10%-O2 (þ) in a time course dependence. PCR array analysis illustrated that the expression of various drug-metabolism genes under 20%-O2 (þ) and 10%-O2 (þ) were closest to those seen in the isolated freshly hepatocytes. These results demonstrate that it is important to satisfy cellular oxygen demand at in vivo-like physiological oxygen concentrations in in vitro hepatocyte culture

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and oxygen permeable membrane provides a simple methodology to realize this condition for in vitro pharmacological and toxicological research.

References [1] I. Martinez. et al. The influence of oxygen tension on the structure and function of isolated liver sinusoidal endothelialcells. Comparative Hepatology. 2008; 7:4. [2] Y. Sakai, M. Nishikawa, et al. Engineering of implantable liver tissues. Liver Stem Cells. 2011; 189–216.

P235. ROLE OF MIRNAS IN DIFFERENTIATION OF HEPATOCYTES AND APPLICATION TO STEM CELL DERIVED HEPATOCYTE PROTOCOLS


Jenna Voellinger Pharmaceutics, University of Washington, Seattle, WA Role of miRNAs in differentiation of hepatocytes and application to stem cell derived hepatocyte protocols Jenna L. Voellinger1, Dagmara Szkolnicka2, David C. Hay2, and Edward J. Kelly1 1Department of Pharmaceutics, University of Washington, Seattle, Washington; 2Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, Scotland, UK The liver is the primary organ of drug metabolism, and as such modeling its function is essential when it comes to predictive evaluation of compounds for efficacy and toxicity. Primary and cryopreserved human hepatocytes are the most relevant model to study this; however, there are limitations to their use in vitro. Hepatocytes derived from pluripotent stem cells may represent a viable alternative. Stem cell derived hepatocytes (SCDHs) generated in our lab display mixed fetal/adult characteristics, with expression of albumin and cytochrome P450 3A4 (CYP3A4) but retention of fetal hepatocyte markers alpha-feto-protein (AFP) and CYP3A7. To enhance hepatocyte differentiation, and to further elucidate mechanisms involved in liver development, we are profiling the expression of microRNAs throughout hepatocyte differentiation, relative to cultured hepatocytes and intact human liver tissue. To establish a database of microRNA expression in human liver we ran microRNA microarrays on the UW School of Pharmacy Liver Bank. The most abundantly expressed microRNA in the liver bank is miR-122, a known liver-specific microRNA. We have evaluated miR-122 expression in SCDHs using RT-qPCR; results demonstrate SCDHs average 4-fold lower expression than select samples of liver tissue from the liver bank, and SCDHs express miR-122 at levels 30–80% of cryopreserved hepatocytes. Select miRNAs, such as miR-122, may enhance hepatocyte differentiation when supplemented into our differentiation protocol. Studies are ongoing to fully characterize the microRNA transcriptome throughout the hepatocyte differentiation process.

P236. TRPC6-ACTIVATING PHLOROGLUCINOL DERIVATIVES OF HYPERFORIN LACK PXR-ACTIVATION AND INDUCTION OF ADME GENE EXPRESSION IN HUMAN HEPATOCYTES Benjamin A. Kandel1, Sean Ekins2, Oliver Burk1, Kristina Leuner3, Christian Harteneck4, Wolfgang E. Thasler5 and Ulrich M. Zanger1 1 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany, 2Collaborations In Chemistry, FuquayVarina, NC, 3Molecular and Clinical Pharmacy, Friedrich-Alexander-University Erlangen-Nuernberg, Erlangen, Germany, 4 Institute of Pharmacology and Toxicology, University Tuebingen, Tuebingen, Germany, 5Surgery, LM University Munich, Hospital Grosshardern, Munich, Germany Hyperforin, the major pharmacological compound of St Johnxs wort (SJW) is supposed to exert its anti-depressive properties by specifically activating the canonical transient receptor potential-6 (TRPC6), thus acting as a potent serotonin reuptake inhibitor. A major problem of therapy with SJW is that hyperforin potently activates PXR, a key transcriptional regulator of genes involved in drug absorption, distribution, metabolism and excretion (ADME), thereby leading to drug-drug interactions (DDI). Recently, phloroglucinol derivatives designed on the basis of the hyperforin core-structure were pharmacologically evaluated and five derivatives were shown to activate TRPC6 with similar potency as hyperforin1. Here we investigated whether these compounds are also activators of PXR. Luciferase reporter gene assays using a CYP3A4 promoter-construct in the presence or absence of cotransfected hPXR expression plasmids were performed in HepG2 cells treated with the PXR agonists, rifampicin, hyperforin or the phloroglucinol derivatives Hyp1 to Hyp9 (Figure 1) in different concentrations. Furthermore primary human hepatocytes (PHH) were treated for 24 h with the agonists and the phloroglucinol derivatives and mRNA expression profiles were analyzed using a high-throughput quantitative real-time PCR microfluidic FluidigmÔ Biomark platform. 3D docking approaches for the phloroglucinol derivatives and PXR as well as pharmacophore studies were applied to predict binding modes and identify relevant structural features of these compounds. Hyperforin and rifampicin treatment resulted in 20- and 12-fold induction of the CYP3A4 promoter in the luciferase-based reporter system, respectively. We determined EC50 values of 0.59 mM and 1.9 mM for hyperforin and rifampicin, respectively. We did not detect a significant PXR-dependent or -independent activation of the promoter by any of the five TRPC6-activating phloroglucinols. Only the TRPC6 non-activating compound Hyp4 showed a


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Figure 1 Structures of the phloroglucinol derivatives tested.

moderate but significant PXR-dependent activation of the reporter construct (5.75 fold, EC50 12 mM). The gene expression changes in PHHs caused by the specific PXR agonist, rifampicin, were highly correlated to those of hyperforin (r240.8), suggesting a PXR-specific regulation of these genes by hyperforin. In contrast, expression changes in response to the phloroglucinol derivatives did not correlate with those of hyperforin or rifampicin and seem to be PXR-independent. Only one derivative was found to map to the original PXR pharmacophore (Hyp3), while several derivatives had docking scores lower than hyperforin, suggesting fewer key interactions of this series of compounds. Taken together our results show that hyperforinmediated changes in ADME gene expression in hepatocytes are highly PXR-dependent. TRPC6-activating phloroglucinols do not activate PXR and should thus be promising new candidates for further drug development. This study was supported by the German Federal Ministry of Education and Research (Virtual Liver grant 0315755) and the Robert Bosch Foundation, Stuttgart, Germany.

Reference 1. Leuner, K. et al. Simple 2,4-diacylphloroglucinols as classic transient receptor potential-6 activators–identification of a novel pharmacophore. Mol. Pharmacol. 77, 368–377 (2010).

P237. MODULATION OF SELECTED ANTIOXIDANT ENZYMES BY CRANBERRIES AND GREEN TEA POLYPHENOLS IN MICE Hana Bartikova, Lucie Stuchlikova, Lucie Vaskova, Vojtech Kvita, Barbora Szotakova and Lenka Skalova Department of Biochemical Sciences, Faculty of Pharmacy, Charles University in Prague, Hradec Kralove, Czech Republic Polyphenols are compounds naturally occurring in plants. They represent important micronutrients in human diet as they are abundant in various fruits, vegetables and beverages. Polyphenols exhibit wide range of biological activities, including antioxidant, antibacterial, antitumor, antiviral, anti-inflammatory and vasodilatory effects. They contribute to the prevention of various diseases, such as cancer, diabetes, obesity, cardiovascular and neurodegenerative diseases. Evidence of beneficial properties on human health has led to the increasing popularity of food supplements and nutraceuticals with flavonoids and other polyphenols. As the nutraceuticals contain concentrated bioactive agents, consumed doses exceed those that could be obtained from food. Therefore, unexpected effects of polyphenols (e.g. interaction with enzymes and transporters) might occur. Modulation of antioxidant enzymes may positively affect defence of organisms against chemicals and oxidative stress. The presented part of our study was focused on the interaction of popular cranberries and green tea polyphenols with selected antioxidant enzymes. For this purpose, mice divided into five groups (10–13 individuals per group) received polyphenols as a part of forage in different schemes of administration: 1) cranberry (Vaccinium macrocarpon Aiton) extract for 4 weeks, 2) nutritional supplement Polyphenon E (green tea extract) in higher dose for 4 weeks, 3) Polyphenon E in lower dose for 4 weeks, 4) Polyphenon E in higher dose for 3 days (in order to reveal the effect of short-term administration of nutritional supplement), 5) control (without polyphenols). Specific activity of selected antioxidant enzymes was assayed in subcellular fractions from liver, small and large intestines of mice using spectrophotometric methods. The attention was focused on peroxidase, glutathione peroxidase, catalase, superoxide dismutase, NAD(P)H:quinone oxidoreductase, thioredoxin reductase

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and glutathione reductase. Modulation of enzymatic activity depended on tested enzyme, its tissue localization, duration of polyphenol administration, source of polyphenols and their dose. Consumption of polyphenols led mostly to the increase of enzymatic activity or had no effect, but the reduction of activity was observed in few cases as well. Although the modulation of enzymatic activity was rather moderate, polyphenols from green tea and cranberries generally proved the ability to influence antioxidant and potential chemopreventive capacity of the organism. However, the results showed that alteration of antioxidant enzymes is not the major mechanism of antioxidant properties of polyphenols from cranberries and green tea. This project was supported by Czech Science Foundation (GACR) - Centre of Drug-Dietary Supplements Interactions and Nutrigenetics (Grant No. P303/12/G163).

P238. INDUCTION OF ANTIOXIDANT/DETOXIFYING SYSTEM IN PRIMARY RAT HEPATOCYTES BY LISOSAN G, A FERMENTED POWDER OF WHEAT


Vincenzo Longo and Margherita La Marca Institute of Agricultural Biology and Biotechnology UOS-IBBA, CNR, Pisa, Pisa, Italy Experimental data indicates that certain vegetables may offer an indirect protection against oxidative stress by activating endogenous cellular defense systems at genetic and protein levels. In recent years a number of studies have shown that whole grain products have strong antioxidant activity and may reduce the risk of coronary heart disease (1). It has been recently shown that Lisosan G, a fermented power of grain of Triticum Sativum has a protective effect against the cisplatin induced toxicity. The authors suggested that the protective effect of Lisosan G could be associated mainly with the attenuation of the oxidative stress and the preservation in antioxidant enzymes (2). Further, Lisosan G does not interfer with drug metabolizing system and protects against CCl4-induced hepatotoxicity (3). Several antioxidant agents trigger Nrf2 signaling with a concomitant repression of NF-kB and its target genes. In this study, primary cultures of rat hepatocytes were used to investigate whether and how Lisosan G is able to modulate antioxidant/detoxifying enzymes, and whether or not it can activate the Nrf2 transcription factor or inhibit the NF-kB activation. All of the antioxidant/detoxifying enzymes studied were significantly up-regulated by 0.7 mg/ml Lisosan G treatment. In particular, NAD(P)H:quinone oxidoreductase and heme oxygenase-1 were induced, although to different degrees, at transcriptional, protein and/or activity levels by treatment. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus after 1 h of Lisosan G treatment was revealed by immunoblotting. Lisosan G was also observed to decrease H2O2-induced toxicity. Taken together, these results show that this powder of wheat is an effective inducer of ARE/Nrf2-regulated antioxidant/ detoxifying genes and has the potential to inhibit the translocation of NF-kB into the nucleus.

References 1. Truswell, A.S., 2002. Cereal grains and coronary heart disease. Eur. J. Clin. Nutr. 56, 1–14 2. Longo, V., et al., 2011. Cisplatin induced toxicity in rat tissues: the protective effect of Lisosan G. Food and chemical toxicology 49, 233–237 3. Longo, V., et al. 2007. Lisosan G, a powder of grain, does not interfer with thw drug metabolizing enzymes and has a protective role on carbon tetrachloride-induced hepatotoxicity. Biotech. Letter 29, 1155–1159

P239. GLAUCARUBULONE GLUCOPYRANOSIDE, A NATURAL QUASSINOID INHIBITS PRO-CARCINOGEN BIOACTIVATING CYP ENZYMES IN VITRO AND DISPLAYS POTENT IMPACT ON BREAST CANCER CELL PROLIFERATION Simone Badal1, George Huang2, Sheena Francis3, Willie Davis4, Eileen Brantley4, Tzuen-RongJeremy Tzeng2, Helen Jacobs5 and Rupika Delgoda1 1 Natural Products Inst, Univ of the West Indies, Mona, Jamaica, 2Department of Biological Sciences, Clemson University, Clemson, SC, 3Natural Products Inst, Univ of the West Indies, Jamaica, 4Dept of Basic Sciences, Loma Linda University, Loma Linda, CA, 5Chemistry, Univ of the West Indies, Mona, Jamaica Quassinoids and coumarins have been associated with anti-inflammatory, antioxidant and anti-proliferative activity in the past. Emerging evidence suggests that they may also act as chemopreventive agents. In this study we investigated the potential for novel quassinoids and a coumarin isolated from the endemic Jamaican Castela macrophylla plant to exhibit anti-proliferative activity using three cancerous and one normal cell lines and to inhibit cytochrome P450s (CYPs) known to convert polyaromatic hydrocarbons (PAHs) into carcinogenic metabolites. The quassinoid, glaucarubulone-15-O-b-D-glucopyranoside (Gg) displayed dose and time dependant reduction on the viability of breast cancer MCF-7 cells with greater potency than tamoxifen and induced cell death significant at 1mM with 24 hour exposure. In addition, Gg inhibited the activities of CYPs1A1 and 1B1 enzymes with Kis 7.1 and 5.6 mM both according to non-competitive kinetics and comparison with structural analogs identifies

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the importance of the glycoside side chain. Gg also attenuated at nano molar values, the ability of PAH benzo[a]pyrene to induce CYP1A gene expression in MCF-7 cells. Taken together, these data suggest that improved chemotherapeutic and chemopreventive regimes can be achieved with the inclusion of the plant isolate Gg.

P240. PRE-CLINICAL EVIDENCE OF LIFE-THREATENING PHARMACOKINETIC INTERACTION BETWEEN QUERCETIN AND METHOTREXATE


Prashant B. Musmade, Shivaji S. Chatur, Ganesh Bhavar, Kranti P. Musmade, M.Trilok Chander and Nayanabhirama Udupa Dept of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences, Manipal, India The purpose of this study was to investigate the effect of oral quercetin on the bioavailability and pharmacokinetics of orally and intravenously administered methotrexate in rats. Quercetin, a flavonoid, is an inhibitor of P-glycoprotein-mediated efflux transport, and its oxidative metabolism is catalyzed by CYP enzymes. Thus, it is expected that the pharmacokinetics of both intravenous and oral methotrexate can be changed by quercetin. The in-vitro metabolism study was carried out using rat liver microsomes. The in-vitro metabolism of methotrexate (10.0 mM) in presence and absence of quercetin (10.0 mM) was carried out. The in-situ single pass intestinal perfusion studies were carried out to understand the role of CYP3A and P-gp in rat intestine. The in-vivo pharmacokinetic parameters of methotrexate were determined in rats after intravenous (2.5 mg/kg) or oral (8.0 mg/ kg) administration to rats in presence and absence of quercetin (10.0 mg/kg). Result of in-vitro metabolism study showed significant (p50.05) difference of % remaining of the methotrexate in control and quercetin treated samples. The % remaining was found to be 76.68 1.09 in quercetin treated samples against 54.83 1.52 for control. The results of in-situ studies revealed that there was significant (p50.05) increase permeability coefficient (Peff) in duodenum and ileum compared to control indicates inhibition of CYP3A and P-gp in intestine. Compare to the control, quercetin significantly (p50.05) increased the area under the plasma concentration-time curve (AUC0–t) by 48.17folds of oral methotrexate. Quercetin also significantly increased the peak plasma concentration (Cmax) of methotrexate by 119.60 folds, while there was significant change in tmax and t1/2 of methotrexate which may be due to inhibition of the metabolizing enzymes in liver and/or intestine results in faster absorption and slower elimination of drug from body. The absolute bioavailability of methotrexate was increased by 41.82 folds which resulted in surprisingly mortalities of 66.66% of rats. These results suggest that the quercetin increases in bioavailability of oral methotrexate can be attributed to enhanced methotrexate absorption in the gastrointestinal tract via quercetin-induced inhibition of P-gp and reduced first-pass metabolism of methotrexate due to inhibition of CYP3A in the small intestine and/or in the liver. The results showed that systemic exposure of the methotrexate was very high results to 66.66% death of rats. The evidences showed the life life-threatening pharmacokinetic drug interaction of methotrexate with quercetin.

P241. IMPACT OF ALOE VERA JUICE ON IN VITRO P-GP TRANSPORT AND CYP3A4 METABOLISM Ane Djuv1, Bent H. Hellum1 and Odd Georg Nilsen2 1 Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway, 2Department of medicine, Norwegian University of Science and Technology, Trondheim, Norway The use of herbal remedies have increasing popularity in the recent decades and are often considered natural and safe [1]. Interaction between conventional drugs and herbal remedies (e.g. St.John‘s wort and cyclosporine) has been shown to significantly modulate drug metabolism and pharmacokinetics increasing the occurrence of side-effects or producing treatment failure [2]. As many patients co-administrate conventional drugs and natural health products [3] drug-herb interactions must be considered a challenge [4]. Aloe vera is an old medicine plant with increased popularity both in cosmetics, dietary supplement and natural medicine and the U.S. Food and Drug Administration (FDA) has called for increased attention to this herb [5]. Hypothesis: The aim of this study was to determine if Aloe vera juice (AVJ) possess any inhibitory effect on P-glycoprotein (P-gp) mediated drug transport or cythochrome-P450 3A4 (CYP3A4) metabolism in vitro. Methods: Caco-2 cells were used for P-gp studies. The 3H-digoxin substrate was added to either the apical or basolateral side of the monolayer, and the net digoxin flux was measured as the difference between the basolateral to apical and the apical to basolateral transport. AVJ was added to both sides of the monolayer. Transport of digoxin was measured for 3 60min periods in both directions. Cell viability, integrity and toxicity were assured by TEER, mannitol and MTT-assay measurements. Recombinant human CYP3A4 supersome metabolic activities were measured by the metabolism of testosterone as substrate. Incubation, added all necessary co-factors, was performed in a 0.1 mM KPO-buffer, PH ¼ 7.4, for 10 min. After centrifugation, buffer aliquotes were transferred to HPLC vials for a validated determination of 6-beta-OH-testosterone. IC50 metabolic inhibition constants of AVJ were calculated. Time and NADPH dependent assays were performed for evaluating a possible mechanistic inhibition of CYP3A4. Results: AVJ, in concentrations from 0.00001 to 1 mg/mL, did not change statistically significant the digoxin flux as compared to control, (13.2 1.1)x106 cm/s. The lowest IC50 inhibition constant for AVJ was estimated to 8.4 0.7 mg/mL. AVJ exerted a time dependent inhibition of CYP3A4 activity, but a decrease in CYP3A4 activity was found when incubated both in the presence and absence of NADPH. Conclusion: AVJ has a low interaction potential against P-gp (transport) and CYP3A4 (IC50) indicating no

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major interference with drug metabolism in man, but the dual mechanistic inhibition of CYP3A4 might be of clinical significance.

References


1. Bent, S. and R. Ko, Commonly used herbal medicines in the United States: a review. The American journal of medicine, 2004. 116(7): p. 478–85. 2. Lynch, T. and A. Price, The effect of cytochrome P450 metabolism on drug response, interactions, and adverse effects. Am Fam Physician, 2007. 76(3): p. 391–6. 3. Bruno, J.J. and J.J. Ellis, Herbal use among US elderly: 2002 National Health Interview Survey. Ann Pharmacother, 2005. 39(4): p. 643–8. 4. Ulbricht, C., et al., Clinical evidence of herb-drug interactions: a systematic review by the natural standard research collaboration. Curr Drug Metab, 2008. 9(10): p. 1063–120. 5. Slikker, W., NCTR FY2006-FY2007 Research Accomplishments and Plans, 2007, National Center for Toxicological Research (NCTR), U.S. Food and Drug Administration (FDA). p. 1–133.

P242. PK/PD EVALUATION OF METABOLISM-BASED SYNERGY: A CLASSICAL HERB PAIR RADIX ANGELICAE DAHURICAE AND RHIZOME LIGUSTICI Xin He, Shan Feng, Peiru Zhong, Jinyi Zhao and Changxiao Liu School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin, China Radix Angelicae dahuricae and Rhizome Ligustici is a classical herb pair used in many prescriptions for the treatment of migraine. Aim: The purpose of this study was to clarify the metabolism-based mechanism for the synergy between total coumarin extract (TCE) of Radix Angelicae dahuricae, which interferes with CYP450s activity, and ligustrazine, a CYP450s substrate possessing migraine relieving effects. Ligustrazine is a phytochemical isolated from Rhizome Ligustici. Methods: The effects of TCE on ligustrazine metabolism in rat liver microsomes (RLM) and human liver microsomes (HLM), were respectively evaluated. TCE was coadministered with ligustrazine to assess its influence on pharmacokinetics (PK) and pharmacodynamics (PD) of ligustrazine in rats. Results: TCE was observed to markedly (p50.05) potentiate ligustrazine AUC and reinforced its anticoagulation activity significantly (p50.05) in rats. The rate of formation of hydroxy-ligustrazine, a metabolite of ligustrazine, was inversely related to the concentration of TCE, imperatorin or isoimperatorin following simultaneous incubation with the parent compound; decreasing in RLM (from 2.5 to 0.2 ng/min) or HLM (from 9 to 1.5 ng/min). Imperatorin and isoimperatorin, two major coumarins in TCE, were shown to be both substrates and inhibitors of CYP450s, suggesting competitive inhibition as a mechanism responsible for the inhibitory effect on CYP450s. Conclusions: TCE could increase ligustrazine plasma concentration and then reinforce its pharmacological effect by inhibiting the CYP450 enzyme-mediated metabolism of ligustrazine. (This work was supported by Program for Changjiang Scholars and Innovative Research Team in University [PCSIRT, No.IRT0973] and Research Fund for the Doctoral Program of Higher Education [No.20121210110011].)

P243. INHIBITORY EFFECTS OF AN AQUEOUS EXTRACT OF CURCUMA XANTHORRHIZA AND ITS CONSTITUENTS ON CYP450 ISOFORMS Sabariah Ismail, Nur Aziah Hanapi and Sharif Mahsufi Mansor Centre for Drug Research, Universiti Sains Malaysia, Pulau Pinang, Malaysia Herbal supplements are commonly used as alternative medicines to treat various diseases which are often administered in combination with therapeutic drugs, raising the potential of herb-drug interactions. The interaction may involve having an herb component causing an increase or decrease in the amount of drug in the blood through induction or inhibition of drug metabolizing enzymes. CYP450s are the major drug metabolizing enzymes as nearly half of the marketed drugs are metabolized by the CYP450 isoforms; CYP2C9, CYP2D6 and CYP3A4. Curcuma xanthorrhiza or commonly known as ‘‘Javanese turmeric’’ is a member of the ginger family (Zingeberacea) and has been traditionally used throughout South East Asia for the treatment of liver damage, hypertension, diabetes and cancer. Therefore, the effects of an aqueous extract of Curcuma xanthorrhiza and its constituents, namely curcumene, curcumin, and xanthorrhizol were investigated in the present study for their inhibitory potential towards expressed recombinant human CYP2C9, CYP2D6 and CYP3A4 isoforms. A concentration range of 0.01 to 1000 mg mL1 of the extract and a concentration range of 0.02 to 200 mM of the constituents were tested on the recombinant human CYP450s using luminogenic substrates. Decrease in luminescence indicates inhibition of the CYP450 isoforms by the extract or constituents. Sulfaphenazole, quinidine and ketoconazole were used as positive controls for CYP2C9, CYP2D6 and CYP3A4 respectively. The IC50 values of percentage inhibition were determined using Graph Pad PrismÕ 5 (Version 5.01). Among the

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constituents, curcumin showed the highest inhibition towards CYP2C9, with an IC50 value of 4.43 0.49 mM. Both curcumin and xanthorrhizol inhibited CYP2D6 and CYP3A4 isoforms, nevertheless, lower IC50values were obtained with xanthorrhizol for both of the isoforms. The aqueous extract of Curcuma xanthorrhiza and curcumene however, did not inhibit the activities of any of the isoforms investigated. These findings show that there is low possibility for herb-drug interaction if Curcuma xanthorrhiza aqueous extract is taken concomitantly with other drugs which are primarily metabolized by the CYP450 isoforms. This study was funded by the NKEA Research Grant Scheme (NRGS), Ministry of Agriculture and Agro-Based Industry, Malaysia.

P244. IN VITRO CYP3A4 INHIBITION BY FRACTIONATED RHODIOLA ROSEA EXTRACTS


Lars H. Bjorneboe, Odd Georg Nilsen and Bent H. Hellum Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway Purpose: To obtain extracts of Rhodiola Rosea using different extraction solvents, and investigate the differences in the extracts’ inhibitory potential towards CYP3A4-mediated metabolism. Also to quantify and correlate assumed biological active constituents in Rhodiola Roseaextracts with the inhibitory potentials of the extracts to come closer in identifying properties of CYP3A4-inhibiting constituents. Method: Two commercial Rhodiola Rosea products (Rosenrot Forte, RF and Artic Root, AR), obtained at a local pharmacy in Norway, were extracted with, and dissolved in ethanol, water, dichloromethane or n-hexane. cDNA baculovirus –expressed CYP3A4 was incubated with herbal extracts for 10 minutes using testosterone as substrate. The formation of 6-OH-testosterone was quantified by a validated HPLC procedure. IC50 –values forRhodiola Rosea extracts were estimated based on inhibition plots using non-linear regression. Rhodiola Roseaextracts were analysed by HPLC methodology in order to quantify the assumed biological active constituents rosavin, rosin, rosarin, salidroside and tyrosol. Results: Ethanolextracted AR and RF gave IC50-values of 135.1 1.7 mg/ml and 125.3 4.5 mg/ml, respectively. Water extract of AR had an IC50-value of 543.7 1.8 mg/ml, while water-extracted RF did not show any significant inhibition towards CYP3A4 in vitro. Dichloromethane-extracted AR and RF produced IC50-values of 336.5 mg 7.5 mg/ml and 652.7 50 mg/ml, respectively. N-hexan extracts of AR and RF did not produce any inhibitory effect on in vitro CYP3A4 mediated metabolism. The assumed active constituents were present in the ethanol- and water-extracts, but there were large variations in the quantities of the constituents. Only small amounts of the constituents were found in the dichloromethane- and n-hexan extracts. There was no correlation between inhibitory potential of the extracts towards CYP3A4 and contents of rosarin, rosin, rosavin, salidroside and/or tyrosol. Conclusion: The ethanol extracts displayed a more potent inhibitory effect on CYP3A4 mediated metabolism than the other extracts. The ethanol extracted constituents may be the main contributor to the inhibitory effect on CYP3A4 by Rhodiola Rosea seen in other studies. No inhibition by the n-hexan extract also points to a polar constituent responsible for the inhibitory properties. Although rosavin, rosin, rosarin, salidroside and tyrosol are extracted mainly by the polar solvents, the lack of correlation between inhibitory effects and the quantified constituents suggests that there are one or more other polar constituent(s) responsible for the observed inhibition.

P245. STUDIES ON LIGNUM DALLBERGIAE ODORIFERAE ASSIST RADIX SALVIAE MILTIORRHIZAE BY OVERLAPPING REGULATION OF P-GP/CYP3A VIA PXR NUCLEAR RECEPTOR PATHWAY Zhihong Yang1, Yuyang You2, Xuehui He3, Shengmin Liu3 and Xiaobo Sun3 1 Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Medicinal Plant Development, Beijing, China, 2Tsinghua University, Beijing, China, 3Institute of Medicinal Plant Development, Beijing, China Guanxin-danshen decoction (GDD, including Radix Salviae Miltiorrhiae, Radix Notoginsen, and Lignum Dalbergiae Odoriferae), a well-known TCM prescription, has been with our systematic pharmacodynamic studies. In the last several years, many new researches in our laboratory on this GDD were undertaken, and some findings indicated that GDD has well protective effects not only on myocardial but also on cerebral ischemia/reperfusion injury via PI3K/Akt, ERK and MAPKs signal pathways. Molecular mechanisms and signal pathways of GDD’s protective effects have been furthered considerably, yet many questions remain as to the compatibility profile of these herbs in GDD in the field of pharmacokinetics, and the corresponding absorption and metabolism mechanisms of herb-herb interaction. Aim: To elucidate the reasonable TCM compatibility theories from the aspect of pharmacokinetic characteristics and the potential effects and mechanism on P-gp and CYP3A in GDD. Methods: Salvianolic acid B, salvianolic acid A, tanshinol and cryptotanshinone are abundant and active hydrophilic or lipophilic components from Radix Salviae Miltiorrhizae, and mainly responsible for its clinically therapeutic effects. Naringenin is one of the flavonoids at the highest concentration in Lignum Dalbergiae Odoriferae. Above five effective components are chosen as the representative ingredients. To study the effect of naringenin on the pharmacokinetic profile of four effective components from Radix Salviae Miltiorrhizae in vivo and in vitro, a rapid and sensitive LC-MS/MS method for the determination them in rat plasma, tissues and Caco-2, MDCK cells is developed. The known inhibitor cyclosporine A, classic


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substrate rhodamine 123 and specific substrate digoxin (not metabolized by CYP3A) were used to assess the functional activity of P-gp. And then the specific substrate midazolam (not efflux transported by P-gp) was applied to investigate the enzymatic activity of CYP3A. Meanwhile, western blot and RT-PCR were used for investigating the protein expression of P-gp and the mRNA level of CYP3A, respectively. Finally, fluorescence polarization detection method was used to gain insight into whether naringenin effect on the four effective components by overlapping regulation of P-gp/CYP3A via PXR nuclear receptor pathway. Results: Naringenin as main flavonoid in Lignum Dalbergiae Odoriferae, not only with a broad spectrum of pharmacological activities just like the most flavonoids in pharmacodynamic aspect, but also could affect the courses of absorption and disposition of salvianolic acid B, salvianolic acid A, tanshinol and cryptotanshinone in rat in vivo and in Caco-2 cells in vitro in pharmacokinetic aspect. In the presence of naringenin, the pharmacokinetic parameters of salvianolic acid B and cryptotanshinone vary significantly (p50.05). The data of t1/2, MRT, AUC(0t), Cmax, Vd and CL in rat plasma are changed from 56.9 min, 73.1 min, 159.3 mgmin/L, 3.0 mg/L, 9.8 L/kg and 0.12 L/min/kg to 47.6 min, 66.5 min, 191.3 mgmin/L, 3.9 mg/ L, 7.0 L/kg and 0.10 L/min/kg, respectively. Naringenin could improve absorption and distribution of four effective components in rat, especially increasing their concentrations in brain tissue. Moreover, it was found that naringenin significantly decreased efflux of Rho123 and digoxin from Caco-2 cells in a concentration-dependent manner, and inhibited metabolism process of midazolam in rat. The protein expression of P-gp and the mRNA level of CYP3A were all decreased accordingly. It was found further that naringenin might inhibit the PXR to recruit the SRC-1. Conclusion: Naringenin in Lignum Dalbergiae Odoriferae could assist the four effective components from Radix Salviae Miltiorrhizae to increase and prolong their absorption process, promote them into the target tissue, and slow their metabolism process by inhibit P-gp and CYP3A simultaneously. Furthermore, the negative regulation of PXR nuclear receptor pathway might be involved in overlapping mechanism of P-gp/CYP3A. Grant sponsor: Key Projects of the National Science & Technology Pillar Program (2008BAI51BOO), and National Natural Science Foundation of China (81102879, 81273654).

P246. DEVELOPMENT OF A MINIATURIZED MECHANISM-BASED INHIBITION ASSAY FOR HIGH-THROUGHPUT MASS SPECTROMETRY ANALYSIS Jing Wang1, Gino Segre2, Temo Bandzara3 and Matthew Greving4 1 Applications, Labcyte, Inc., Sunnyvale, CA, 2Marketing, Labcyte, Inc., Sunnyvale, CA, 3R&D, Labcyte, Inc., Sunnyvale, CA, 4 Nextval, San Diego, CA Mechanism-based inhibition (MBI) of CYP450 enzymes often results in clinically significant drug-drug interactions. The evaluation of MBI is critical but it also presents many challenges including complex experimental design, numerous incubation time points, consumption of expensive reagents, and lengthy mass spectrometry data collection and analysis. This makes MBI evaluation costly and time consuming. To meet these challenges a miniaturized, high throughput MBI assay with mass spectrometry readout has been developed. By combining the nanoliter scale, non-contact dispense capability of a Labcyte Echo liquid handler with the sensitivity and high throughput of the Nextval MassInsight process, high quality data was obtained. The results demonstrate MBI and other ADME assays can be high throughput and miniaturized on a routine basis.

P247. STATE OF THE ART IN VITRO ADME ASSAYS AND WORK FLOW IN PHARMACEUTICAL DISCOVERY Yau Yi Lau, Stella Doktor, Kelly Desion, Tom Suhar, Sue Swanson, Ellen Roberts and Sonia De morais Department of Drug Metabolism and Pharmacokinetics, Abbvie Inc, North Chicago, IL Enabling multiparametric optimization and generation of IVIVCs are essential to drive the optimization of drug-like properties and enhance productivity at the Discovery stage. With the help of various technologies, Drug Metabolism has developed and implemented a panel of high-throughput ADME assays to screen all project related compounds synthesized. The panel of ADME assays include human (HLM) and rat (RLM) microsomal stability, microsomal protein binding (Fu mic), cell based permeability (cell perm), solubility and eLogD. These assays have a capacity of 752 compounds per week and the median cycle time is one day from receipt. A number of state of the art technologies were employed to achieve the objectives of high quality, high capacity and short cycle time. These include 96- and 384-well platforms, fast LC-MS-MS, enabling proprietary software, robotic automation and plate reader analytics. In order to achieve the high capacity and short cycle time while keeping cost low, all assays were automated using either a 96- or 384-well format. Ultra fast, high sensitivity and specificity LC/MS instruments are used for sample analysis. The specificity of MS detection allows the analysis of samples in a cassette fashion. The highly efficient software DiscoveryQuant is used to automatically determine the LC/MS parameters for all compounds analyzed, create the MS run method, submit the sample batches and generate a user friendly peak review interface. This poster will detail each assay and the technologies used that enabled the characterization of all newly synthesized compounds in these high quality, high capacity in vitro ADME assays.

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P248. ULTRAFAST PLASMA PROTEIN BINDING ANALYSIS USING A HIGH-THROUGHPUT SPE/TOF-MS SYSTEM


Kari E. Schlicht1, Vaughn P. Miller2, William A. LaMarr1 and Can Ozbal1 1 Agilent Technologies, Wakefield, MA, 2Agilent Technologies Inc., Wakefield, MA To enable earlier optimization of drug candidates, there has been increased demand for higher throughput and faster turnaround of in vitro ADME analyses. One in vitro ADME analysis that is key to assessing both the pharmacokinetics and pharmacodynamics of a drug candidate is plasma protein binding (PPB). LC/MS/MS has become the method of choice for determining PPB due to its selectivity and sensitivity. However, disadvantages include the need to develop individual MRM methods for every compound and analysis times of several minutes per sample. In the present study, we sought to overcome these time-consuming disadvantages by developing a single, ultrafast method for analysis utilizing an online SPE/TOF-MS system. Human plasma was spiked with individual test compounds at a concentration of 5 mM and subjected to rapid equilibrium dialysis (RED). More than forty compounds with diverse chemical properties including a xLogP range of 0.4 to 7.1 and molecular weight range of 160–733 g/mol were evaluated in triplicate. Following a 5 hour incubation, samples were precipitated with the addition of acetonitrile containing 0.1% formic acid and internal standard. Samples were then centrifuged, diluted with water, and transferred to a microwell plate for analysis. Identical samples were analyzed using a High-Throughput RapidFire Mass Spectrometry System interfaced with a Q-TOF as well as by HPLC interfaced with a QQQ mass spectrometer. The SPE/MS-TOF system analyzed the samples at a rate of 9 seconds per sample while the LC/MS/MS methodology provided sampling rates of 3 minutes per sample. The percentage bound was calculated for each compound from both analytical analyses. The analytical results for the SPE/TOF-MS method were found to be comparable to traditional LC/MS/MS despite the differences in methodology. There was excellent correlation between the two methods with a R2 value of 0.977. However, the SPE/TOF-MS method provided 20 times greater throughput compared to LC/MS/MS with a capacity of 400 samples/hour. This method also provided increased efficiency by eliminating the need for offline MRM method development. This ultrafast, high resolution approach may be useful for the analysis of other ADME assays.

P249. A HIGH THROUGHPUT ASSAY FOR ISOFORM-SPECIFIC P450 INHIBITION IN HUMAN HEPATOCYTES Albert P. Li1, Utkarsh Doshi1, Qian Yang1, Kari E. Schlicht2, Nikunj Parikh2 and Vaughn P. Miller2 1 In Vitro ADMET Laboratories LLC, Columbia, MD, 2Agilent Technologies Inc., Wakefield, MA P450 inhibition is a major mechanism for pharmacokinetic drug-drug interactions. Screening new chemical entities (NCE) for P450 inhibitory potential is now routinely practiced in early phases of drug development. Traditionally, P450 inhibition evaluation is performed with human liver microsomes (HLM). Recently, it is recognized that P450 inhibition with intact human hepatocytes may offer data that are more relevant to humans in vivo due to the presence on intact cell properties (ICP) including an intact plasma membrane which controls bioavailability, uptake transporters for bioaccumulation, physiological levels of enzymes and cofactors to minimize artifacts (e.g. ubiquitous binding to microsomal proteins), and the presence of complete drug metabolizing pathways for metabolic inactivation or activation. Previously, we have reported a higher throughput screening assays for CYP3A4 inhibition using luciferin IPA (LIPA) which allows the employment of plate reader for rapid quantification of luciferin formation (Doshi U, Li AP. J Biomol Screen. 16(8):903–9, 2011). This approach, however, cannot be extended towards nonCYP3A4 isoforms due to the lack of effective luciferin-based substrates for the nonCYP3A4 isoforms. We thereby explore the effectiveness of the High-throughput RapidFire/MS system for isoform-specific P450 inhibition screening. The RapidFire/MS system allows ultra-fast sample analysis (cycle time of 7–16 seconds per sample). Cryopreserved human hepatocytes from five donors were individually thawed and pooled for the study. The 384-well plate format was employed with the following sequential steps: 1. Addition of 10 uL of 4X concentrated inhibitors; 2. Addition of 10 uL of 4X concentrated isoform-selective substrate; 3. Addition of 20 uL of a 2X concentrated hepatocyte suspension; 4. Incubation at 37 deg. C for 60 minutes; 5. Addition of 40 uL acetonitrile containing internal standards to terminate the reaction. The 384-well plates were then stored at 80 C until RapidFire/MS analysis. One 384well plate was used for each substrate which allows the evaluation of 16 test articles at 8 concentrations (solvent control plus 7 concentrations) in triplicate. One 384 well plate was used for the quantification of cell viability to account for decrease in activity due to cytotoxicity. Over 15 model inhibitors were evaluated for their inhibitor effects in human hepatocytes using the following isoform-selective substrates (tacrine (CYP1A2); bupropion (CYP2B6); amodiaquine (CYP2C8); diclofenac (CYP2C9); s-mephenytoin (CYP2C19); dextromethorphan (CYP2D6); midazolam (CYP3A4). Deuterium labeled hydroxylation metabolites were used as internal standards. Robust signals were observed for the substrates except for s-mephenytoin. Both time- and dose-dependent isoform-specific inhibition of P450 activities were observed with the model inhibitors, with results qualitatively and quantitatively similar to published results for HLM. The HTS RapidFire-Human Hepatocyte P450 Inhibition Assay described here should be useful for routinely screening of NCEs to minimized drug-drug interaction liability due to P450 inhibition.

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P250. TOXICITY OF THE DIARRHETIC MARINE PHYCOTOXINS OKADAIC ACID AND ITS ANALOGUES DINOPHYSISTOXIN-1 AND -2 IN THE HUMAN HEPATIC HEPARG CELL LINE


Pierre-Jean Ferron1, Hogeveen Kevin2, Roger Rhamani3, Georges De Suza4, Le Hegarat Ludovic2 and Fessard Vale´rie2 1 Unite´ de toxicologie des contaminants, ANSES, Fouge`res, France, 2ANSES, Fouge`res, France, 3UMR 1331 TOXALIM, Equipe 10: Toxicologie Cellulaire & Molećulaire des Xeńobiotiques, INRA, Antibes, France, 4INRA, Antibes, France Lipophilic toxins produced by various species of marine microalgae have been shown to accumulate in edible bivalve shellfish leading to human intoxications following consumption. In Europe, the polyether toxin okadaic acid (OA) is largely involved in the diarrheic shellfish poisoning (DSP) inducing severe gastro-intestinal effects including diarrhea, nausea, vomiting and abdominal pain. Two other related toxins, dinophysistoxins 1 and 2 (DTX-1, DTX-2), which differ from OA in the number or position of methyl groups, have been also involved in DSP syndrome. The principal mechanism mediating the toxicity of OA and DTX is generally thought to be the inhibition of protein phosphatases, however recent studies have brought into question this paradigm, and hypothesize that several other pathways could be involved. Further investigation is therefore needed to clearly elucidate the molecular mechanisms of action. Therefore, in order to investigate how structural differences between OA and its analogues affect their toxicity, we assessed various toxicity endpoints in human hepatic cell lines. We assessed cytotoxicity, apoptosis and DNA damage in HepaRG cells treated with OA, DTX-1 and DTX-2 using a high content screening approach. We then evaluated the activation of the human Pregnane X Receptor (PXR) by OA and its analogues in a HepG2 cell model expressing a PXR-responsive luciferase reporter. Results from high content screening studies and PXR gene reporter assay showed effects with lower concentration of DTX-1 compared to OA and DTX-2. The modulation of CYP450 activities by DSP toxins in HepaRG may affect the difference of toxicity between OA, DTX-1 and DTX-2. Based on our results, we discuss the role of hepatic metabolism in the evaluation of the toxicity of the different analogues of okadaic acid, and how this could contribute to a more accurate hazard assessment of diarrheic marine phycotoxins.

P251. PREDICTION OF PLASMA PROTEIN BINDING USING GENETIC ALGORITHMS AND LOCAL LAZY REGRESSIONS Narayanan R, Vijay Singh and Sitarama Gunturi Life Sciences R & D Division, Tata Consultancy Services Ltd., Hyderabad, India Plasma protein binding profile of drugs plays a crucial role in determining the fraction of free drug that is distributed through the various tissues and consequently, the resultant pharmacological response [1]. Thus, an early estimate of the plasma protein binding profile of new drug candidates, even before chemical synthesis will be very useful in the selection and optimization of drug candidates. Researchers have employed different methods such as Machine Learning techniques, Multiple Linear Regression (MLR) etc for the prediction of plasma protein binding based on the publicly available data. However most of these approaches suffer from i) use of small datasets ii) insufficient validation and iii) use of several descriptors The limitations of the reported approaches, prompted us to employ an integrated approach to build robust and easily interpretable protein binding models. We describe herein, the applications of a number of variable selection, data division and regression methods and the best models are generated using the combinations of a) Kennard Stone approach for data division [2] b), Genetic Algorithms (GA) for variable selections [3] and Local Lazy Regression (LLR) method [4] for model generation based on a large dataset of 1639 drugs and drug-like compounds [5] and 445 number of physico-chemical descriptors. The best model has the following impressive statistical parameters: r2 ¼ 0.7086, Q21 ¼ 0.6798 and Q22 ¼ 0.6876. Based on the in-depth analyses it is inferred that a) the performances of LLR based models are superior to the reported models b) the best model is based on less number of descriptors, six and c) the models are easily interpretable as they are generated using least squares principle in the neighborhoods.

References 1. Van de Waterbeemd, H.; Gifford, E. ADMET in silicomodelling: Towards Prediction Paradise? Nat. ReV. Drug DiscoVery 2003, 2, 192–204. 2. Kennard RW, Stone LA. 1969. Computer aided design of experiments. Technometrics 11:137–148. 3. J.H. Holland, Adaptation in Natural and Artificial Systems, MIT Press, Cambridge, MA, 1975. 4. lazy package, R 2.9.0, R Project Home Page. http://www.r-project.org 5. Xiang-Wei Zhu & Alexander Sedykh&Hao Zhu &Shu-Shen Liu & Alexander Tropsha, The Use of PseudoEquilibrium Constant Affords Improved QSAR Models of Human Plasma Protein Binding, Pharm Res (2013) 30:1790–1798.

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P252. A MECHANISM BASED MODEL OF STEROIDS METABOLIC SITES PREDICTION FOR CYTOCHROME P450 3A4


Ziru Dai, Chunzhi Ai, Guangbo Ge, Huizi Man, Yan Jia and Ling Yang Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, DaLian, China Cytochrome P450 3A4 metabolizes nearly 50% of the drugs currently available on the market. Early prediction of xenobiotic metabolism can guide optimization process in drug discovery and development. But as CYP3A4 has a large active site cavity with a broad range of substrate specificity, it is of great challenge to predict its metabolic sites, especially for the kind of steroids compounds with rigid structures, which are difficult to change their conformation to accommodate to the CYP3A4 active cavity. Actually, the selectivity of the metabolic sites is primarily based on the binding conformation and molecular reactivity. Hence, we established a mechanism-based prediction model by evaluating binding accessibility and activation energy simultaneously. We collected 36 steroids compounds including three major metabolic reactions, aliphatic hydroxylation, N-dealkylation and aromatic hydroxylation reaction as our training set, and 14 well-known other structures of substrates as test set for the prediction model validation. On the basis of the generation of the bioactive pose for the molecules in training set, two representative interaction features were refined to establish the criteria for rational conformations: (1) hydrogen-bond interaction with three water molecules and five important amino acids Arg105, Arg106, Ser119, Ile301 and Glu308; (2) lipophilic interaction with Phe-cluster or toward the heme, The conformations satisfying one of the two feathers were considered as the possible binding conformation, and then were submitted to evaluate the molecular activation energy. The metabolic sites with lowest energy were defined as the potential reaction sites. As a result, by evaluating the distance between the preferred site and the heme, chemscore and activation energy, our model can reliably identify at least one observed site of metabolism in the top three rank-positions for 75% of the substrates in training set and 92.85% for the test set. We also find that the model show superiority in predicting aliphatic hydroxylation and Ndealkylation over aromatic hydroxylation. This should be attributed to the different catalytic process that the former followed a radical mechanism while the latter demonstrated a mechanism of aromatic ring addition. In conclusion, we got a metabolism sites prediction model based on combination with binding and the catalytic mechanism with high predictive accuracy and scalability, which could be implement to not only unpredictable rigid steroids substrates but also other structure of compounds.

P253. COMPUTATIONAL INSIGHT INTO SUBSTRATE BINDING DETERMINANTS OF CYTOCHROME P450 2A6 Yan Jia, Chunzhi Ai, Yong Liu, Huizi Man, Ziru Dai and Ling Yang Dalian Institute of Chemical Physics, Dalian, China It is well known that cytochrome P450 2A6 (CYP2A6) has been shown great selectivity in substrates due to its small and compact active pocket. However, with the increase of its substrates recently identified, structural diversity of CYP2A6 substrate is paid more attention to. Previously, we have identified the lipophilic/electrostatic potential as well as the orbital energies as key features in determining the binding specificity of naphthalene inhibitors of CYP2A6. In the present study, a detailed summary of CYP2A6 substrates was described followed by a series of computational analysis. Based on the bioactive conformation generated by 1Z10 (CYP2A6 combined with coumarin), the dependence of MLP (molecular lipophilic potential), MEP (molecular electrostatic potential) and orbital energy analysis upon the binding affinity were carried out to explore the common characteristics among substrates as well as enzyme-substrate interactions. Quantum Mechanic (DFT method) was further employed to illustrate the electrostatic /pi-pi interaction intensity between special residues and the substrate. The results turned out that substrate tended to form stable H-bond with Asn297, T-shape pi-pi interaction with Phe107/480/118, and a paralleldisplaced pi-pi interaction with Phe209. Comparatively, the H-bond or electrostatic interaction with Asn297 plays a more crucial role in selecting substrate rather than inhibitor for its special role in governing a proper conformation of metabolism. Hydrophobic interaction or pi-pi stacking between Phe and substrate also strengthen the binding affinity. The specified binding determinants of 2A6 can be a good application in predicting substrate out of similar structures. This was verified by an in vitro experiment that identified tanshinone IIA as a selective substrate of 2A6 out of its structurally similar analogs. The proposed study takes a step toward understanding the substrate selectivity of CYP2A6 enzyme.

P254. DYNAMIC MODELLING OF HEPATOCYTE REGULATORY NETWORKS USING NOVEL PETRI NET-LINKED FLUX BALANCE ANALYSIS Ciaran P. Fisher1, Andrzej M. Kierzek2, Nick J. Plant3 and J. Bernadette Moore1 1 Nutrition and Metabolism, University of Surrey, Guildford, United Kingdom, 2Micorbial and Cellular Science, University of Surrey, Guildford, United Kingdom, 3Biochemistry and Physiology, University of Surrey, Guildford, United Kingdom


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Objectives: Comprehensive, hepatocyte specific, genome scale metabolic network reconstructions have been published, but are limited to steady-state simulation. As such, they cannot reproduce the liver’s capacity to dynamically regulate the expression of its metabolic machinery and so modulate its response to stimuli. Building on these established models, we have developed a novel modelling strategy combining Petri net formalism, to model signalling molecules and pathways, with metabolic flux balance analysis. The objective of this work was to validate Quasi steady-state Petri nets (QSSPN) through the reconstruction and dynamic simulation of hepatic bile acid synthesis in silico. Methods: Signalling pathways were reconstructed in an extended Petri net format, from databases and the published literature, and linked to a hepatocyte specific flux balance model of metabolism. Briefly, the novel PNFBA algorithm sets flux bounds through the status of two Petri net node classes: constraint nodes, that establish the reaction bounds within the flux balance model and substrate nodes, that translate metabolite producibility into Petri-net status. Reaction bounds and metabolite producibility are calculated at each time step of the simulation, reproducing a dynamic metabolic response. Using our model, we simulated (1000 trajectories) an acute increase in intracellular cholesterol and monitored subsequent changes in bile acid synthesis, cholesterol and bile acid clearance and the expression of enzyme and transport proteins. Results: In response to increased intracellular cholesterol, the model demonstrated a sharp increase in intracellular bile acids. Importantly, the in silico hepatocyte model was able to modulate both bile acid synthesis and transport and ultimately, the clearance of the cholesterol. Key enzymes in the bile acid synthesis pathway were down-regulated in response to the increase in bile acids. While export proteins mediating secretion into the biliary compartment were up-regulated, hepatic bile acid uptake mechanisms were down-regulated transiently in response to elevated bile acids. Conclusions: This regulatory competent, in silico, hepatocyte model of bile acid homeostasis demonstrates PNFBA as a novel and robust strategy for the dynamic modelling of regulatory networks in hepatocytes, as well as other cells.

P255. SIMULATING THE EFFECT OF UGT, SULT AND MRP2 INDUCTION AND/OR INHIBITION ON THE DISPOSITION OF RESVERATROL AND ITS CONJUGATED METABOLITES Aneesh Arvind Argikar1 and Swati V. Nagar2 1 Pharmaceutical Sciences, Temple University, Philadelphia, PA, 2Dept of Pharmaceut Sciences, Temple University School of Pharmacy, Philadelphia, PA To evaluate the effect of human and mouse UGT, SULT and MRP2 inhibition/ induction on the disposition of resveratrol and its conjugated metabolites. SAAM II v.2.1 was used to perform all simulations. A comprehensive model generated for studying the disposition of resveratrol and its major conjugated metabolites was used (1). 1) Simulations were first performed with this model assuming linear elimination kinetics. The disposition of resveratrol, resveratrol-3-glucuronide (R3G), and resveratrol-3-sulfate (R3S) was evaluated with the following scenarios: i) inhibition or induction of mouse ugt1a ii) inhibition of sult1a, and iii) induction of mouse ugt1a and mrp2. 2) Next, non-linear Michaelis-Menten elimination kinetics were assumed for resveratrol glucuronidation. Clearance was calculated as Vmax/Km from published in vitro data (2, 3). Simulations were performed as a first attempt to correlate in vitro and in vivo glucuronidation data. 3) Finally, the role of hMRP2 (BD Biosciences) in resveratrol transport was investigated. The transport of probe MRP2 substrate, CDCF [5(6)-Carboxy-20 , 70 -dichlorofluoresccein] (santa cruz biotechnology, inc.), was investigated using hMRP2 inside out vesicles. Kinetic parameters for CDCF uptake were obtained. Inhibition of CDCF uptake by curcumin was evaluated. The possibility of MRP2 inhibition by resveratrol or resveratrol-3-glucuronide is currently being investigated. 1) Mouse ugt1a inhibition or induction caused no change in simulated resveratrol concentration profiles. R3S exposure was predicted to decrease 9-fold upon 2-fold ugt1a induction, with no change upon ugt1a inhibition. R3G exposure was predicted to decrease 2-fold and increase 1.9-fold upon 2-fold ugt1a inhibition or induction respectively. Sult inhibition was predicted to only decrease R3S exposure proportionally, with no change in resveratrol or R3G profiles. Mrp2 induction modulated R3S and R3G exposure. 2) Human in vitro glucuronidation data predicted lower resveratrol exposure than in the mouse, with higher R3G being formed. Mouse in vitro data were unable to predict in vivo mouse pharmacokinetics. 3) CDCF Vmax and Km were 263.6 42.7 pmol/min/mg and 38.5 14.8 mM respectively (data presented as estimate SE, n ¼ 2). Curcumin (5 mM) inhibited MRP2 activity by 29–43% (n ¼ 2). Simulations of mouse ugt inhibition and induction, sult induction and mrp2 induction were used to predict the changes in disposition of resveratrol and its conjugated metabolites. In vitro glucuronidation data did not correlate with in vivo mouse pharmacokinetics. The role of MRP2 in resveratrol disposition is being evaluated. Interplay between glucuronidation, sulfation and transport will be characterized in future studies. This work was funded in part by NIH grant 1R03CA159389 to SN.

References 1. Sharan S et.al DMD 2012: 40(10):1993–2001. 2. Iwuchukwu et.al DMD 2008: 36(2): 322–330. 3. Maier-Salamon et.al Drug Metab. Pharmacokinet. 2011: 26(4): 364–73

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P256. EXAMINING THE RELATIVE ROLES OF APICAL VERSUS BASOLATERAL TRANSPORT IN DETERMINING DRUG FATE


Joanna H. Sier and Nick J. Plant Biochemistry and Physiology, University of Surrey, Guildford, United Kingdom The role of active drug transport in determining chemical fate is becoming increasingly clear, with efflux transporters such as MDR1, MRP2 and BCRP recognised as key determinants of drug action. These efflux transporters may impact upon both compound bioavailability, through first-pass effects, and drug resistance, through their up-regulation following chronic drug exposure. While the role of these transporters is well established, there is less information on other transporters, such as MRP3 and MRP4, which may act to move chemicals back into the systemic circulation rather than target them for elimination. We have reconstructed the molecular interaction network of active drug transporters, passive transport and drug metabolising enzymes that exists in the liver and determines the biological fate of 17b-estradiol and it’s metabolites. The mode was generated using CellDesigner (v4.0.1; Systems Biology Institute, http://celldesigner.org/index.html), a graphical front-end for creating process diagrams of biochemical networks in systems Biology Markup Language. Each individual chemical or protein is identified as a species (s1. . .. . .sn), while interactions between species are identified as reactions (r1. . ..rn). For each reaction, a kinetic term can be included, detailing the mathematics underlying the interaction between the species. For more complex analysis, CellDesigner-generated models were transferred to COPASI (v4.6, build 32), which is another Systems Biology Markup Language-compliant programme, but with a wider variety of analysis options. The model is paramaterised using specific kinetic values derived from the literature, or biologically realistic estimates that allow replication of the known biological behaviour of the system. As such, it represents, to the best of our knowledge, the situation occurring in the liver. Under steady-state conditions and physiologically relevant concentrations of 17b-estradiol we are able to demonstrate that network flux is predominantly via passive processes, with active transport a minor component. However, as higher concentrations of 17b-estradiol are simulated then the role of active transport becomes increasingly important, until it at micromolar substrate concentrations (equivalent to plasma range seen with many therapeutic compounds) it represents a significant proportion of total trans-membrane flux. Using a gene essentiality screen we predict that MRP2 is the most sensitive node in the network with respect to determining compound apical:basolateral flux ratio. In summary, we present a computational approach to examine the relative input of network components in determining chemical fate. Such computational approaches provide novel systems to examine the impact of genetic variation or transporter-specific inhibitors on drug fate and, hence, efficacy.

P257. MIDAZOLAM CLEARANCE IN HUMAN HEPATOCYTES IS RESTRICTED COMPARED WITH HUMAN LIVER MICROSOMES BUT NOT BY CELL PERMEABILITY OR COFACTOR AVAILABILITY Faraz Kazmi1, Phyllis Yerino1 and Andrew Parkinson2 1 XenoTech, LLC, Lenexa, KS, 2XPD Consulting, Shawnee, KS In our previous work we confirmed previous reports that the clearance of midazolam, but not dextromethorphan, in cryopreserved human hepatocytes (CHH; n ¼ 50) was an order of magnitude less than that in pooled human liver microsomes (HLM; n ¼ 200). We also demonstrated that this was not due to low levels or activity of CYP3A4/5 in CHH because CYP3A4/5 activity in microsomes isolated from the pooled CHH was comparable to that in pooled HLM (within 20%). Previous investigators have proposed that the restricted clearance of midazolam (and other high clearance drugs) in hepatocytes may be due a limitation imposed by membrane permeability or the availability of cofactor (NADPH); two factors that would not impact midazolam clearance by HLM. In the present study, we investigated if these factors were involved in the greatly reduced clearance of midazolam in CHH relative to HLM. Pooled CHH (1 106cells/mL) were incubated with 1 mM midazolam for 0–120 min at 37 C with the following treatments: (A) intact cells, (B) intact cells þ exogenous NADPH (0.1 mM), (C) cells sonicated for 60 sec, (D) cells sonicated for 60 sec þ exogenous NADPH (0.1 mM), (E) cells treated with 0.01% saponin for 5 min, and (F) cells treated with 0.01% saponin for 5 min þ exogenous NADPH (0.1 mM). Additionally, whole system (WS) versus media loss (ML) of midazolam (1 mM) was examined with pooled CHH (1 106 cells/mL) incubated for 0–60 min at 37 C, and reactions were stopped directly with organic solvent for WS samples, whereas ML samples were centrifuged and aliquots of the supernatant fraction were added to organic solvent. All samples were analyzed by LC/MS for midazolam loss as well as 10 -hydroxymidazolam formation. Permeabilizing the hepatocytes with saponin (with or without exogenously added NADPH) had little or no impact on midazolam metabolism. Likewise, sonicating the hepatocytes (with or without exogenously added NADPH) had little or no impact on midazolam metabolism. These results suggest that neither the rate of uptake of midazolam into hepatocytes nor the amount of NADPH available to support CYP3A4/5 in hepatocytes is the reason why midazolam clearance in human hepatocytes is restricted compared with that in human liver microsomes.

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P258. TEST SYSTEM-DEPENDENT CLEARANCE OF CYP2D6 AND CYP3A4/5 SUBSTRATES: A COMPARISON OF HUMAN LIVER MICROSOMES AND CRYOPRESERVED HUMAN HEPATOCYTES


Faraz Kazmi1, Phyllis Yerino1 and Andrew Parkinson2 1 XenoTech, LLC, Lenexa, KS, 2XPD Consulting, Shawnee, KS The in vitro to in vivo extrapolation (IVIVE) of drug clearance involves the determination of intrinsic clearance in vitro (CLint), based on in vitro measurements of Vmax/Km or half-life (t1/2) in human liver microsomes (HLM) or cryopreserved human hepatocytes (CHH), which are then scaled to predict hepatic clearance in vivo (CLH,int). Although in vitro values of CLint often underpredict in vivo values of CLH,int, the values of CLint determined with HLM would be expected to match those determined in CHH for drugs predominantly cleared by cytochrome P450 (CYP). However, there are reports, in the case of drugs rapidly cleared by CYP3A4, showing that CLint values determined in HLM are much greater than those determined in CHH whereas the opposite is true for drugs that are slowly cleared by CYP2D6. In the present study we examined the clearance of the CYP3A4/5 substrates midazolam (high intrinsic clearance) and the CYP2D6 substrate dextromethorphan (low intrinsic clearance) in HLM and CHH. Midazolam and dextromethorphan were incubated at 1 mM or Cmax with CHH (pool of 50 at 1 106 cells/mL) or the equivalent concentration of HLM (pool of 200 at 0.33 mg/mL). The samples were analyzed for loss of parent drug and formation of the major metabolite over 0–15 min (HLM) and 0–120 min (CHH). Additionally, microsomes were isolated from the pooled CHH so that CYP3A4/5 and CYP2D6 activities could be compared with those in pooled HLM based on rates of formation of 10 hydroxymidazolam and dextrorphan, respectively. In CHH, dextromethorphan t1/2 values at 1 mM and Cmax were approximately 56 and 81 min, respectively. Midazolam t1/2 values at 1 mM and Cmax were approximately 75 and 63 min respectively. The scaled value for dextromethorphan CLH, int determined with HLM (115 L/h) agreed well with the scaled value ofCLH,int determined with CHH (1 mM, CLH,int ¼ 147 L/h; Cmax, CLH,int ¼ 102 L/h). For midazolam the scaled value of CLH,int determined with HLM (2130 L/h) was significantly different from that determined with CHH (1 mM, CLH,int ¼ 110 L/h; Cmax, CLH,int ¼ 129 L/h). Microsomes isolated from the pooled CHH had CYP3A4 and CYP2D6 activity comparable (within 20%) to those in pooled HLM, confirming previous reports that, with rapidly metabolized substrates like midazolam, CYP3A4 activity in hepatocytes is somehow restricted compared with CYP3A4 activity in HLM.

P259. PREDICTION OF UGT-MEDIATED GEMFIBROZIL CLEARANCE USING IN VITRO DETERMINED INTRINSIC CLEARANCE Anil Kolur, Jessica F. Rehmel, Stephen D. Hall, Yingying Guo and Michael A. Mohutsky Drug Disposition, Eli Lilly & Company, Indianapolis, IN The relative contributions of non-cytochrome P-450 enzymes such as the uridine 50 -diphosphoglucuronsyltransferase (UGT) to metabolic drug clearance are often not well characterized. Glucuronidation was found to be the major clearance mechanism for 10% of the top two hundred prescribed drugs, so there is a need to predict the quantitative contribution to clearance by different UGT isoforms and determine the substrates that are likely to be cleared through each enzyme. In vitro to-in vivo extrapolation (IVIVE) for UGTs is hindered by the absence of information regarding abundance, relative activity, and the lack of a specific probe or inhibitor for each enzyme. We validated a standardized in vitro screening approach with positive controls for metabolite formation through each enzyme using human recombinant UGT matrices (rUGT) as well as human liver, intestine, and kidney microsomes (HLM, HIM, and HKM respectively) to identify the isoforms responsible for the clearance of our model compound, gemfibrozil. A subset of incubations also incorporated 2% bovine serum albumin (BSA) to account for any reduction in clearance due to inhibition by free fatty acids. The rUGT panel identified UGTs 2B7, 1A3, 1A9, 2B4, and 2B17 as contributors to gemfibrozil glucuronidation in the absence of BSA and UGT2B7 in the presence of BSA. We employed an IVIVE approach to determine tissue-specificity of the metabolic activity observed in vitro. The predominant UGT in human kidney microsomes is UGT1A9 (75 pmol/ mg microsomal protein), and consequently a relative activity factor (RAF: 2.5) was generated to convert rUGT rates to microsomal rates per pmol of UGT. Assuming the enzymatic activity to be the same in the liver and kidney, we were able to use the RAF to determine the individual fractions of gemfibrozil metabolized by each isoform in the liver by considering liver-specific UGT abundance values. UGT2B7 was found to be the dominant enzyme, accounting for more than 90% of the UGT-mediated clearance. Scaling of total intrinsic clearance of gemfibrozil glucuronidation using the well-stirred model predicted that the total contribution of the kidney to the overall clearance was negligible. Scaled hepatic clearance from HLM was 1.5 ml/min/kg, in good agreement with the observed value of 1.3 ml/min/kg. This scaling method accurately estimated the hepatic clearance of gemfibrozil and the dominant role of UGT2B7 in gemfibrozil glucuronidation.

P260. MATHEMATICAL MODELING OF IN VITRO HEPATIC DISPOSITION OF MYCOPHENOLIC ACID AND ITS GLUCURONIDE IN SANDWICH-CULTURED HUMAN HEPATOCYTES Norikazu Matsunaga1, Sho Wada1, Takeo Nakanishi1, Miho Ikenaga1, Mikio Ogawa2 and Ikumi Tamai1 1 Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan, 2Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., Tsukuba, Japan


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Recently, safety testing for circulating metabolites has been significantly impacting on drug discovery and development. In terms of drug development, accordingly, methods for identification of major metabolites and their disposition must be developed in non-clinical animals and humans. Hepatocytes are ideal in vitro materials for evaluating the hepatic disposition of endogenous and exogenous compounds. Sandwich culture technique with hepatocytes has advantages in long-term incubation, maintenance of metabolic enzyme and transporter activities, and development of canalicular networks sealed by tight junctions. Therefore, sandwich-cultured hepatocytes are a powerful tool for a comprehensive estimation of the hepatic disposition of drugs. Mycophenolic acid (MPA), a selective inosine-50 -monophosphate dehydrogenase inhibitor, is glucuronidated and the enterohepatic circulation of MPA-glucuronides is important in the continuous systemic exposure of MPA. On the other hand, administered MPA is finally excreted as phenyl-glucuronide of MPA (MPAG) into urine up to 90% of the dose in humans. Moreover, plasma concentration of MPAG is much higher than that of MPA. These outcomes indicate that MPAG formed in human hepatocytes is effluxed into bloodstream across basolateral membrane as well as excreted into bile. In the present study, we performed metabolic/hepatobiliary transport studies of MPA and MPAG with sandwich-cultured human hepatocytes (SCHH) to construct mathematical models of their hepatic disposition. In addition, we performed vesicular transport studies to identify human MRPs involved in the basolateral efflux of MPAG in human hepatocytes. The intrinsic uptake clearance (CLuptake,int) of MPAG via active transporter was approximately one-third of the first-order rate constant of accumulation for apparently nonsaturable component (Pdif) in SCHH. Furthermore, we found that MPAG is a favorable substrate of the canalicular efflux transporter MRP2 and the basolateral efflux transporters MRP3 and MRP4 in humans from the conventional and metabolic/hepatobiliary transport studies using SCHH and vesicular transport studies using human MRP-expressing membrane vesicles. Moreover, the constructed mathematical model revealed that the basolateral efflux process plays an important role in the hepatic disposition of MPAG formed in human hepatocytes. Our findings suggest that mathematical modeling approach using the data obtained from metabolic/hepatobiliary transport studies with SCHH should provide useful information for prediction of in vivo fate of metabolites formed in human hepatocytes.

P261. SUBSTRATES-DEPENDENT INFLUENCE ON PROBING METABOLIZING ENZYME-MEDIATED DRUG INTERACTIONS POTENTIALS IN VIVO Jingjing Wu, Yong Liu and Ling Yang Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China It is well documented that the inhibitory effects of some potent inhibitors of CYP3A can not be observed in vivo when typical CYP3A substrates were used at therapeutic doses, though significant inhibitory effects are predicted based on the theoretical ratio of inhibitor concentrations to Ki (I/Ki) recommended by FDA with the same inhibitors and substrates. The underlying reasons are still unknown. In this study, the time-course influence of enzyme concentration, substrate concentration and substrate type on possibility of in vivo inhibitory potential was investigated. Nifedipine (NIF), quinine (Qui) and ketoconazole (KCZ) were employed as the in vitro/in situ substrate, in vivo substrate and inhibitor of CYP3A respectively. The results from in vitro experiments with rat liver microsomes indicated that with the increase of microsomal protein concentrations, shorter durations for generating significant inhibitory effects were observed, while the increase of the substrate concentration could significantly prolonged these durations. Similar phenomena were observed in both in situ rat liver perfusion study and in vivo experiment. The experimental results were also consistent with the mathematical inference based on enzyme kinetic equation for different type of substrates. In conclusion, probe substrates at suitable dosage and rational sampling time in drug-drug interactions (DDI) might be required to avoid the occurrence of false negative outcomes. Moreover, the selected substrate with appropriate kinetic behaviors should also be considered in the in vivo enzyme-mediated DDI assessments.

P262. ESTIMATION OF IN VIVO SKIN AND SYSTEMIC EXPOSURE FOR AROMATIC AMINE HAIR DYES: EXTRAPOLATION FROM KERATINOCYTE AND HEPATOCYTE METABOLISM KINETICS IN VITRO John D. Manwaring1, Cindy M. Obringer1, Helga Rothe2 and Carsten Goebel2 1 Central Product Safety, Procter & Gamble Co., Mason, OH, 2Central Product Safety, Procter & Gamble Co., Darmstadt, Germany Approaches to assess the role of absorption, metabolism and excretion of cosmetic ingredients, such as hair dyes, that are based on the integration of different in vitro data are important for their safety assessment due to the ban on animal testing in the European Union. In order to estimate systemic exposures (AUC) of the parent hair dye after dermal application, firstly, keratinocyte and hepatocyte metabolism studies were conducted to assess the metabolic conversion. Then, the determination of the amount of the applied dose that would penetrate the skin and survive metabolism was made, and second, prediction of the systemic exposure of the compound was conducted, suggesting pharmacokinetic values such as elimination rate constants and half-lives plus AUCs and Cmax values. The amount of the parent hair dye that would survive metabolism in the viable epidermis

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and reach the general circulation unchanged was estimated using: a) Michaelis-Menten kinetics parameters to quantitate the metabolic processes, b) the estimated keratinocyte cell density in the viable epidermis, c) the penetration rate determined by in vitro skin penetration, d) the calculated Mean Residence Time of the compound in the viable epidermis (represented by keratinocytes), e) the viable epidermis thickness and f) the skin permeability coefficient. Based on these pharmacokinetically relevant parameters the internal dose of the compound (amount surviving dermal penetration and skin metabolism) was estimated. Next, the systemic exposure (AUC and Cmax) of the compound in the general circulation was approximated using the internal dose and scaled hepatic clearance (based on the liver-based Km and Vmax values and using reported values for whole liver mass and cell density) and with an estimated volume of distribution. The elimination rate constant and half life were also calculated using standard pharmacokinetic relationships. The individual steps, description, rationale and discussion of these processes with the resultant data will be shown. Not only are the estimated skin bioavailability values in agreement with independent ex-vivo skin penetration/metabolism data, but the extrapolated systemic exposure levels are in agreement with existing human in vivo data.

P263. APPLICATION OF IN VITRO-IN VIVO EXTRAPOLATION (IVIVE) TO INVESTIGATE THE RATE-DETERMINING STEPS IN THE HEPATIC CLEARANCE OF HIV PROTEASE INHIBITORS IN RAT Tom De Bruyn, Patrick Augustijns and Pieter Annaert Drug Delivery & Disposition, KU Leuven, Leuven, Belgium Hepatic drug clearance of xenobiotics comprises a complex interplay between sinusoidal uptake, intracellular sequestration, protein binding, metabolism and biliary excretion. This study aimed to determine which of these processes constitutes the rate-limiting step in the overall hepatic elimination of HIV protease inhibitors in rats. Therefore, the hepatic in vivo clearance of these drugs was predicted based on in vitro uptake, metabolism or a combination of both. In vitro uptake and metabolic clearance values were determined in suspended rat hepatocytes and rat liver microsomes, respectively. The unbound plasma fraction and blood plasma partition coefficient were measured with equilibrium dialysis. Using the in vitroin vivo extrapolation, a linear correlation was found between predicted and observed hepatic clearance values when combining hepatic uptake and metabolism processes. Sensitivity analysis revealed a significant role of plasma binding. Our results indicate that mechanistic conclusions with respect to the rate limiting step in hepatic drug clearance can be drawn from in vitro – in vivo extrapolations based on individual disposition processes. Modelling efforts are ongoing to gain better quantitative insight into the relative roles of transport, metabolism and plasma protein binding in the hepatic clearance of HIV protease inhibitors in rat.

P264. TRANSLATIONAL PBPK MODELING AND SIMULATION OF THE HEPATIC DISPOSITION OF ACTIVELY TRANSPORTED DRUGS BASED ON INDIVIDUAL TRANSPORTER ASSAYS Heleen M. Wortelboer, Sieto Bosgra, Evita van de Steeg, Marijn LH Vlaming, Kitty CM Verhoeckx and Miriam Verwei Kinetics Research for Food & Pharma, TNO, Zeist, Netherlands Insight in the hepatic disposition of drugs and the role of influx and efflux transporters is important throughout the drug development process. A translational PBPK modeling approach is presented to extrapolate the transporter activity from in vitro to in vivoand animal-to-human based on individual transporter assays. The use of this model to simulate various factors influencing active transport is demonstrated for the HMG-CoA-reductase inhibitor rosuvastatin. The activity of the relevant rat and human in- and efflux transporters towards rosuvastatin was measured in stably transfected cell-lines overexpressing these individual transporters.1 The abundance of these transporter proteins in the outer membranes of the cell-lines and in liver tissue samples were quantified by LCMS/MS to scale up transport from in vitro to in vivo. A rosuvastatin PBPK model in rat was built based on pharmacokinetic and tissue distribution studies, embedding scaled transporter activities to predict hepatocellular drug concentrations. The rosuvastatin PBPK model was scaled to human by adjusting physiological parameters and incorporating scaled activities of human transporters. Predictions of hepatic clearance based on scaled transporter assay data were comparable to observations in rat and human. The rat PBPK model was able to reconstruct hepatic intracellular concentrations and was successfully scaled to predict human pharmacokinetics. It was then shown how this human PBPK model can be used as a tool to simulate the effects on hepatic disposition of (1) subpopulation variation in transporter expression; (2) transporter polymorphisms; (3) transporter drug-drug interactions (DDI). Modeling hepatic disposition based on individual transporter assays provides a more complete picture than just scaling from primary hepatocytes, and allows simulation of various factors influencing transporter activity. We demonstrate how this PBPK modeling approach can be useful in various stages of drug development, including early preclinical prediction of hepatic intracellular drug concentrations, first-in-man and clinical trial simulation, simulation of DDI and personalized medication.

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Reference 1. Van de Steeg E, Greupink R, Schreurs M, et al. Drug-Drug Interactions between Rosuvastatin and Oral Antidiabetic Drugs Occurring at the Level of OATP1B1. Drug Metab Dispos 2013;41:592–601.

P265. CHARACTERIZATION OF HUMAN COLONIC CARCINOMA CELL LINES FOR STUDYING INTESTINAL PERMEABILITY AND FIRST-PASS METABOLISM AS AN ALTERNATIVE TO CACO-2 CELLS


Yoshiyuki Yamaura1, Zhican Wang1, Jonathan Himmelfarb2 and Kenneth Thummel1 1 Pharmaceutics, University of Washington, Seattle, WA, 2Nephrology, University of Washington, Seattle, WA Caco-2 cell monolayer grown on permeable filter insert is one of the most commonly used in vitro models to study intestinal absorption of drugs. However, Caco-2 cells cannot be used evaluate intestinal first-pass metabolism, since they are deficient in drug metabolism capabilities and enzyme inducibility. To develop an in vitro model that can simultaneously assess intestinal absorption and first-pass metabolism, we evaluated two candidate human colonic carcinoma cell lines, LS180 and T84. Although LS180 cells exhibit basal and inducible CYP3A4-dependant drug metabolism activity, it is not known whether this cell line forms tight junctions in culture. In contrast, T84 cells are known to form tight junctions, but CYP3A4 expression and its inducibility have not been reported. After prolonged culture of LS180 and T84 cells on permeable filter support, transepithelial electrical resistance (TEER), midazolam (a typical CYP3A4 substrate) hydroxylation activity and its permeability across cell monolayer were determined. In addition, immunofluorescence microscopy was conducted to determine tight junction formation and cell polarity. The TEER values of LS180 cells were low (10–30 cm2), whereas that of T84 cells were comparable to that of Caco-2 cells as reported (4350 cm2). Interestingly, the apparent permeability coefficients (Papp) of midazolam across LS180 and T84 cell monolayers were at the same level, despite a large difference in TEER value. T84 cells showed basal CYP3A4 metabolic activity comparable to that in LS180 cells. However, the inducibility of CYP3A4 in T84 cell by vitamin D and prototypical xenobiotic inducers was much lower than that seen with LS180 cells. The tight junction protein zonula occludens-1 (ZO-1) was well organized into cell circumscribing strands in T84 cells. In contrast, ZO-1 was also detected in LS180 cells, however its localization was more diffused and less tight than that of T84 cells. These results suggest that both cell lines are promising candidates for studying intestinal permeability and first-pass metabolism as an alternative to Caco-2 cells, but that additional work is needed to address their respective deficiencies. Supported in part by a grant from NIH: UH2 TR000504.

P266. IN VITRO INVESTIGATION OF BILE RELEASE DYNAMICS OF PRIMARY HUMAN HEPATOCYTES AND HEPARG CELLS Marc Lu¨bberstedt1, Christin Schneider1, Franziska Schulze1, Thao Arslan1, Marco Richter1, Christoph Ertel1, Antje Schulz2, Georg Damm3, Tommy B. Andersson4 and Katrin Zeilinger1 1 Experimantal Surgery / Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charite´ University Medicine Berlin, Berlin, Germany, 2Chirurgie, Visceral- und Gefa¨bchirurgie, Vivantes Humboldt-Klinikum, Berlin, Germany,3Department for General, Visceral and Transplantation Surgery, Charite´ University Medicine Berlin, Berlin, Germany, 4AstraZeneca R&D Mo¨lndal, DMPK Innovative Medicines, Mo¨lndal, Sweden Transport processes in hepatoytes are strongly dependent on the polarization of the cells and require the formation of bile canaliculi. Bile transport studies are primarily performed with radioactively labeled substances in animal models. The availability of a reliable in vitro model based on human hepatocytes could improve predictability, and reduce the use of animals.We use a 3D four-compartment bioreactor system, which is based on a hollow-fiber membrane technology with independent capillary systems for counter-current medium perfusion and decentralized oxygenation. The system allows for high-density culture of hepatic cells in a perfused environment, which enables reorganization of liver cells to tissue-like structures including polarization of hepatocytes and formation of bile canaliculi. The preservation of specific functions of primary human hepatocytes such as urea production and cytochrome P450 activity for at least 10 days in the bioreactor was shown. To characterize the dynamics of bile release in vitro, spontaneous and induced bile release dynamics of primary human hepatocytes were initially studied in sandwich cultures using live cell imaging. As an alternative source to primary hepatocytes for these studies, the usability human hepatic cell line HepaRG was investigated. The immunohistochemical detection of hepatic transporter proteins in 2D cultures in combination with results of the live cell imaging studies indicate that HepaRG cells could provide a suitable model for bile extraction studies. Results from bile release studies were used to develop a method for bile extraction in the 3D bioreactor system. Initial results with HepaRG cultures in the 3D bioreactor system show the possibility of induced bile release and extraction from the cultures.

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P267. EVALUATION OF TRANSIL HIGH SENSITIVITY BINDING ASSAY FOR HIGHLY PLASMA PROTEIN BOUND COMPOUNDS


Heather Eng1, Li Di1, Jian Lin1, Thomas S McDonald1 and Hinnerk Boriss2 1 Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, CT, 2Sovicell GmbH, Leipzig, Germany Plasma protein binding (PPB) is an important property of drug candidates. PPB values are widely applied in drug discovery and development for scaling of in vitro intrinsic clearance measured from liver microsomes or hepatocytes to in vivo plasma clearance for development of IVIVC and for prediction of human clearance. Species difference in PPB can affect free drug exposure between toxicity species and human and impact therapeutic index and safety window calculations. The inaccuracy of PPB measurement can lead to mis-prediction of human PK and clinical drug-drug interaction. Determination of fraction unbound (fu) for highly plasma protein bound compounds poses a great challenge in drug discovery, due to high non-specific binding, poor recovery, or long time-to-equilibrium. Measuring PPB for these compounds using the existing methods, such as equilibrium dialysis, ultrafiltration, or ultracentrifugation proved to be problematic. A new approach, the TRANSIL High Sensitivity Binding (HSB) assay, was developed to measure PPB of highly lipophilic and bound compounds. This TRANSIL HSB assay assesses PPB via partition between silica beads coated with immobilized phosphate lipid membranes and plasma (any species). By varying the ratio between the beads and plasma, the binding constant to plasma and membrane affinity can be derived. The TRANSIL HSB assay design overcomes the issues of non-specific binding and the need for analytical sensitivity due to the large concentration difference between bound and unbound for high PPB compounds. In this study, twelve very highly bound structurally diverse compounds with reported fu ranging from 0.0002 to 0.012 were tested in the TRANSIL HSB assay using pooled human plasma to evaluate the accuracy and precision of the assay. The fu values were comparable with those obtained from the traditional methods reported in the literature or measured in-house. The results were highly reproducible from run to run and from day to day. The data demonstrated that TRANSIL HSB assay has a great potential for PPB measurement of very sticky and highly bound compounds. It can be applied to compounds that are not amenable to traditional PPB methods due to high non-specific binding, instability, analytical challenges, or extremely long time-to-equilibrium.

P268. AN IN VITRO SCREENING METHOD TO EVALUATE THE DRUG-DRUG INTERACTION DUE TO INHIBITION OF SULFOTRANSFERASE ENZYMES (SULTS) Mohammed Taimi and Jon Gilbert Cyprotex, Watertown, MA Ethinyl estradiol (EE), an active semi-synthetic steroidal estrogen, is one of the most commonly prescribed oral contraceptives. EE is well absorbed but has poor oral systemic bioavailability due to its first pass metabolism in both the intestine and the liver. Thus, EE is prone to interactions with co-administrated drugs due to this high first pass metabolism. Sulfation of EE is a major pathway of biotransformation in humans, mainly by sulfotransferaseenzymes (SULT1E1). The cytosolic sulfotransferase (SULT) enzyme family catalyzes the transfer of a sulfate group from the universal 30 -phosphoadenosine 50 -phosphosulfate (PAPS) donor to a wide variety of acceptor molecules bearing a hydroxyl or an amine group. Inhibition of SULT enzymes may cause impairment of EE metabolism, leading to the surge of the plasma concentration levels and consequently increasing the potential for serious adverse reactions including hypertension and vascular diseases. In this study, in vitro SULT inhibition assays using human recombinant enzymes (SULT1A1, SULT1E1) and LC/MS/MS analysis were developed as screening approaches to assess the impact of new chemical entities on altering the exposure of drugs metabolized by sulfotransferase eznymes. Using para-nitrophenol as substrate; we evaluated protein and incubation time linearity, co-factor concentration effects, and enzyme kinetics (Km andVmax). Using the optimized conditions, we then established the profile of the enzyme inhibition using quercetin, a known inhibitor of SULT enzymes. In conclusion, understanding of a compound’s potential for SULT interference may be useful tool for limiting or avoiding drug-drug interaction liabilities in humans.

P269. A HUMAN IN VITRO MODEL FOR THE INVESTIGATION OF INFLAMMATORY REACTIONS IN XENOBIOTIC MEDIATED HEPATOTOXICITY Georg Damm1, Victoria Kegel1, Theresa Schulz1, Britta Burkhardt2, Daniel Seehofer1 and Andreas K. Nu¨ssler2 1 Department of General-, Visceral- and Transplantation Surgery, Charite´ University Medicine Berlin, Berlin, Germany, 2 Deptartment of Traumatology, Eberhard Karls University Tu¨bingen, Tu¨bingen, Germany Hepatic tissue damage can occur due to surgical interventions, infections or xenobiotic mediated hepatocyte damage. Kupffer cells (KCs) play an important role in initiating tissue damage but also in regenerative processes. KC activation by lipopolysaccharides (LPS), antigens or endogenous proteins can lead to an activation of the NF-kB signaling pathway


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transmitted by reactive oxygen intermediates (ROI). Once activated, KCs release different cytokine patterns that support inflammation or induce tolerance reactions. Aim of the present study was the establishment of a human in vitro model which enables the investigation of KCs and their role in xenobiotic mediated hepatotoxicity. Primary human hepatocytes (PHH) and KCs were isolated from human liver resectates using a two-step collagenase perfusion technique. The isolated KC yield was 1.23 0.89 106 cells/g liver tissue with a purity of 480% measured by immunofluorescence staining of CD 68. Additionally, the isolated KC population was characterized by the ability for phagocytosis of fluorescent Latex Beads. KC activation was investigated measuring the intracellular formation of ROI using the DCF-assay with and without LPS treatment. The cell viability and mitochondrial activity was measured using the XTT-assay. In order to simulate the activation of KCs following hepatocyte damage, KCs were incubated with supernatants of PHH that had previously been treated with diclofenac or acetaminophen, respectively. KC reaction was determined by measurement of cytokine concentrations using specific cytokine ELISA sets. Initial ROI levels in KCs showed high donor variability and were dependent on the condition of the liver tissue. The stimulation of KCs with LPS caused a concentration-dependent increase in ROI formation. Incubation of KCs with supernatants from xenobiotic-treated PHH increased mitochondrial activity and the formation of ROI compared to untreated KCs. Furthermore, compound dependant changes in the secreted cytokine profiles could be observed upon exposure of KCs to supernatants from xenobiotic-treated PHH. Taken together, most of the isolated KC populations showed high basal ROI levels, which are linked to the isolation process and to the quality of the donor liver tissue. Nevertheless, LPS and xenobiotic-treated PHH induced KC activation and leads to compound specific reactions. Thus our human in vitro model could be a suitable tool for the investigation of xenobiotic mediated inflammatory reactions in the liver. First attempts to transfer this method into a transwell coculture system are in progress. This study was supported by the Virtual-Liver-Network, BMBF:0315741

P270. IN VITRO METABOLISM STUDY OF DESIGNER DRUGS USING A HEPATOCYTE SPHEROID ARRAY KIT Tatsuyuki Kanamori, Tadashi Yamamuro, Kenji Kuwayama, Kenji Tsujikawa, Yuko T. Iwata and Hiroyuki Inoue National Research Institute of Police Science, Kashiwa, Japan [Introduction] Cell-ableÔ (Transparent Inc., Inzai, Japan) is a hepatocyte spheroid array kit that enables simple threedimensional hepatocyte culture. In the present study, we evaluated the usefulness of this kit as a tool to predict the metabolic fate of designer drugs by comparing the metabolic pattern of designer drugs in rat hepatocyte culture to that of in vivo rat studies. [Method] Mouse fibroblast cells (3T3-Swiss albino) were used as feeder cells and incubated at 37 C in a Cell-able 24-well plate for 3 days. After the monolayer of feeder cells was formed, rat primary hepatocytes were seeded (2 105 cells/mL, 0.5 mL/well) in the plate and incubated for 2 or 6 days. The spheroids of the hepatocytes were formed on the feeder cells. The hallucinogenic designer drug, 2C-T-2, 2C-T-4 or 2C-T-7 (analogs of 2,5-dimethoxy-4-alkylthiophenethylamine), was added to the plate at a concentration of 10 mM and incubated for 24 or 48 hours. The medium was collected, and the metabolites were extracted by liquid-liquid extraction. The extracts were analyzed by LC-MS/MS to quantitate each metabolite. [Results and Discussion] In the case of 2C-T-7 (2,5-dimethoxy-4-propylthiophenethylamine), various metabolic reactions, such as sulfoxidation, N-acetylation, hydroxylation of S-propyl chain, S-depropylation followed by S-methylation and deamination followed by oxidation, occurred on the substrate. Similar metabolic reactions were observed in the case of 2C-T-2 (2,5-dimethoxy-4-ethylthiophenethylamine) and 2C-T-4 (2,5-dimethoxy-4-isopropylthiophenethylamine). The main 2C-T-7 metabolites found in rat urine1 were also detected in the culture medium of Cell-able, indicating that the in vivo metabolic pattern of 2C-T-7 was successfully reproduced using the Cell-able hepatocyte spheroid array kit. Feeder cells were absolutely essential to maintain the metabolic activity of the hepatocytes in Cell-able because the activities of hydroxylation of S-propyl chain and S-depropylation declined significantly in the hepatocyte culture without feeder cells. In conclusion, Cell-able is considered a useful tool for predicting the in vivo metabolism of designer drugs. [Reference] 1. T. Kanamori et al., In vivo metabolism of 2,5-dimethoxy-4-propylthiophenethylamine in rat, Xenobiotica, 37, 679–692 (2007).

P271. PERMEABILITY OF PIPERINE ACROSS MDCK CELL MONOLAYERS COMPARED WITH IN SILICO PREDICTION BY USING ADMET PREDICTORä SOFTWARE Chatsiri Jesadakultavee1 and Korbtham Sathirakul2 1 Pharmaceutics, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand, 2Pharmaceutics, Mahidol University, Bangkok, Thailand The aim of this study was to investigate the permeability of piperine, the major constituent of black pepper (Piper nigrum Linn.), across MDCK cell monolayers. In addition, the results from the experiment were compared to the predicted permeability from ADMET PredictorÔ software for further building the oral absorption model of piperine by using GastroPlusÔ software. Cytotoxicity of piperine was examined by MTT assay. Bidirectional transport, apical to basal (A–B) and basal to apical (B–A) direction, of piperine was performed for 3 hour on MDCK cell monolayers growth on transwell insert. Lucifer yellow was used

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as a paracellular leakage marker. Quantitation of piperine was performed by LC-MS/MS. Piperine concentration ranging from 5– 50 mM resulted in viability more than 96 percent. The accumulated amount of piperine transported across MDCK cell monolayers was increased proportional with started concentration ranging from 5, 25, and 50 mM. Papp values in A–B direction of piperine 5, 25, and 50 mM were 2.36 0.7 105, 2.72 0.78 105 , and 3.49 0.69 105 cm/sec, respectively. While, Papp values in B–A direction of piperine 5, 25, and 50 mM were found out to be 2.99 0.32 105, 3.5 1.18 105, and 3.21 0.27 105 cm/sec, respectively. Papp values of piperine for each concentration was not significantly different for both A–B and B–A direction, p50.05. Efflux ratio at 5, 25, and 20 mM were 1.27, 1.29, and 0.92, respectively. Lucifer yellow measurement exhibited Papp value of 6.15 1.12 106 cm/sec confirming that the monolayer integrity was intact at the end of the study. By using ADMET PredictorÔ with only input chemical structure of piperine to the software, the predicted MDCK Papp of piperine was found to correlate well with the mean Papp value from experimental study (3.59 105 and 2.86 0.73 105 cm/sec, respectively). Thus, either predicted or measured MDCK Papp of piperine was able to integrate into the oral absorption model in GastroPlusÔ software.


P272. USE OF DEXTRAN-COATED CHARCOAL METHOD FOR MEASURING THE RATE OF DISSOCIATION (KOFF) OF SMALL MOLECULES FROM PLASMA PROTEINS Kamelia Behnia1, Qi Wang2, Susan Johnghar2, Mark Witmer2, James Smalley2, Punit Marathe2 and David Rodrigues2 1 Department of Drug Metabolism and Pharmacokinetics, Bristol Myers Squibb Pharmaceutical Research Institute, Princeton, NJ, 2Bristol Myers Squibb Pharmaceutical Research Institute, Princeton, NJ The avidity of binding and rate of dissociation from plasma proteins are known to alter the pharmacokinetics of small molecules. We established a methodology based on the adsorption of the free (unbound) molecules todextran-coated charcoal to determine the dissociation rate constant (koff) of the compounds from plasma proteins. The utility of this methodology was investigated by determining the koff for a series of commercial compounds (Midazolam, propranolol, UCN-01, Meloxicam, Gleevec) from human a1-acid glycoprotein (hAGP), human serum albumin (HSA) and human plasma. Protein solutions (hAGP, HSA or plasma) were spiked with individual compounds and incubated for 15 min at 37 C prior to addition of dextran coated charcoal solution. Samples were collected during a 2h incubation, following addition of the DCC, and centrifuged to separate the charcoal bound molecules. Concentration of the compounds in the supernatant (protein-bound fraction) measured by LC/MS/MS and compared to initial concentrations. Koff’s were determined from the kinetic of decline in the concentration of the drug in the protein-bound fraction over-time and compared to the koff from the Surface Plasmon Resonance analysis (SPR) employing individual proteins (HSA or hAGP). UCN-01 showed the slowestkoff from hAGP (0.0009 and 0.004 S1, with DCC vs. SPR, respectively) which was similar to koff from plasma (0.0006 and 0.007 S1, respectively) suggesting that the dissociation from plasma is governed by its dissociation from hAGP. Propranolol, another AGP binder however, had a fast off rate from hAGP (40.08 S1). UCN-01, and propranolol dissociated rapidly from human albumin (koff 0.1–0.8 S1) suggesting minimal impact of albumin binding on their dissociation from plasma. Meloxicam and Midazolam were albumin binders and rapidly dissociated from hAGP. The dissociation of Midazolam from albumin was slower and similar to plasma. There was a good agreement in rank ordering of compounds based on their koff values from DCC vs. SPR analysis. Clear advantage of DCCmethodology is the applicability to measure the koff from pool of proteins (plasma) as compared to SPR where individual proteins are utilized. Slow Koff from hAGP is known to be the culprit for the long half life of UCN-01 in human the DCC method was able to capture the differentiated profile of this compound. In conclusion DCC adsorption is a practical in vitro method for rank ordering based on rate of dissociation from plasma proteins and for identifying compounds with potential for altered pharmacokinetics due to slow off rates from proteins.

P273. DEVELOPMENT OF A LC/MS/MS SCREENING METHOD TO EVALUATE THE IMPACT OF DRUGS ON CORTISOL BIOSYNTHESIS IN VITRO Mohammed Taimi and Jon Gilbert Cyprotex, Watertown, MA Cortisol, an essential glucocorticoid, plays an important role in the regulation of metabolism and cardiovascular functions, modulates the function of the immune system, and contributes to stress responses. Cortisol is synthesized primarily in the adrenal cortex through a series of biochemical reactions mediated by multiple cytochrome P450 enzymes and dehydrogenases. Drug exposure can alter the activity of these enzymes and subsequently the production of cortisol, which might lead to serious side effects. In this study, an in vitro assay using the human adrenocortical carcinoma cell line H295R was developed as a screening platform to assess the impact of new chemical entities (NCEs) on altering cortisol biosynthesis. To this end, a robust LC/MS/MS method was established to determine the levels of cortisol and its precursor 11-deoxycortisol (11-DOC) in cultured H295R cells. We first developed the model to describe the biosynthetic pathway for the formation of 11-DOC catalyzed by 21-hydroxylase (P450c21; CYP21A2) and its conversion to cortisol by 11b-hydroxylase (CYP11B1). Then, we investigated the inhibition of

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11-DOC and cortisol using etomidate, a sedative drug known to suppress cortisol levels in vivo. In addition, we evaluated the impact of 18 marketed drugs on biosynthesis of 11-DOC and cortisol in H295R cells. These results demonstrate the feasibility of using an in vitro 11-DOC and cortisol inhibition assay to help define mechanisms of action of NCEs on cortisol biosynthesis and to limit or avoid potential liabilities in humans that are not often detected by standard preclinical animal models.

P274. INVESTIGATION OF AMINOGLUTETHIMIDE-INDUCED CHANGES IN NEUTROPHILS: IMPLICATIONS FOR IDIOSYNCRATIC DRUG-INDUCED AGRANULOCYTOSIS


Winnie Ng and Jack P. Uetrecht Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada Background: Aminoglutethimide (AMG) is an aromatase inhibitor associated with idiosyncratic agranulocytosis which is characterized by a severe decrease in granulocytes, mainly neutrophils, that markedly increases one’s susceptibility to serious infections and can be fatal. AMG contains an aromatic amine moiety, and virtually all aromatic amine drugs are associated with a high incidence of idiosyncratic drug reactions (IDRs). Animal models of IDRs represent essential tools to study these reactions; however, like humans, treating animals with a drug that can cause IDRs does not usually induce an IDR. Previously, mice treated with AMG developed leucopenia;1 however, this is not representative of what occurs in patients and is not a true model of AMG-induced agranulocytosis. A limited number of valid animal models of IDRs have been developed in rats,2 and thus it is hypothesized that rats may present a better opportunity to develop a more realistic model of AMG-induced agranulocytosis. Methods: Male Brown Norway rats were treated orally with 125 mg/kg/day AMG for up to 14 days and the blood and bone marrow were monitored to determine the effect of AMG on neutrophils. Results: Blood levels of AMG in the rat after a single dose of AMG were similar to the drug levels in AMG-treated patients. Paradoxically, AMG increased peripheral blood neutrophils as early as 24 hr, and this elevation was sustained throughout the study. This corresponded to an increase in the release of new neutrophil from the bone marrow as determined by BrdU labelling. There was also a decrease in neutrophil apoptosis as measured by Annexin V/propidium iodide, but this may be caused by a greater number of younger neutrophils expressing less phosphatidylserine rather than prolonged survival induced by the drug. Treatment with AMG also led to bone marrow hypercellularity, specifically an increase in myeloid cells and stimulated the proliferation of granulocyte progenitors. In the blood, AMG significantly decreased the expression of CD62L and increased the expression of CD11b on neutrophils after 7 and 12 days of treatment, and these changes suggest neutrophil activation. Conclusions: The increase in neutrophils and changes in the bone marrow by AMG in rats is opposite to what occurs with agranulocytosis. However, this is similar to the initial neutrophilia observed in patients and rats treated with other drugs that induce idiosyncratic agranulocytosis such as clozapine.3 Therefore, the rat may be a good model for the early changes induced by AMG that can be a precursor to agranulocytosis in some patients. Further research will be required to determine how these changes may be related to the mechanism of AMG-induced agranulocytosis.

References (1) Coleman, M. D., Khalaf, L. F., and Nicholls, P. J. (2003) Aminoglutethimide-induced leucopenia in a mouse model: effects of metabolic and structural determinates. Environmental Toxicology and Pharmacology,15, 27–32. (2) Ng, W., Lobach, A. R. M., Zhu, X., et al.(2012) Animal models of idiosyncratic drug reactions. Advances Pharmacol., 63, 81–135. (3) Alvir, J. M., Lieberman, J. A., and Safferman, A. Z. (1995) Do white-cell count spikes predict agranulocytosis in clozapine recipients? Psychopharmacol. Bull., 31, 311–314.

P275. MECHANISTIC AND EPIDEMIOLOGIC INSIGHTS INTO UPREGULATION OF GENE OF INFLUENZA VIRUS NS1 PROTEIN BY DIOXIN IN HOST CELL Ilya B. Tsyrlov1, Charles Woo2, Irina N. Shur3 and Vladimir S. Rumak4 1 Xenobiotical Virology, XENOTOX, Inc., Scarsdale, NY, 2Bioinformatics/In Silico, XENOTOX, Inc., Scarsdale, NY, 3Clinical, ISI Medicine, Bronx, NY, 4Dioxin Toxicities, RAS TROPOCENTER, Hanoi, Vietnam Cognate DRE sites within DNA enhancer epitomizes wide range of mammalian genes expression mediated via the Ah receptor pathway. Earlier we postulated the same for DRE-containing viral genes transactivation caused by dioxin in human cells infected with HIV-1, HBV and HCMV. Here, such mechanistic concept applied to type A influenza virus NS1 binding protein in human and avian (G. gallus gallus) host cells. The NS1 is known to prevent transcriptional induction of antiviral interferons, to inhibit splicing and dsRNA-mediated signal transduction in target cells. Presenting data range from the cellular to population levels. It was shown that gene encoding the NS1 possessed multiple DREs (core nucleotide sequence 30 A-CGCAC 50 ), two of which were identified within the promoter area, namely at positions 7942 and 687. SITECON, an established computational tool for

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detecting transcriptional factor binding site recognition, proved the above sites as potentially active. SITECON-selected adjacent variable sequences were used to detect properties of the DRE site, and conformational similiarity score threshold of 0.95 was utilized to rank identified DRE. On the cellular level, Western blot analysis of lysates of infected or DNA-transfected confluent HeLa cells pretreated with 10 ppt dioxin for 36 h revealed several-fold increase of NS1-specific polypeptide. As the NS1 promoter contains two potentially active DRE, an extrapolation from the data on HIV-1 (1 DRE) and HCMV (10 DRE) also suggests that concentration of dioxin upregulating NS1 gene should be moderately above current dioxin levels in general population (4 ppt). Presumably, elevated dioxin level in the host cells might lead to enhanced ability of NS1 to diminish antiviral interferons. That can bring new insights to the fact that resistance of highly virulent H5N1 to antiviral effects of IFN-x and TNF-alpha directly associated with the NS1. On the population level, the data on wild birds and domestic poultry (G. gallus gallus) dying from H5N1 in Guangdong province of China, and Long An, Tieng Giang and Ben Tre provinces of Vietnam, all relate to the fact that water and soil in these regions are highly contaminated with dioxin-like compounds. Eventually, human cohorts from the above regions of China and Vietnam are exposed to elevated concentrations of dioxin, which might serve as a promotional factor for seasonal influenza outbreaks. Moreover, the sub-nanomolar body burden dioxin might strongly facilitate spreading of the H5N1 in case avian flu pandemic were to occur.

P276. A PREVIOUSLY UNEXPLORED LINK BETWEEN CANNABINOID RECEPTORS AND UDP-GLUCURONOSYLTRANSFERASES IN CANCER Anna Radominska-Pandya1, FeAna FrancisDevaraj1, Centdrika Dates1, Aleksandra K. Greer1, Stacie M. Bratton1, Sebastian Pyrek1, Tariq Fahmi2, Fabricio Medina-Bolivar3, Randy Haun4 and Paul L. Prather5 1 Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, 2Medicine Genetics, University of Arkansas for Medical Sciences, Little Rock, AR, 3Arkansas Biosciences Institute and Biological Sciences, Arkansas State University, Jonesboro, AR, 4Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, 5Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR There is growing evidence that human UDP-glucuronosyltransferases (UGTs) can regulate cellular homeostasis by controlling the levels of biologically active endo- and xenobiotics within the cell. The importance of this regulation is especially evident in cancer cells where UGTs possess the ability to interfere with the availability of chemicals that drive cell proliferation through the glucuronidation of these compounds. It is also recognized that UGTs can effectively metabolize the majority of anticancer drugs, further complicating our understanding of the role of these enzymes in cancer. In this study, the anticancer potential of several known UGT substrates, Tamoxifen (Tam) and its hydroxylated and/or chlorinated derivatives [4OHTamoxifen (4OHTam), Toremifene (Tor) and 4OHTor], gambogic acid (GA), and prenylated stilbenoids (transArachidins; tAs), was demonstrated in cultured pancreatic and breast cancer cell lines. GA was the most cytotoxic with IC50 values of 0.5, 2.3, and 0.9 mM in Panc1, MCF7 and MDA231 cells, respectively. Unexpectedly, Tam and 4OHTam were found to be equally cytotoxic in ER-positive MCF-7 cells, ER-negative MDA-231 cells, and Panc1 pancreatic cancer cells (IC50 values from 27–35 mM). Based on this information, we postulate that another mechanism of action, separate from the estrogen receptor must be involved in this cytotoxicity. We had previously found that GA and tAs are ligands for cannabinoid receptors (CBRs). Therefore, we have investigated whether the effects of all these compounds may be in part due to their binding to CBRs, causing perturbations in the downstream signaling cascade. We have shown for the first time that Tam and its derivatives bind to both CBRs (CB1 & CB2). 4OHTam has the highest affinity for both CB1 and CB2 with Ki values of 1.4 mM and 0.6 mM, respectively. These drugs also decreased G-protein activity, which suggests they function as inverse agonists at these receptors. Based on these data, we hypothesize that the observed cytotoxicity of these compounds may be regulated by UGTs, which control their availability. Their action may be mediated by the binding of these drugs to CB1 and/or CB2, which activate or suppress downstream effects linked to cell proliferation. In order to fully understand the link between these classes of proteins, we have begun to correlate the varied levels of UGT and CBR1/2 expression using qPCR, Western blot, and enzymatic activity assays and to establish whether they affect the availability and efficacy of these anti-cancer cannabinoids. If our hypothesis is correct, CBRs could constitute a novel molecular target to which effective, non-toxic, natural and synthetic cannabinoids might be developed for treatment of cancer. [DoD-W81XWH1110795 and UAMS Translation Research Institute (CTSA Grant Award #UL1TR000039) ABCRP funds to AR-P; AR-Center for Plant Powered Production-P3 to FMB; VA Merit Award 01BX000828-01A1 to RH]

P277. TRANSCRIPTOMIC AND METABONOMIC EFFECTS OF TAMOXIFEN METABOLIZATION BY CYP3A4 AND CYP2D6 IN HEPG2 CELLS Aurelie J. Krol1, Anne Bolopion1, Mary Dutheil1, Ce´line Lepeigneux1, Ahmad Al Ali1, Monique Diry1, Gildas Bertho2, Philippe Beaune3 and Isabelle de Waziers1 1 INSERM U775, Paris, France, 2Umr 8601, Uninersite´ Paris Descartes, Paris CX 06, France, 3U775, INSERM, Paris, France Tamoxifen (TAM), an antagonist of the estrogen receptor used against breast cancer, is known to be mainly metabolized by CYP3A4 and CYP2D6. To better understand its mechanism of action, we investigated the effects of CYP3A4- or


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CYP2D6-metabolized TAM, as well as the effects of non metabolized TAM. We used HepG2 cell lines stably expressing GFP (control), or CYP3A4, or CYP2D6 under the control of the hepatic Phospho Glycerate Kinase promoter (PGK). The catalytic activity of each CYP was checked in the different cell lines using specific known substrates of each CYP. We treated these cell lines with DMSO as a control and TAM at concentrations of 1 mM and 10 mM. At 24 hours cells were harvested cells. Analysis of the transcriptome were performed using pangenomic affymetrix microarray GeneChip Human Gene 1.0 ST. We also harvested the culture medium at 24 h and cells at 24 h and 48 h for metabonomic analysis. RMN(1H) analysis were performed. Non supervised analysis of the transcriptomic data showed a good separation of the different cell lines implicating that the expression of both CYP genes has a specific and different effects. Supervised analysis showed that non-metabolized TAM down-regulated genes linked to mitosis, and up-regulated genes linked to metabolism (carbohydrates, steroids and oxydoreduction), RNA processing, and nucleotides binding. CYP2D6-metabolized tamoxifen regulates similar kind of genes (down-regulation of genes linked to mitosis, up-regulation of genes involved in metabolism, RNA processing) and some genes involved in xenobiotic metabolism. However CYP3A4-metabolized tamoxifen regulates less genes and has no effect on mitosis, but does regulate genes linked to different metabolisms (carbohydrates, steroids, fatty acids and oxydo reduction). The preliminary RMN results on 24 and 48 hours culture medium showed that the TAM at 1 mM has no effect. At 10 mM, it caused an increase concentration of valine and alanine in the GFP and CYP3A4 cell lines. On the CYP2D6 cell lines, the 10 mM TAM caused an increase concentration of valine and glutamine, and a decreased concentration of lactate and pyruvate. We showed that depending on its metabolization, the effect of TAM is different. We are now working to validate the results and integrate the different kind of data.

P278. COVALENT INTERACTION OF AN IMINIUM ION METABOLITE OF RIMONABANT WITH BIOLOGICAL TARGETS Annika Thorsell1, Emre M. Isin1, Katarina Breitholtz2 and Ulrik Jurva1 1 DMPK Design and Biotransformation CVMD iMED DMPK, AstraZeneca, Mo¨lndal, Sweden, 2In Vitro & In Silico ADME, Global DMPK, AstraZeneca, Mo¨lndal, Sweden Iminium ions are reactive drug metabolites that have been shown to bind covalently to proteins and they are proposed to be linked to adverse drug reactions. The aim of this study is to investigate the reactivity of iminium ion metabolites derived from drug molecules towards biological targets. In this work rimonabant, a cannabinoid receptor 1 (cb1) antagonist, is used as a model drug. Previously, high levels of covalent binding of radiolabelled rimonabant to microsomal proteins has been demonstrated1 as well as cytotoxicity towards SV40-T-antigen-immortalized human liver derived (THLE) cell lines selectively expressing P450 3A42.The iminium ion is the major metabolite produced from rimonabant in in vitro microsomal incubations. One-dimensional gel electrophoretic separation of microsomes incubated with the radioactive rimonabant in the presence of NADPH revealed two distinct radiolabeled protein bands by phosphoimaging. These radioactive bands did not correspond to the most intense protein bands on the gel, suggesting selective covalent binding of rimonabant metabolites to certain specific proteins. No radioactive protein bands were observed when the incubations were carried out in the absence of NADPH. In-gel digestion by trypsin followed by nano LC-MS analysis identified several potential protein targets in each band. The digested sample corresponding to the most intense band was fractionated by LC into a top count plate yielding one radioactive peak which strengthens the hypothesis of covalent binding to specific proteins. In order to understand the kinetics of covalent binding in comparison to the rimonabant turnover, the concentration of rimonabant and iminium ion in the supernatant and levels of covalent binding at different time points during the microsomal incubation was measured. Prolonged incubations ensured that, changes in iminium ion concentration and covalent binding post enzymatic activity was also assessed when the microsomes were expected not to be active any more. The disappearance of rimonabant correlated with generation of the iminium ion. The covalent binding is time dependent and levels off after approximately 4 hours. The profile of covalent binding seems to follow the rate of iminium ion disappearance. A relatively high concentration of iminium ion is present in the incubation when the covalent binding levels off, suggesting that the covalent binding is specific and does not seem to be due to a simple stoichometric reaction with all microsomal proteins present in solution. Additional experiments will be performed to get a better understanding of the kinetics of covalent binding and to identify the biological targets of rimonabant.

References 1. Bergstro¨m et al. (2011) Bioactivation pathways of the cannabinoid receptor 1 antagonist Rimonabant Drug metab Dispos, 39, 1823 2. Foster et al. (2012) Bioactivation of the cannabinoid receptor antagonist Rimonabant to a cytotoxic iminium ion metabolite Chem. Res. Toxicol, 26, 124

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P279. DETECTION OF XENOBIOTIC-INDUCED HEPATOTOXICITY IN HUMAN IPSC-DERIVED HEPATOCYTES


Shannon Einhorn1, Jingtao Lu2, Paul B. Watkins3, Edward LeCluyse4 and David A. Mann1 1 Cellular Dynamics International, Madison, WI, 2The Hamner Institutes for Health Sciences, Research Triangle Park, NC, 3 University of North Carolina Institute for Drug Safety Sciences, Hamner, Research Triangle Park, NC, 4Triangle Research Laboratories at the Hamner Institutes for Health Sciences, Research Triangle Park, NC Drug induced liver injury (DILI) is a leading cause of drug withdrawal from the market, highlighting the fact that current preclinical models of toxicity are not universally predictive of drug effects in humans. The manifestations of hepatotoxicity are highly variable and range from intrinsic toxic effects to the enzymatic production of toxic metabolites. Development of a more predictive in vitro model system that can elucidate the underlying mechanisms of hepatotoxicity early in the drug development process is critical to subverting unanticipated DILI in the clinic. To that end, we have developed human induced pluripotent stem cell (iPSC)-derived hepatocytes (iCellÕ Hepatocytes) that exhibit high purity (495%) and biologically relevant functions necessary for hepatotoxicity studies. To demonstrate the functional utility of iCell Hepatocytes in assessing drug-induced hepatotoxicity, we tested their response to a set of known hepatotoxins: amiodarone, acetaminophen (APAP), menadione, and isoniazid (INH). The cellular responses were evaluated using multiple read outs of activity, including luminescence-based assays (Promega), High Content Analysis (ImageXpress, Molecular Devices), and the XF96 Extracellular Flux Analyzer (Seahorse Biosciences) to assess direct and metabolism-mediated toxicities. At 24 hours post treatment, amiodarone (18 mM) induced a 50% increase in phospholipid accumulation coupled with a decrease in ATP production. A 75% decrease in glutathione (GSH) production was observed in response to APAP at the AC50 (32 mM) concentration. At 24 hours, menadione (24 mM) showed an oxidative stress response using the XF96eExtracellular Flux Analyzer and the GSH/GSSG-GloÔ Assay, through reduced levels of GSH similar to primary human hepatocytes (22 mM). Lastly, a four hour exposure to INH elicited a dose-dependent decrease in respiratory capacity and 48 hours exposure reduced ATP production (AC50 33 mM). Responses in the iPSC-derived Hepatocytes were observed for all compounds at AC50s comparable to those seen with primary human hepatocytes. Mechanism-based toxicity was evaluated by analyzing cell metabolism, oxidative stress, and lysosomal phospholipase activity. This multiple platform approach for functional analyses demonstrated how iPSC-derived Heaptocytes provide a biologically relevant human model system for investigating drug-induced hepatotoxicity.

P280. LACK OF METABOLIC ACTIVATION, THE PRIMARY REASON RESPONSIBLE FOR NON-TOXICITY OFPLATYNECINETYPE PYRROLIZIDINE ALKALOIDS Jianqing Ruan1, Cangsong Liao2, Yang Ye2 and Ge Lin3 1 School of Biomedical Sicences, The Chinese University of Hong Kong, Hong Kong, Hong Kong, 2State Key Laboratory of Drug Research & Natural Products Chemistry Department, Shanghai Institute of MateriaMedica, Chinese Academy of Sciences, Shanghai, China, 3School of Biomedical Sicences, The Chinese University of Hong Kong, Hong Kong, China Pyrrolizidine alkaloids (PAs) poisoning is well-known because of the intake of PA-containing plant-derived natural products and PA-contaminated foodstuffs. Based on different structures of the necine base, PAs are classified into three types: retronecine type, otonecine type, and platynecine type. The former two type PAs possessing an unsaturated necine base with a 1,2-double bond are hepatotoxic due to the P450-mediated metabolic activation to generate pyrrolic ester, a reactive metabolic intermediate, which interacts with cellular macromolecules leading to hepatotoxicity. Whereas, having a saturated necine base, platynecine type PAs are reported to be non-toxic, however, the mechanism responsible for their non-toxic nature is largely unknown. In the present study, to delineate the metabolism-based mechanism underlying the non-toxicity of platynecine type PAs, hepatic metabolism of platyphylline (PLA), a representative platynecine type PA, was investigated and directly compared with the representatives of two toxic types of PAs in the parallel study. Our results demonstrated that, the metabolic rates of three different types of PAs tested were similar with about 40%, but unlike the toxic PAs,PLA did not undergo metabolic activation to generate the reactive metabolic intermediate. More interestingly, its three hepatic microsomal metabolic pathways identified, including dehydrogenation, N-oxidation, and C-oxidation, were all mediated by P450, especially CYP3A isozyme, and the predominant dehydrogenation pathway formed a novel metabolite, dehydroplatyphylline acid, which was aqueous soluble, readily excreted, and could not interact with cellular macromolecules. In conclusion, for the first time, our study revealed the mechanism underlying non-toxic feature of platynecine type PAs. The structural differences in the necine bases caused significant differences in hepatic metabolism of different PAs. Metabolic activation only occurred in two toxic types of PAs possessing the unsaturated necine base. Whereas, having the saturated necine base, the lack of metabolic activation and the predominant formation of an aqueous soluble and readily excreted metabolite governed the non-toxic nature of platynecine type PAs. [Supported by Research Grant Council of Hong Kong (GRF Grants no. 471310 and 469712) and CUHK (Direct Grants no. 2041744 and 4054047)].

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P281. FECAL MICROBIOME PROFILING REVEALS ASSOCIATION OF B-GLUCURONIDASE PRODUCING BACTERIA WITH SUSCEPTIBILITY TO TACRINE-INDUCED HEPATOTOXICITY


Lian Yee Yip1, Jessalyn Mei Xuan Chan1, Aparna Sampathkumar1, Chiu Cheong Aw2, Woei Shin Chen3, Edward Browne2, Ratha Mahendran4, Yuan Kun Lee5 and Eric Chun Yong Chan1 1 Department of Pharmacy, National University of Singapore, Singapore, Singapore, 2GlaxoSmithKline R&D Centre, Singapore, Singapore, 3GlaxoSmithKline R&D Center, Singapore, Singapore, 4Surgery, National University of Singapore, Singapore, Singapore, 5Microbiology, National University of Singapore, Singapore, Singapore The human gut microbiome, sometimes referred to as a forgotten organ, is known to possess metabolic activities capable of influencing drug disposition and toxicity. Tacrine, the first acetylcholinesterase inhibitor approved for treatment of Alzheimer’s disease, causes hepatotoxic transaminitis in 49% of the patients. However it is unclear what mechanism underlies the differential susceptibility to tacrine-induced transaminitis. In an in-house study conducted on 28 tacrine-dosed Lister-hooded (LH) rats (given a single oral dose of 20 mg tacrine/kg body weight), 42% of the rats was found to exhibited transaminitis. They were classified as non-responder (Non-R, n ¼ 16), mild responder (MilR, n ¼ 5), moderate responder (ModR, n ¼ 3) or extreme responder (ExtR, n ¼ 4) based on their degree and duration of transaminitis. Among rats exhibiting extreme transaminitis (ExtR), we observed a double Cmax pharmacokinetic profile linked to a significantly greater system exposure to tacrine (direct hepatotoxicant) that was previously unreported for tacrine. Enterohepatic recirculation is a common underlying mechanism resulting in double Cmax profile. We hypothesized that the gut microbiome, known to be involved in enterohepatic recycling, is associated with the observed inter-individual variations to tacrine-induced transaminitis. To test this hypothesis, we aimed to profile and elucidate the differences in the intrinsic and post-dose gut microbiome of these tacrine-dosed rats belonging to the different toxicity phenotypes. Literature review was performed to identify a panel of predominant bacterial groups in the rat gut microbiome. Quantitative polymerase chain reaction was employed to profile the target bacterial groups in the pre-dose (24 to 0 h) and post-dose (8 to 24 h) fecal samples of these LH rats. A panel of 6 bacterial groups namely Enterobacteriaceae, Lactobacillus, Bacteroides-Prevotella-Porphyromonadaceae, Erysipelotrichaceae, Clostridium coccoides-Eubacterium rectale and Clostridium leptum were selected for microbiome profiling. We observed no statistical difference in the intrinsic levels of the profiled gut microbiome across rats of the various toxicity phenotypes, except for Clostridium coccoides where ModR has a higher level of this bacteria group than Non-R (p50.05, ANOVA). Enterobactericeae target group associated with b-glucuronidase activities showed a significant increase in count following tacrine administration, particularly among ExtR (pre-dose 1.18 107 1.21 107 cells/g feces vs. post-dose 1.11 108 8.67 107 cells/g feces; p50.001 for Non-R vs. ExtR, ANOVA). A general decline in Lactobacillus abundance across all rats in the post-dose profile was detected. Lactobacillus has been reported to inhibit b-glucuronidase productivity of the gut microbiome and is associated with hepatoprotection1. Ratio analysis of pre-dose Enterobactericeae:Lactobacillus (E/L) revealed a trend of higher E/L ratio for tacrine-dosed rats with more severe transaminitis. Post-dose E/L analysis detected significantly higher E/L ratio in ExtR vs. Non-R (p50.01, ANOVA). Altogether, we speculated the post-dose decrease in Lactobacillus may potentially favor the outgrowth of Enterobacteriaceae in ExtR with intrinsically higher E/L compared to other responder groups. No significant findings were observed for other bacterial groups. In conclusion, we observed an association of b-glucuronidase related gut microbiome with the differential susceptibility to tacrine-induced transaminitis.

References 1. Han SY et al. Hepatoprotective effect of lactic acid bacteria, inhibitors of beta-glucuronidase production against intestinal microflora. Archives of pharmacal research. 2005;28(3):325-9. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed.

P282. TOXICITY, GENOTOXICITY AND PROINFLAMMATORY EFFECTS OF AMORPHOUS NANOSILICA IN THE HUMAN INTESTINAL CACO-2 CELL LINE Adeline Tarantini, Kevin N. Hogeveen, Rachelle Lanceleur, Annick Mourot, Gerard Jarry and Vale´rie Fessard Contaminant Toxicology, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), Fouge`res, France While amorphous silica (SiO2) in its nanosized form is currently used as an additive in the food industry, the potential risks for human health need to be evaluated in further detail. In the current study, the uptake and toxicity of 15 and 55 nm colloidal SiO2 nanoparticles (NPs) was investigated in the human intestinal Caco-2 cell line. The ability of these NPs to induce cytotoxicity, genotoxicity, apoptosis, oxidative stress and proinflammatory effects was evaluated. Size- and concentration-dependent cytotoxic and genotoxic effects, accompanied by the generation of reactive oxygen species (ROS), were observed following

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treatment with SiO2 nanoparticles. Moreover, increases in IL-8 secretion were observed following treatment with SiO2-15 nm at the highest tested dose (32 mg/mL). TEM analysis demonstrated that both NPs were present within the cytoplasm of treated Caco-2 cells, primarily within lysosomal and endocytic compartments. Although NPs were detected at the nuclear periphery, NPs were not observed within the nucleus. SiO2-15 nm, and to a lesser extent SiO2-55 nm, exerted toxic effects in Caco-2 cells. The genotoxicity observed following treatment with these NPs is like to be mediated through oxidative stress rather than a direct interaction with DNA. Our results suggest that exposure to SiO2 NPs induce adverse effects on the intestinal epithelium in vitro.

P283. PERSONALIZED DOSING OF DICHLOROACETATE (DCA) BASED ON HAPLOTYPE VARIATIONS IN GLUTATHIONE TRANFERASE ZETA 1


Albert L Shroads1, Taimour Langaee2, Natalia Felitsyn1, Monica Abdelmalak1, and Peter W. Stacpoole1 1 College of Medicine, University of Florida, Gainesville, FL, 2Center for Pharmacogenomics, University of Florida, Gainesville, FL Dichloroacetate (DCA), a xenobiotic used in the treatment of various mitochondrial diseases, pulmonary arterial hypertension and certain cancers, is dehalogenated by the bifunctional enzyme glutathione transferase zeta 1 (GSTZ1)/ maleylacetoacetate isomerase (MAAI), the penultimate enzyme in the phenylalanine/tyrosine catabolic pathway. Polymorphisms in GSTZ1/MAAI have been shown to influence the pharmacokinetics of DCA and result in a wide variation in the biotransformation of the drug. Healthy adult volunteers, (age ¼ 26 4.5) who lacked the wildtype EGT allele showed markedly delayed DCA plasma clearance and higher urinary accumulation of potentially toxic tyrosine metabolites after receiving 25 mg/kg/day of DCA for five days.1 However, little is known of the long term biotransformation of DCA by individuals possessing the slowest forms of GSTZ1. We postulated that long-term DCA administration will demonstrate altered drug kinetics and tyrosine metabolism based on GSTZ1 genotype. Methods: We determined the GSTZ1 diplotype in 14 children with primary mitochondrial diseases, previously enrolled in a randomized controlled trial of DCA (age 6.2 5.1 yrs at entry).2 Subjects received 25 mg/ kg/day of DCA for 6 months for up to 15 yrs. At 6 month intervals, plasma (12 hr trough) and urine (12 hour composite) were collected and DCA, its metabolites and the tyrosine intermediate maleylacetone (MA) were measured by GC-MS. Urinary delta-aminolevulinate (d-ala), a heme precursor and reactive molecule that accumulates in patients with inhibition of the distal tyrosine catabolic pathway, was determined by LC-MS. Each patient also received a single dose of 12.5 mg/kg of 1,2-13CDCA for plasma pharmacokinetic analysis by WinNonLin software (Version 5.1, Pharsight Corp). Results: Patients lacking the EGT allele showed dramatically slower clearance of DCA. The average DCA plasma elimination half-life after 6 months of DCA treatment was 19 17 hours for the non-EGT group vs. 6.8 6.9 for the EGT group (p ¼ 0.048). DCA plasma trough levels were almost 6 fold higher (141 mg/ml 125) in the non-EGT group, than the EGT group (24 mg/ml 48; p ¼ 0.04). However, plasma DCA appeared to plateau over time and DCA did not accumulate, regardless of GSTZ1 genotype. Plasma DCA trough concentrations at 6 months correlated with the accumulation of urinary d-ala, (r2 ¼ 0.64) and MA (r2 ¼ 0.78), with the highest values corresponding to the non-EGT group. In sampling intervals up to 30 months, plasma DCA trough levels became more highly correlated with urinary d-ala (r2 ¼ 0.84) and MA (r2 ¼ 0.86). We conclude that, in chronically treated children with primary mitochondrial disorders, DCA plasma clearance and accumulation of MA and dala are directly related to GSTZ1 polymorphism. Those individuals lacking the EGT allele may be at heightened risk for the accumulation of toxic tyrosine metabolites and the possible development of adverse clinical effects, unless dose adjustments are made.

References 1. Shroads AL, Langaee T, Coats BS, Kurtz TL, Bullock JR, Weithorn D, Gong Y, Wagner DA, Ostrov DA, Johnson JA, Stacpoole PW. Human polymorphisms in the glutathione transferase zeta 1/maleylacetoacetate isomerase gene influence the toxicokinetics of dichloroacetate. J Clin Pharmacol. 2012 Jun;52(6):837–49. 2. Stacpoole PW, Kerr DS, Barnes C, Bunch ST, Carney PR, Fennell EM, Felitsyn NM, Gilmore RL, Greer M, Henderson GN, Hutson AD, Neiberger RE, O’Brien RG, Perkins LA, Quisling RG, Shroads AL, Shuster JJ, Silverstein JH, Theriaque DW, Valenstein E. Controlled clinical trial of dichloroacetate for treatment of congenital lactic acidosis in children. Pediatrics. 2006 May;117(5):1519–31.

P284. EVALUATION OF CRYOPRESERVED HEPARG CELLS AS AN IN VITRO MODEL FOR DRUG-INDUCED BILIARY TOXICITY Rita Ciurlionis1, Gerard D. Gagne2, Jonathan M. Maher2, Michael J. Liguori1 and Eric A.G. Blomme1 1 Cell, Molecular, & Exploratory Toxicology, Abbvie, Inc., North Chicago, IL, 2Investigative Toxicology and Pathology, Abbvie, Inc., North Chicago, IL


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Drug-induced biliary toxicity is challenging to characterize and predict due to the likely involvement of multiple mechanisms. Preclinically, biliary toxicity is typically seen in vivo after chronic dosing and the current biomarkers are not sensitive to monitor the toxicity. Furthermore, there are currently no robust in vitro models to reliably identify biliary toxicants early and to characterize the mechanism of biliary toxicity. The hepatocyte-like features of HepaRG cells have been widely reported. However, the concurrent presence of biliary epithelial cells in this culture system has been less well examined. In this study, we explored whether this unique dual biliary/hepatocellular nature of HepaRG cells along with their ability to sustain long term culture could aid in the prediction of biliary toxicity. Cryopreserved HepaRG cells (Invitrogen) were plated on duplicate 24-well collagen coated plates at 600 K/well. Cells were dosed with 9 prototypical biliary toxicants representing diverse mechanisms, 2 known hepatocellular toxicants and 2 benign control compounds for 24 hr, 72 hr and 10 days. Concentrations were selected based on literature and in-house data. Endpoints included clinical chemistry of conditioned media and imaging by brightfield and immunofluorescence microscopy. The cholangiocyte marker CK19 and hepatocyte specific marker HepPar1 were used to distinguish the two cell populations, while Hoechst nuclear stain was used to monitor total cell number. In all treatments including vehicle, nuclear staining was observed where no CK19þ nor HepPar1þ cells were present, suggesting the presence of a third population, possibly progenitor cells. Only ALT, AST, GLDH, LDH changed appreciably with any treatment. These parameters appeared to detect general cytotoxicity and no parameter was able to reliably and specifically distinguish biliary injury. A number of treatments resulted in weak nuclear staining, but very intense CK19 staining in cells with normal appearance by brightfield microscopy. These observations suggest that HepaRG cells are a dynamic system in which culture and/or treatment conditions may affect the differentiation/remodeling state. Gene expression analysis may provide a better understanding of changes observed in the culture system. Based on the conditions and endpoints employed in this study, HepaRG cells provide no clear advantage for either the identification or the mechanistic differentiation of compounds inducing biliary toxicity. Further testing, however, is needed to determine if cells from varying sources, including non-cryopreserved cells, yield a similar result. RC, GG, JM, ML, EB are employees of Abbvie, Inc. This study was sponsored by Abbvie. Abbvie contributed to the study design, research, and interpretation of data, writing, reviewing and approving this abstract.

P285. POTENTIAL MOLECULAR CYTOTOXIC MECHANISMS OF METHOTREXATE IN ISOLATED RAT HEPATOCYTES Abdullah Al Maruf1 and Peter J. O’Brien2 1 Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada, 2Leslie Dan Facuty of Pharmacy, University of Toronto, Toronto, ON, Canada Methotrexate (MTX) is a folic acid antagonist that is widely used to treat a variety of diseases. The most serious side effect of MTX therapy is hepatotoxicity. The potential molecular cytotoxic mechanisms of MTX towards isolated male Sprague Dawley rat hepatocytes were investigated in this study using ‘‘Accelerated Cytotoxicity Mechanism Screening’’ (ACMS) Techniques. A concentration-dependent increase in cytotoxicity (as as measured by the trypan blue exclusion assay) and reactive oxygen species (ROS) formation (as measured with dichlorofluorescin diacetate) was observed for methotrexate compared to control hepatocytes. We hypothesized that MTX produced hepatic necrosis through reactive oxygen species (ROS) formation towards isolated rat hepatocytes. Incubation of isolated hepatocytes for 2 hr with 300 mM methotrexate induced an approximate 50% loss in hepatocyte viability (LC50, according to ACMS). A significant increase in MTX cytotoxicity and ROS formation was observed when glutathione (GSH) depleted hepatocytes were used and this toxicity was reversed by the addition of l-Cysteine and N-Acetyl-l-cysteine (GSH precursors). Catalase inactivation with sodium azide (4 mM) also resulted in an increase in MTX-induced cytotoxicity, while the direct addition of catalase (0.25 mg/mL) to the hepatocyte suspension delayed cytotoxicity, suggesting that H2O2 generated from the MTX caused cytotoxicity. Nontoxic concentrations of the non-selective cytochorome P450 inhibitor, 1-aminobenzotriazole (100 mM), aldehyde oxidase inhibitor, menadione (50 mM) and xanthine oxidase inhibitor, allupurinol (50 mM) were also used. Menadione caused significant decrease in MTX-induced cytotoxicity and ROS formation. Xanthine oxidase inhibition and cytochrome P450 inhibition also decreased toxicity but was not statistically significant. These results suggest that aldehyde oxidase is mainly involved in the metabolism of MTX in rat hepatocytes. It has been proposed that 7-hydroxy methotrexate is the major metabolite which is found to be more cytotoxic than MTX formed by aldehyde oxidase in rat. Similar results were observed in our study as inhibition of aldehyde oxidase significantly decreased MTX cytotoxicity and ROS formation. Ascorbate (200 mM) is a known antioxidant and cofactor for many enzymes and resulted in a significant delay in cytotoxicity when added to the hepatocyte suspension with MTX. The vitamin E analogue, trolox (200 mM), also significantly reversed MTX–induced cytotoxicity and ROS formation. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) (200 mM), a known ROS scavenger and superoxide dismutase mimic, also prevented hepatic cell death. Scavenging free ferrous iron with desferoxamine (500 mM) protected hepatocytes, likely by inhibiting the Fenton reaction to take place. From the observed results, it can be suggested that MTX-induced cytotoxity in isolated rat hepatoctes involved ROS and toxic metabolite formation leading to oxidative stress.

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P286. INVESTIGATION OF ISONIAZID DILI MECHANISMS IN HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED HEPATOCYTES


Jingtao Lu1, Imir G. Metushi2, Jack P. Uetrecht2, Shannon Einhorn3, David A. Mann3, Paul B. Watkins1 and Edward L. LeCluyse1 1 The Hamner Institutes for Health Sciences, Reseach Triangle Park, NC, 2Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada, 3Cellular Dynamics Internationalm Madison, WI Isoniazid (INH), the preferred medication for prevention and treatment of tuberculosis, has been associated with idiosyncratic drug induced liver injury (IDILI). INH causes benign ALT elevations (43 X ULN) in about 10% patients, and causes clinically important DILI in about 0.1% patients. Formation of INH-protein adducts in human liver microsomes, detection of antibodies targeting INH and INH-adduted proteins in IDILI patients’ serum, and positive lymphocyte transformation test (LTT) in IDILI patients all support the hypothesis that INH-induced IDILI is involves an adaptive immune response. However, the mechanism underlying these rare immune reactions remains unclear. Prior studies support a genetic basis for susceptibility to INH hepatotoxicity. In the current study, hepatocytes derived from induced pluripotent stem cells (iPSC), which carry the donor’s genetic information, were characterized for studying INH hepatotoxicity. The Vmax of CYP2E1 in iPSC-derived hepatocytes were 16.0 3.0 or 10.8 2.2 pmol/min/106 cells using 4-Nitrophenol or chlozoxazone as substrate, respectively. Parallel experiments in primary human hepatocytes (pHH) were performed with 4-Nitrophenol and activity was calculated as 21.2 0.5 pmol/min/106 cells. INH-adducted proteins were assessed by western blot using rabbit antiserum raised agaist an INH protein adduct. Both iPSC-derived hepatocytes and pHH exhibited dose-dependent INH-protein adduct formation, with the adducts being more prominent in iPSC-derived hepatocytes. The major bands of INH-adducted proteins in both cell types were similar qualitatively but not quantitatively. The viability of iPSC-derived hepatocytes incubation with increasing concentrations of INH was assessed by measuring cellular ATP content or Lactate dehydrogenase (LDH) release in to the culture medium. INH, concentrations up to 100 fold higher than the mean Cmax in patients serum (10 mM), did not cause noticeable ATP loss, LDH release or morphological changes in iPSC-derived hepatocytes. However, release of microparticles into the medium was observed iPSC-derived hepatocytes after 5 mM INH treatment. The majority of the particles sized round 0.1 um with minimum side scatter readings but a portion of the particles sized 0.1 um to 1um and had relatively higher SSC readings. INH-adducted proteins were detected in both the conditioned medium and microparticles. Untreated control cells had very few particles released. At non-cytotoxic concentrations, INH also induced a concentration-dependent decrease of oxygen consumption rate as well as mitochondrial membrane potential, measured by seahorse and high content imaging, respectively. In summary, the iPSC-derived hepatocytes exhibited CYP2E1 activity comparable to primary hepatocytes. At non-cytotoxic INH concentrations, iPSC-derived hepatocytes formed INH-adducted proteins and released microparticles, which may function in neoantigen presentation and generation of the ‘‘danger signal’’ that is required for an adaptive immune response. We conclude that iPSC hepatocytes derived from patients who have experienced IDILI due to INH are a promising tool to define the underlying mechanisms.

P287. THE ANTI-HIV DRUG EFAVIRENZ INCREASES THE PROTEIN EXPRESSION AND ACTIVITY OF IRE1A IN PRIMARY HUMAN HEPATOCYTES Jennifer VanAusdall and Namandje´ Bumpus Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD Efavirenz (EFV) is among the most widely prescribed drugs for the treatment of HIV. Long-term use of EFV has been linked to hepatotoxicity, but the mechanism(s) underlying this are largely unknown. The present study tested whether EFV has the ability to modulate the activity of ER stress responsive pathways. In primary human hepatocytes, protein expression of IRE1a (inositol-requiring enzyme 1a), which is a major signal transducer of the unfolded protein response (UPR), was increased up to 3-fold and 5-fold over vehicle control following 3 and 6 hours of incubation with 10 mM EFV, respectively. The protein expression of the other signal transducers of the UPR, ATF6 (activating transcription factor 6) and PERK (PKR-like ER kinase), were not changed significantly following treatment with 10 mM EFV. Activation of IRE1a via the UPR results in the association with TRAF2 (TNF-receptor associated factor 2) as well as splicing of XBP1 (X-box binding protein 1) mRNA. Immunoprecipitation of TRAF2 followed by immunoblotting for IRE1a revealed that the association of IRE1a with TRAF2 was increased 2-fold after 6 hours and 3-fold after 12 hours of EFV treatment, as compared to vehicle control. Further, EFV treatment stimulated marked splicing of XBP1 following 3 and 6 hours of treatment, in that unspliced transcript was marginally detectable in these samples as compared to vehicle controls in which the XBP1 detected was mostly the unspliced transcript. These results indicate that the IRE1a pathway is activated by EFV in primary human hepatocytes and should be explored further as potential mediator of EFV-induced hepatotoxicity.

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P288. INTESTINAL ABSORPTION OF PYRROLIZIDINE ALKALOIDS AND THEIR N-OXIDES


Mengbi Yang1, Jianqing Ruan1, Yang Ye2 and Ge Lin1 1 School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong, 2State Key Laboratory of Drug Research & Natural Products Chemistry Department, Shanghai Institute of MateriaMedica, Chinese Academy of Sciences, Shanghai, China Pyrrolizidine alkaloids (PAs) are a group of naturally occurring toxic phytochemicals present in various herbal medicines and contaminate many food stuffs. There are two types of toxic PAs, namely retronecine type and otonecine type. The former type PAs have their corresponding N-oxide form. Previous studies on rodent models demonstrated similar toxicological mechanism but different toxic potency between PAs and their N-oxides. TheN-oxides showed obvious but much less toxicity comparing with their parent PAs. However, the reasons responsible for the significantly less hepatotoxic potency of PA N-oxides are unknown. The objective of the present study was to unveil the mechanism underlying the differences in toxic potency between PAs and PA N-oxides from the perspective of intestinal absorption. Caco-2 cell monolayer model was employed for the evaluation of intestinal absorbability of five pairs of retronecine-type PAs and their N-oxides. Two otonecine-type PAs were also tested for the comparison between these two type PAs. Our results demonstrated a poor-median absorption of five PA N-oxides (Papp A to B ¼ (0.33 1.00) 106 cm/s), and mediangood absorption of five parent PAs (Papp A to B ¼ (1.21 14.35) 106 cm/s). For the otonecine type PAs, median permeability was found (Papp A to B ¼ (3.96 5.06) 106 cm/s). The permeability of all compounds tested correlated with their lipophilicity. Active transport was only observed for senecionineN-oxide with a weak efflux ratio of 3.3, which was completely inhibited by a P-gp inhibitor verapamil. No biotransformation was found in the Caco-2 cell model for both PAs and their N-oxides. Our study investigated the absorbability of different types of PAs and their N-oxides and demonstrated, for the first time, that the significantly lower intestinal absorption of PA N-oxides was one of the reasons responsible for their remarkably lower hepatotoxicity comparing with the corresponding PAs. [Supported by Research Grant Council of Hong Kong (GRF Grants no. 471310 and 469712) and CUHK (Direct Grants no. 2041744 and 4054047)].

P289. INCREASED DNA METHYLATION OF SCAVENGER RECEPTOR CLASS B TYPE I MEDIATE INHIBITORY EFFECT OF PRENATAL CAFFEINE INGESTION ON FETAL ADRENAL CHOLESTEROL UPTAKE Dong-mei Wu, Zheng He, Liang-peng Ma, Jie Ping Sr. and Hui Wang Pharmacology, Medical School of Wuhan University, Wuhan, China Prenatal caffeine ingestion is one of the most definite risk factor for intrauterine growth restriction (IUGR). Adrenal plays a pivotal role, mainly through steroidogenesis, in the regulation of intrauterine homeostasis and in fetal development and maturation. Our previous studies proved that prenatal caffeine exposure can inhibit fetal adrenal corticosterone production, however, the underlying mechanism is unknown. Within the adrenals, a constant supply of free cholesterol is required to serve as precursor for steroidogenesis. Scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters from HDL, which serves as the major route for the uptake of cholesterol in adrenal steroidogenesis. This study was designed to investigate the effects of prenatal caffeine ingestion on cholesterol uptake in fetal adrenal in rats, and explore the underlying mechanism by observing the DNA methylation and expression of SR-BI. Pregnant Wistar rats were intragastrically treated with 60 mg/kg caffeine from gestational days 7–17. Concentrations of free cholesterol and cholesteryl esters in fetal adrenals were determined using enzymatic colorimetric assays. Whole genome expression arrays and DNA methylation arrays were performed to investigate for alterations in the expression and the promoter methylation state of genes, respectively, in fetal adrenals. The mRNA and protein expressions of SR-BI were measured by real-time RTPCR and immunohistochemistry, respectively. The methylation state of CpG sites within the SR-BI promoter region were detected by bisulfite-sequencing PCR (BSP). The results showed that prenatal caffeine ingestion-induced IUGR rate was 61.2%. The contents of free cholesterol and cholesteryl esters in cytoplasm were decreased significantly in fetal adrenal after caffeine treatment. The conjoint analysis of methylation array with expression array indicated that SR-BI contribute to caffeine-inhibited cholesterol uptake. Furthermore, caffeine reduced both protein and mRNA expression of SR-BI in fetal adrenal. BSP analysis uncovered notably increased total methylation rate in SR-BI promoter (478 703). In conclusion, prenatal caffeine ingestion was able to induce increased DNA methylation of SR-BI promoter in rat fetal adrenal. This effect results in inhibited SR-BI expression and cholesterol uptake, which may contribute to caffeine-induced fetal developmental toxicity. Supported by the National Natural Science Foundation of China (No. 81173138, 81273107, 30800931) and Outstanding Youth Science Fund of Hubei Province (No. 2012FFA017); *Correspondence author: [email protected]

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P290. THE INVOLVEMENT OF THE NEUTROPHIL OXIDATIVE BURST PATHWAY IN THE COVALENT BINDING OF AMODIAQUINE TO RODENT NEUTROPHILS IN VIVO: IMPLICATIONS FOR DRUG-INDUCED AGRANULOCYTOSIS


Alexandra R. Lobach and Jack Uetrecht Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada Introduction: Amodiaquine (AQ) is an antimalarial agent associated with idiosyncratic agranulocytosis, a reaction that is suspected to involve haptenation of neutrophil proteins with the drug. Previous studies have shown that AQ is oxidized to a reactive quinone imine in vitro by components of the neutrophil oxidative burst pathway, specifically myeloperoxidase, and that this reactive species is capable of covalently binding to protein. Covalent binding of AQ was also detected to human neutrophils in vitro, but only upon activation of the cells, suggesting that the neutrophil oxidative burst is a key pathway in the mechanism of this binding. In order to demonstrate the importance of this pathwayin vivo, studies were carried out in rodents to detect and characterize the nature of this binding. Methods and Results: In order to detect the covalent binding of AQ to protein using immunochemical methods, AQ-specific polyclonal antiserum was generated in rabbits using AQ-conjugated bovine serum albumin (BSA). The antiserum was determined to be specific for AQ by ELISA and immunoblot. Neutrophils were isolated from the bone marrow of rats and mice treated with AQ (62.5 and 300–350 mg/kg/day, respectively) and covalent binding to neutrophil proteins was detected by immunoblot. Binding to neutrophils in AQ-treated myeloperoxidase knockout mice decreased 2.5-fold but there were still significant levels of binding. More surprising was that when the entire neutrophil oxidative burst pathway was absent in vivo (gp91 knockout mice) there was no decrease in binding compared to wild type. When bone marrow and whole blood were incubated with AQ in vitro in the absence of activation, significant binding to leukocyte proteins was also observed. Furthermore, lymphocytes and megakaryocytes, but not the erythrocytes, were found to bind AQin vivo. Conclusions: These results indicate that neutrophil activation is not essential for AQ to covalently bind to neutrophils in vivo and that myeloperoxidase is only partially responsible for the binding. A significant portion of neutrophil binding in vivo, and likely reactive metabolite generation, appears to be occurring independent of the oxidative burst pathway. The ability of other cells to bind AQ in vivo suggests that the neutrophil is not the only source of reactive metabolites in the blood and bone marrow. These findings indicate that screening drugs for the potential to cause agranulocytosis may be difficult in some cases, as cells and pathways independent of the neutrophil may be playing an important role. To confirm this, other drugs that cause agranulocytosis that have been shown to be metabolized to reactive species in vitro by neutrophils should be tested. These studies were funded by the Canadian Institutes of Health Research.

P291. PHARMACOKINETICS OF ORAL DICHLOROACETATE IN DOGS Peter W. Stacpoole Medicine, University of Florida, Gainesville, FL We characterized the pharmacokinetics and dynamics of dichloroacetate (DCA), an investigational drug for mitochondrial diseases, pulmonary arterial hypertension and cancer. Adult beagle dogs were administered 6.25 mg/kg q12h DCA for 4 weeks. Plasma kinetics were determined after 1, 14 and 28 days. The activity and expression of glutathione transferase zeta 1 (GSTZ1), which biotransforms DCA to glyoxylate, were determined from liver biopsies were determined at baseline and after 27 days. The maximum DCA plasma concentration (C max) (p50.01) and the area under the curve [AUC (0–t)] (p50.05) were higher on day 14 compared to day 1, while plasma clearance was lower (p50.02) (Table 1 and Fig. 1). The differences between day 1 and 28 of drug administration were also significant for plasma half-life (t1/2) (p50.02), C max (p50.02), and clearance (p50.02). Liver biopsies demonstrated almost complete inhibition of GSTZ1 protein expression and activity (p50.01). Dogs demonstrate much slower clearance and greater inhibition of DCA metabolism than rodents and most humans. Indeed, the plasma kinetics of DCA in dogs is similar to humans with GSTZ1 polymorphisms that confer exceptionally slow plasma clearance. Dogs may be a useful model to further investigate the toxicokinetics and therapeutic potential of DCA.

P292. HIGH-RESOLUTION MASS SPECTROMETRY FOR RAPID METABOLITE CHARACTERIZATION AND REACTION PHENOTYPING: A CASE STUDY WITH REPAGLINIDE Joanna E. Barbara, Sylvie Kandel, Forrest A. Stanley and David B. Buckley XenoTech, LLC, Lenexa, KS Assessing the involvement of specific cytochrome P450 (CYP) enzymes in biotransformation of a drug is an important step in evaluating victim potential for drug interactions. Current methods for biotransformation pathway identification (reaction phenotyping) rely on prior knowledge of metabolites, biotransformation routes, and availability of metabolite reference standards


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or radioisotopically-labeled drug. Synthesis of metabolite standards and radiolabeled compounds is time-consuming and expensive and the material is typically not available in early preclinical testing. Reaction phenotyping can be achieved by monitoring only the parent drug. That approach, however, suffers from a lack of specificity as only a general evaluation of all enzymes contributing to the loss of parent drug is made, and the individual enzymes associated with specific biotransformation pathways cannot be identified. Liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) is a powerful tool for a priori metabolite profiling and characterization because complex data sets comprising information on all of the components in a sample, within a specified mass range, are acquired. When HRMS data are employed for relative quantitation of metabolites, reaction phenotyping information can also be derived without metabolite standards. In the present study, repaglinide (1 or 10 mM) was incubated for up to 30 min (37 C; pH 7.4) with NADPH-fortified human liver microsomes (HLM; 0.1 mg protein/ mL; pooled or from individual donors) or recombinant CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, or 2J2 (10 pmol/incubation). Incubations were subjected to metabolite profiling with reverse-phase gradient ultra-performance LC and HRMS on a Waters Synapt quadrupole time of flight mass spectrometer. An unrelated internal standard was employed. Data were processed for metabolite profiling, relative metabolite quantitation, and correlation of metabolite formation with CYP activity. Ten repaglinide metabolites were characterized. Five were the well-established metabolites M0-OH and M4 (hydroxylation metabolites), M1 (the N-dealkylated primary amine metabolite), M5 (O-desethyl repaglinide) and M2 (the ring-opened dicarboxylic acid). M2 is the major human metabolite formed in vivo and M4 is an in vivo probe substrate employed for investigating CYP2C8 inhibition in clinical studies. Major roles have been established for CYP2C8 and CYP3A4 in the metabolism of repaglinide. Consistent with published literature, the approach resulted in the identification of CYP2C8 as the primary mediator of M0-OH and M4 formation and CYP3A4 as primarily involved in formation of M1 and M5. Although only CYP2C8 and 3A4 were implicated in formation of M2, the data were ambiguous as to the extent of involvement of either enzyme. M2 was detected in incubations with recombinant CYP3A4 but not with recombinant CYP2C8, but the HLM correlation data implied a role for both enzymes in formation of M2. Roles for CYP2C8 and CYP3A4 in mediation of several unexplored minor biotransformation pathways of repaglinide were also established. The HRMS approach was appropriate for simultaneous metabolite profiling and reaction phenotyping without the need for metabolite standards for the test compound repaglinide. The approach is, however, unsuitable for the derivation of kinetic parameters such as Km and Vmax as absolute quantitation cannot be performed.

P293. APPLICATION OF GAS PHASE ION-MOLECULE REACTIONS TO CHARACTERIZE 2AMINOTHIAZOLE OXIDIZED METABOLITES Minli Zhang1, Ryan Eismin2, Xiong Hui3 and Doug Burdette1 1 Dmpk, AstraZeneca, Waltham, MA, 2Chemistry and Biochemistry, University of Arizona, Tucson, AZ, AstraZeneca, Waltham, MA

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Chemistry,

Work describing the characterization of chemical functional groups via Gas-Phase Ion-molecule Reactions (GPIMR) using a modified LTQ ion trap mass spectrometer (Habicht et al. Anal. Chem. 2008, 80, 3416–3421) indicated their potential application in characterizing metabolite functional groups and their reactivities, especially when metabolites are unstable and difficult to synthesize. Characterization of oxidized 2-aminothiazole metabolites presents such a challenge due to the lack of unique ms/ms fragmentation patterns from these chemical moieties and the fact that many of these metabolites are unstable and difficult to synthesize. We tested a novel approach to this problem based upon the comparison of GPIMR profiles of a series of standards to that of a 2-aminothiazole oxidized metabolite generated by dog microsomes. The reference GPIMR ion adduct profiles were generated against aliphatic and aromaticN-oxides, nitroso-compounds, and N-hydroxyl anilines using three reagents, dimethyl disulfide (DMDS), tert-butyl peroxide (TBP) and tri(dimethylamino)borane (TDMAB), that have been shown to form unique GPIMR adducts and to generate distinct fragment patterns when reacting with ions of oxidized nitrogen functional groups derived from aliphatic and aromatic N-oxide or nitroso- compounds. Because 2-aminothiazoles are often thought to be mainly oxidized in the anime group, we also generated DMDS, TBP and TDMAB GPIMR profiles for a series of N-hydroxyl anilines as the references. Our results showed that DMDS generated a specific [MH þ 31]þ ion adduct with nitrosobenzene and N-hydroxyl aniline compounds, but did not form diagnostic adducts with N-oxides. TBP formed a unique [MH þ 56]þ ion with nitrosobenzene and a distinct [MH þ 220]þ ion with N-hydroxyl anilines, but no specific adducts were observed withN-oxide compounds. TDMAB formed a unique [MH þ 98]þ ion adduct with N-oxide and N-hydroxyl aniline compounds but not with nitrosobenzene. Further fragmentation of the [MH þ 98]þ ion adducts yielded a neutral loss of 116 amu from aliphatic N-oxide adducts and a m/z 115 product ion from aromatic N-oxide adducts but no unique fragments from N-hydroxyl aniline adducts. Reaction of DMDS with 2-aminothiazole oxidized metabolite did not generate the [MH þ 31]þ ion adducts nor were [MH þ 56]þ or [MH þ 220]þ ions seen in the presence of TBP, data which were inconsistent with the N-hydroxyl aniline GPIMRs, but more consistent with the profile of the N-oxides tested. When reacted with TDMAB, the 2-aminothiazole oxidized metabolite formed a [MH þ 98]þ ion which lost 116 amu upon fragmentation, consistent with the aliphatic N-oxide GPIMR patterns. In summary, this research (1) indicates that the combination of ion-adduct and fragmentation patterns from the three reagents could clearly differentiate the oxidized nitrogen containing compounds, demonstrating the feasibility of fingerprinting the chemical nature of oxidized nitrogen functional groups via multi-reagent GPIMR profiling and (2) suggests that the chemical reactivity and potentially the toxicological liability of the oxidized 2-aminothiazole-containing compound tested could be very

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different from those of an N-hydroxyl aniline as might have been presumed from prevailing thought about the oxidation of 2-aminothiazoles.

P294. METABOLITE PROFILING ANALYSIS OF CLINICAL SAMPLES - ADDRESSING THE CHALLENGES OF MIST


Caroline Anderson1 and David Lankester2 1 Metabolism, Covance Laboratories Ltd, Harrogate, United Kingdom, 2Department of Metabolism, Covance Laboratories Limited, Harrogate, United Kingdom Following publication of the FDA MIST guidelines and revision of ICH M3, there is increasing interest in obtaining quantitative metabolic profiling data for clinical samples. Limitations on the level of radioactivity administered in the clinical dose, present challenges for the sensitivity of radio-detection. Whereas the trend to perform radio-labelled ADME studies later in the development of a drug, has increased pressures to characterise metabolites in Phase I clinical samples prior to the synthesis of the radiolabelled compound. This review evaluates innovative approaches used in our laboratories to prepare and analyse clinical plasma samples containing low levels of radioactivity, and samples originating from non-radiolabelled clinical studies. Standard approaches for the preparation of radiolabelled clinical plasma samples for metabolite profiling use solvent extraction for the precipitation of plasma proteins. Sample extracts are then typically concentrated by evaporation to increase the level of radioactivity injected, and therefore maximise the detection level of metabolites. Samples are then analysed by HPLC using fraction collection and off-line radio-detection. However the degree of sample concentration is generally limited to 10–20 fold due to the detrimental effects on the chromatography from material originating from the sample matrix, which is also concentrated to the same level. For radio-labelled clinical plasma samples containing metabolites for which reference standards were available, solid-phase extraction (SPE) methods were successfully developed and used to concentrate plasma samples 90-fold prior to radio-HPLC analysis. For plasma samples with unknown metabolite profiles SPE is not appropriate, as selective extraction of metabolites can not be discounted. In such samples, analytical procedures were developed to concentrate samples 60-fold using repeated injections into 96-well plates. These approaches have enabled AUC pooled plasma samples with levels of radioactivity down to 200 DPM/g to be successfully profiled. For plasma samples originating from non-radiolabelled clinical studies, a combined approach using quantitative determinations of the parent compound and notable metabolites (410% of total drug related exposure), has been used together with a qualitative screen for unknown and minor metabolites using accurate mass LC-MS. A qualified quantitative method was used to determine any disproportionate exposure of significant circulating metabolites in human. The samples were then further analysed using a high resolution mass spectrometer to identify any unique human metabolites, and assess the relative exposure of metabolites in human and the preclinical species using matrix matched samples.

P295. THE CHANGE OF PLASMA LIPID METABOLISM AT THE ORAL ADMINISTRATION OF ROUSUVASTATIN IN HUMAN Jong Min Choi1, Byung Hwa Jung2 and Joo-Youn Cho3 1 Molecular Recognition Center, Korea Institute of Science and Technology, Seoul, South Korea, 2Molecular Recognition Research Center, Korea Institute of Science and Technology, Seoul, South Korea, 3Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine, Seoul, South Korea Rosuvastatin is a cholesterol-lowering agent used to treat hypercholesterolemia, it reduces cholesterol level in plasma and affects global lipid metabolism. Therefore overall change of lipid profile can be closely related to the drug’s effects or adverse effect. In this study, a simple and rapid lipid profiling method using UPLC-QTOF-MS to detect and identify multiple classes of lipids from human plasma was optimized and metabolic change of lipid after rosuvastatin treatment was investigated. Among various lipids, phosphatidylcholine (PC), in particular, is one of the modest phospholipids and it is involved in metabolism, inflammation and cell signaling in mammalian cells. So PC profile was specially checked using PC retention time index. First, sample extraction and mobile phase in the liquid chromatographic analysis were tested with representative standards of 8 lipid classes to set up the conditions for lipid profiling. Second, a retention time index of PC was constructed and utilized to discover the role of PC in drug response. Finally, the chromatographic conditions were decided as gradient elution using 10 mM ammonium acetate in 40% acetonitrile and 10 mM ammonium acetate in acetonitrile: isopropanol ¼ 10:90 (v/v). For sample preparations, liquid-liquid extraction (LLE) method with a mixture of chloroform/methanol (1:2, v/v) and a 0.15 M of NaCl additive was selected. In the results of lipid profiling, the level of triglyceride, sphingomyelin, phosphatidylinositol decreased significantly after drug treatment. LysoPC(20:4) and LysoPC(18:1) were increased, while LysoPC (16:0) and LysoPC (18:0) decreased. This pattern of change also appeared in PC. The level of plasma increased in PC(36:5), PC(38:6), PC(32:1), PC(38:5), PC(38:4) and PC(36:1), decreased in

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PC(36:4), PC(34:2), PC(36:3), PC(40:6), PC(32:0), PC(34:1), PC(36:1). The results indicated that cholesterol lowering effect of rosuvastatin affected membrane fluidity following increase of phospholipase A2 activity which is related to phosphatidylcholine degradation. In conclusion, it was found that our lipidomic methodology and PC retention time index is useful to provide not only overall lipidomic information but also profiled the specific constitution of PC.

P296. PROFILING AND CHARACTERIZATION OF METABOLITES OF [14C]PONATINIB IN PLASMA, URINE, BILE AND FECES OF RATS AFTER ORAL ADMINISTRATION


Yihua Emily Ye, Qurish Mohemmad and Narayana Narasimhan Dmpk, Ariad Pharmaceuticals, Inc, Cambridge, MA Ponatinib (IclusigÕ ) is an oral multi-targeted tyrosine kinase inhibitor (TKI) with potent activity against native BCR-ABL and its mutants. Recently, IclusigÕ received accelerated approval from US FDA for the treatment of adult patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Phþ ALL) who are resistant or intolerant to prior TKI therapy. The objectives of this study were to determine the metabolic profile and to characterize the metabolites of ponatinib. Following an oral dose of 10 mg/kg [14C]ponatinib to male, bile duct-cannulated (BDC) rats and normal (non-cannulated) rats bile, whole blood, urine and feces were collected at several time points/intervals. Total radioactivity (TRA) was determined by direct liquid scintillation counting (LSC) or by combustion followed by LSC. Metabolite profiling and characterization were carried out using LC/MS/MS, and radioactivity analysis of the HPLC eluate. The radioactive dose was excreted mainly in feces (88.4%) and to a much lower extent in urine (10.2%) in normal rats. In BDC rats, 54.4% of the dose was recovered in bile. Ponatinib underwent amide hydrolysis to generate AP24600 (an acid), which was the major radioactive component in rat plasma (50.2%); other drug-related components in rat plasma included AP24600-methyl ester (AP25047, 12.1%) and ponatinib (19.1%). In feces, ponatinib and N-desmethyl ponatinib (AP24567) accounted for 39.9% and 26.1% of the total radioactivity, respectively. Other minor metabolites were AP24600, AP24600 S-methyl ether, and two methylated metabolites contributing 1.2, 1.9, 4.6 and 3.2% to the total fecal radioactivity, respectively. In urine, there was very little ponatinib, with most of the metabolites identified as AP24600 (30.4%) or AP24600 sulfoxide (30.9%) and AP24600-glucuronides (5.3%). In bile, several metabolites were observed with AP24600 (8.4%) and its methyl ester (23.0%) as major components. Overall, fecal elimination via biliary excretion was the major clearance pathway for ponatinib in rats. Approximately 35% of the radioactive dose was excreted as ponatinib in feces. The major metabolic pathways of ponatinib in rats were N-demethylation, methylation, and amide hydrolysis.

P297. AN ACYL GLUCURONIDE STABILITY SCREENING APPROACH IN DRUG DISCOVERY Sara Amberntsson1, Anna Noveń1 and Emre M Isin2 1 Global DMPK, In Vitro In Silico ADME, AstraZeneca R&D, Mo¨lndal, Sweden, 2CVGI iMed DMPK, DMPK Design and Biotransformation, AstraZeneca R&D, Mo¨lndal, Sweden Acyl glucuronide (AG) conjugates are metabolites formed from drug compounds containing carboxylic acids. Reactivity of AGs has been proposed to lead to covalent binding to macromolecules and hence involved in adverse drug reactions1. For this reason it is of importance to determine the stability of AG when carboxylic acid moieties are included in the design of new drug candidates during the drug discovery process. Based on a published model2 we have further developed a Formation-Degradation AG screening assay that is used for estimating the stability of AG conjugates formed from carboxylic acids containing drug candidates. Isomers formed via acyl migration during the degradation phase are separated and analyzed using UPLC-TOFMS. Percent of isomerization is estimated for every incubated time point during a 180 min degradation incubation. Half-life is calculated by fitting a single exponential curve to the percent of isomerization versus time. Hydrolysis of the AG is estimated by calculating the half-life for degradation of the b-1-O-AG isomer. A set of 105 in-house drug candidates and 20 drug compounds is used for examining structure-reactivity relationships for the corresponding AGs. Poor correlation between pKa of the drug molecules and half-life for the corresponding AG indicate that both electronic and steric effects influence the AG stability. However, when the structures of the compounds only differ at the a-carbon, pKa can be used to rank order the stability of AGs. In our data set carboxylic acids that have an electron-withdrawing group attached on the a-carbon form unstable AGs with short half-lives. Compounds containing a benzoic acid moiety mostly form stable AGs. The observation is independent of chemical series (drug project).

References 1. Sophie L. Regan, James L. Maggs, Thomas G. Hammond, Craig Lambert, Dominic P. Williams, B. Kevin Park Acyl Glucuronides: The Good, The Bad and The Ugly Biopharmaceutics & Drug Disposition 31: 367–395 (2010).

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2. Zhesheng Chen, Tom G. Holt, James V. Pivnichny, Kwan Leung A simple in vitro model to study the stability of acyl glucuronides Journal of Pharmacological and Toxicological Methods 55: 91–95 (2007).

P298. BMS-820836: STRUCTURAL ELUCIDATION OF HUMAN CIRCULATING N-GLUCURONIDE METABOLITES


Van T. Ly1, Barry Brock1, Sarah C. Traeger2, Bethanne M. Warrack2, Holly Shackman3, Mark Bolgar3 and Ramaswamy A. Iyer4 1 Biotransformation, Bristol-Myers Squibb, Princeton, NJ, 2Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb, Princeton, NJ, 3Bristol-Myers Squibb, New Brunswick, NJ, 4Biotransformation, Bristol-Myers Squibb Company, Princeton, NJ The metabolism and excretion of [14C]BMS-820836 were evaluated after a single oral administration of [14C]BMS-820836 to six healthy subjects (3 mg, 80 mCi). The observation of 14C drug-related material in excess of parent plasma levels suggested that metabolites contributed to the discrepancy between BMS-820836 plasma levels and total drug-related radioactivity. Radioprofiling of plasma samples showed the presence of two peaks which shared retention times with previously observed direct glucuronic acid conjugated metabolites. To elucidate the structure of these metabolites, they were generated in vitro by incubating BMS-820836 with recombinant UGT1A4, isolated with liquid chromatographic separation and subjected to analyses by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The structure of two out of four metabolites were elucidated as quaternary N-glucuronide metabolites by NMR. The other two minor glucuronide metabolites were proposed to be tertiary Nglucuronides. The structures of these metabolites were further confirmed with a specific LC-MS method that can distinguish between tertiary and quaternary amine glucuronides based on their clustering behavior with trifluoroacetic acid anions. Additionally, the structures were further confirmed by in vitro enzymatic and chemical hydrolysis experiment.

P299. HUMAN ADME STUDIES: A REVIEW OF OUTCOMES AND THE IMPACT OF STUDY DESIGN VARIABLES Iain Shaw1, Lloyd Stevens1, Stuart Mair1, Andrew McEwen2, Gill Ford2 and Stuart G. Wood2 1 Quotient Clinical, Nottingham, United Kingdom, 2Quotient Bioresearch Limited, Rushden, United Kingdom Background: At the 12th European ISSX Meeting in June 2012, McEwen et al1 compared mass balance data from human ADME studies performed pre and post the MIST guidance2. This paper develops the discussion initiated at that time by looking in greater detail at the impact that different aspects of human ADME study design have on mass balance recovery. Aims: This paper reviews the mass balance data obtained from human ADME studies performed by Quotient post the MIST guidance and looks specifically at the impact of dose route, formulation, amount of radioactivity administered, duration of collection period and route of excretion on mass balance recovery. Methods: Data are collated from human mass balance studies conducted at Quotient from 2010 to 2013. Results: Data from 420 completed studies each with different drug products and with many different modes of action will be presented and reviewed. Summary/Conclusion: The impact of different variables in study design on mass balance recovery will be presented. Conclusions on the requirements to address these impacts when designing a human mass balance study will be discussed.

References 1. Human ADME studies: A post-MIST analysis, McEwen et al. (2012). 2. US FDA guidance for industry. Safety testing of drug metabolites (2008).

P300. STABILITY OF GLUTATHIONE ADDUCTS – INFLUENCE ON THE ASSESSMENT OF THE BIOACTIVATION POTENTIAL OF NCES AT EARLY STAGE OF DRUG DEVELOPMENT Joachim Blanz1, Philippe Ramstein2, Werner Gertsch2, David Pearson2, Thierry De´le´monte´2, Jerome Dayer2, Reiner Aichholz2, Grit Laue2, Claudia Betschart3, Samuel Hintermann3, Dirk Behnke3 and Silvio Ofner3 1 Metabolism and Pharmcokinetics, Novartis Institutes for Biomedical Research, Basel, Switzerland, 2Metabolism & Pharmacokinetics, Novartis Institutes for Biomedical Research, Basel, Switzerland, 3Global Discovery Chemistry, Novartis Institutes for Biomedical Research, Basel, Switzerland Reactive metabolite trapping with glutathione (GSH) in incubations with liver microsomes, screening for GSH-related metabolites in hepatocytes or in samples obtained from the bile-duct cannulated rat model are considered to be valuable screening methods during lead optimization. Substances that form GSH adducts are often deprioritized since reactive intermediate formation is seen as a risk factor for the formation of covalent protein adducts of essential cellular targets.


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Carcinogenesis, potential risk for clinical toxicities or idiosyncratic adverse drug reactions are discussed as consequences of bioactivation. Mass spectrometric (MS) detection using electrospray ionization (ESI) is the current method of choice for structural characterization and semi-quantitative assessment of GSH adducts of unlabeled drug candidates. It is well-known that GSH adducts may be subject to peptide bond cleavage by y-glutamyltranspeptidase and dipeptidase(s) to form the corresponding cysteine conjugates in cell based and in in vivo assays. However, the lack of enzymes necessary for degradation of GSH adducts in incubations with cellular fractions may lead to the assumption that these adducts are stable during sample workup and analysis. During the investigation of GSH adduct formation of indole derivatives it was found that some adducts are unstable even in liver microsomal incubation samples stored at 80 C. GSH adducts clearly detected in a first analysis were not detectable any more after re-thawing of the same sample. An investigation was performed to assess the influence of workup and analysis conditions. A hypothesis for the metabolic pathways that led to the formation of these adducts will be provided. The currently used methodology that is widely used in early pharmaceutical research to support the safety assessment of drug candidates can hardly take into account that trapping with nucleophilic agents may lead to adducts with short in vitro or in vivo half-lifes. Sample stability assessment is impossible in a setup that intends ‘‘high-throughput’’ metabolism investigations. The combination of chemical instability, difficult chromatography that often leads to broad peak profiles and a low response factors of GSH adducts in ESI detection may lead to significant underestimation of the formation of these reactive intermediates. This may lead to arbitrary or even false-negative results with respect to bioactivation.

References 1. A S Kalgutkar, J R Soglia, Minimising the potential for metabolic activation in drug discovery. Expert Opin. Drug Metab. Toxicol. 1(1): 91–142 (2005) 2. U A Argikar et al., Strategies and chemical design approaches to reduce the potential for formation of reactive metabolic species. Current Topics in Medicinal Chemistry 11: 419–449 (2011)

P301. DRIED MICROSOMAL SPOT (DMS): EXPLORING THE FEASIBILITY OF MICRO VOLUME SAMPLING AND DRIED BLOOD SPOT TECHNIQUE FOR IN-VITRO MICROSOMAL METABOLIC STABILITY ASSAYS Raghava Chaudary Palacharla, Vishwottam Kandikere, Rajesh Kumar Boggavarapu, Vijaya Bhargava Kanamarlapudi, Gopinadh Bhyrapuneni and Ramakrishna Nirogi Discovery Research, Suven Life Sciences Ltd, Hyderabad, India Dried Blood Spot (DBS) technique is a form of biosampling where blood samples are blotted and dried on filter paper. The DBS technique was actively evaluated as replacement for plasma in pharmacokinetic / toxicokinetic studies and is gaining importance in pharmaceutical industry 1, 2, 3. DBS is known for its advantages like low sampling volume, reduction in sample storage space and shipment costs4. The objective of the study is to explore the feasibility of using the DBS technique to metabolic stability studies where a low volume of microsomal sample was spotted on DBS cards. Microsomal metabolic stability studies were routinely carried high throughput in drug discovery to rank order the compounds that will go through animal testing for determination of pharmacokinetic parameters. A total of 15 compounds were selected with low, moderate and high degree of metabolism and evaluated for their stability in human liver microsomes. Microsomal incubations were prepared by mixing human liver microsomes (0.5 mg/mL) and test compound (2.5 mM) in phosphate buffer (100 mM, pH 7.4). The reactions were initiated by NADPH. At predefined time points, for analysis of metabolic stability samples using dried spot technique, an aliquot of the sample was spotted on DBS cards (FTA, DMPK-C). An equal volume of quenching reagent was added immediately on to the spot to terminate the reaction. The cards were dried and stored at ambient temperature. The cards were analyzed using the procedure described elsewhere5. For analysis of metabolic stability samples using 96-well plate method, an aliquot of the sample was quenched with acetonitrile followed by centrifugation and analyzing the supernatants. Half life and intrinsic clearance values were calculated and were compared between the two methods of analysis. The results indicate that there is a good correlation between the intrinsic clearance values obtained between the two methods of analysis. The results also showed that the use of dried spot technique, besides its own advantages, can increase the number of compounds that can be screened in a single vial of microsomes. The results and feasibility of applying the dried spot technique to low, moderate and highly metabolized compounds will be discussed.

References 1. Patel P, Mulla H, Tanna S, Pandya H. Facilitating pharmacokinetic studies in children: a new use of dried blood spots. Arch Dis Child, 2010, 95(6): 484–487 2. Neil Spooner, Rakesh Lad and Matt Barfield. Dried blood spots as a Sample Collection Technique for the Determination of Pharmacokinetics in Clinical Studies: Considerations for the validation of a Quantitative Bioanalytical Method. Anal Chem, 2008, 81 (4), 1557–1563

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3. Malcolm Rowland and Gary T Emmons Use of Dried Blood Spots in Drug Development: Pharmacokinetic Considerations, AAPS J, 2010;12(3):290–293. 4. Peter van Amsterdam and Clare Waldrop, The application of dried blood spot sampling in global clinical trials, Bioanalysis, 2010, 2(11): 1783–1786. 5. Nirogi R, Ajjala DR, Kandikere V, Aleti R, Srikakolapu S, Vurimindi H. Dried blood spot analysis of iron chelatorDeferasirox and its potential application to therapeutic drug monitoring. J Chromatogr B, 2012, 907; 65–73.

P302. METHODS FOR IMPROVING IN VITRO PREDICTION OF INTRINSIC CLEARANCE UTILISING 3D CELL CULTURE MODELS


Phil Butler, Katie Fox and Joanne Shaw Cyprotex, Macclesfield, United Kingdom Accurate assessment of low clearance compounds is a particular challenge within drug discovery. Short term metabolism studies in microsomes or suspensions of hepatocytes are limited by assay sensitivity for low clearance compounds resulting in a large variability in estimated intrinsic clearance (CLint). Plated monolayer cultures of cryopreserved human hepatocytes have been proposed as more suitable models for more accurate determination of CLint of low clearance compounds, allowing for longer incubation times. 3D cell culture models are in use within the industry for a range of biological applications and may also potentially provide a useful tool for improving CLint prediction. The aim of this work was to assess the potential application of 3D cellular models for prediction of CLint for low clearance compounds, in comparison to 2D monolayer cultures of plated cryopreserved hepatocytes. The 3D cellular models utilised were InSphero’s 3D InSightÔ Human Liver Microtissues and Reinnervate’s AlvetexÕ Scaffold with cryopreserved human hepatocytes. The following compounds were screened for metabolic stability in all 3 systems: diazepam, tolbutamide, warfarin, propranolol, antipyrine, caffeine and verapamil. Compounds were incubated in duplicate at 1 mM with each cell system for two differing incubation periods; up to 24 h (0, 1, 2, 4, 8, 24 h), and up to 72 h (0, 2, 8, 24, 48, 72 h); to assess the potential application of 3D cell cultures in accurate prediction of CLint for low clearance compounds. At the end of each incubation time, supernatant was removed into pre-chilled methanol to precipitate the protein. The viability of each cell system was measured over the incubation period using a Cell Titer-GloÕ Luminescent cell viability assay. Substrate depletion of each compound was monitored by LC-MS/MS. Data generated from these assays were then utilised in the prediction of in vivo human CLint to assess the suitability of each system in determining accurate CLint of low clearance compounds.

P303. PHARMACOKINETICS OF ISOLIQUIRITIGENIN AND ITS METABOLITES IN RATS: LOW BIOAVAILABILITY IS PRIMARILY DUE TO THE HEPATIC AND INTESTINAL METABOLISM VIA GLUCURONIDATION Young-Won Chin, Young Hee Choi, Yu Kyung Lee, You Jin Kim and Jin Kyung Bae College of Pharmacy, Dongguk University, Goyang, South Korea Isoliquiritigenin (isoLQ), a chalcone found in licorice has shown a variety of biological activity including antioxidative, antiinflammatory, estrogenic, chemopreventive and antitumor effects. Thus, pharmacokinetics of isoLQ and its metabolites [liquiritigenin (LQ), glucuronidated isoLQ (M1) and glucuronidated LQ (M2)] after intravenous and oral administration of isoLQ was evaluated in rats. The pharmacokinetics of isoLQ, LQ, M1 and M2 showed dose-independent manner after both intravenous and oral administration of isoLQ. Although approximately 88.7% of the oral isoLQ was absorbed, F value was only 11.3% of oral dose. The low F value of isoLQ might be due to considerable metabolism of isoLQ in liver and small intestine. It was supported that isoLQ was disappeared and M1 and M2 were formed mainly in S9 fractions of liver and small intestine. Also the affinities of isoLQ, LQ, M1 and M2 were high in liver, small intestine, large intestine and/or kidney. This study was supported by GRRC program of Gyeonggi province (GRRC-DONGGUK2013-B03), Biological Applications of New Therapeutic Target Modulators and the grant of National Research Foundation of Korea (NRF) funded by the government of Korea.

P304. CYP2C8 IS A PREVIOUSLY UNREPORTED IMPORTANT CONTRIBUTOR TO OLANZAPINE OXIDATIVE METABOLISM Porntipa Korprasertthaworn1, Andrew J. McLachlan2, Thomas M. Polasek1, Geoffrey T. Tucker3, John O. Miners1 and Andrew Rowland1 1 Clinical Pharmacology, Flinders University, Adelaide, Australia, 2Faculty of Pharmacy, University of Sydney, New South Wales, Australia, 3University of Sheffield and Simcyp Ltd, Sheffield, United Kingdom


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Olanzapine (OLZ; 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-B][1,5]benzodiazepine) is an atypical antipsychotic used to treat schizophrenia and related psychoses. The clinical utilisation of OLZ is associated with large inter-individual variability in OLZ pharmacokinetics, which leads to variability in response, frequently resulting in poor compliance due to either a lack of efficacy or drug-related adverse effects. The metabolism of OLZ is complex and relatively poorly understood. Although UDP-glucuronosyltransferase 1A4 (UGT1A4), cytochrome P450 1A2 (CYP1A2), and flavin-containing mono-oxygenase 3 (FMO3) have been shown to contribute to glucuronidation and to the oxidative metabolic pathways, respectively, to date no study has screened a full panel of drug metabolising enzymes for activity toward OLZ. Indeed, most studies have focused on the role of CYP1A2 (and its polymorphic variants) in determining variability in OLZ response. However, these studies found no association between OLZ disposition and CYP1A2 phenotype. CYP3A4 is also known to contribute to the oxidative metabolism of OLZ. This enzyme shares significant overlap substrate selectivity with CYP2C8, but no study has reported the contribution of the latter enzyme to OLZ oxidative metabolism. In addition, CYP2C8 polymorphisms are known to result in variability in substrate pharmacokinetics for many clinically used drugs (e.g. rosiglitazone, pioglitazone, and repaglinide). Thus, the aim of this study was to screen a full panel of drug metabolising enzymes (CYPs, UGTs, and FMO), with a particular focus on elucidating the contribution of CYP2C8 to OLZ oxidative metabolism. An Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) method was developed and validated to quantify OLZ, its four oxidative metabolites (N-desmethyl-, 2-hydroxy-, 7-hydroxy-OLZ, and OLZ-N-oxide), and OLZ 10-N-glucuronide. Currently, screening experiments have been performed with recombinant human CYP 1A1, 1A2, 2C8, 2C9, and 3A4, UGT1A4, and FMO3. Consistent with previous reports, UGT1A4, CYP1A2, and FMO3 were found to catalyse the formation of OLZ 10-N-glucuronide, 7-hydroxy-OLZ, and OLZ-N-oxide, respectively. In addition, a previously uncharacterised contribution of CYP2C8 to OLZ N-demethylation was demonstrated. For incubations conducted in the presence of bovine serum albumin (BSA; 2% w/v), Km values for OLZ N-demethylation by CYP1A2 and CYP2C8 were 66 7 and 61 20 mM, respectively, while Vmax values were 2.3 0.3 and 1.3 0.1 pmol/min/ pmol P450, respectively. The SimCYP population-based simulator was used to predict the in vivo clearance of OLZ from the in vitro albumin adjusted kinetic data. Simulation of OLZ clearance, particularly CYP-mediated oxidative pathways, demonstrated a 32% contribution of CYP2C8 to CYP-mediated OLZ oxidative metabolism. These data indicate that CYP2C8 contributes to OLZ oxidative metabolism through catalysis of OLZ N-demethylation. As significant correlation between N-desmethyl OLZ metabolic ratio with OLZ clearance has been reported. It is plausible that inter-individual variability in CYP2C8 expression and activity may contribute to the inter-individual variability in OLZ disposition and response.

P305. THE CONTRIBUTION OF HUMAN UDP-GLUCURONOSYLTRANSFERASE ENZYMES TO THE GLUCOSIDATION OF MYCOPHENOLIC ACID Nuy Chau, Peter I. Mackenzie and John O. Miners Clinical Pharmacology, Flinders University, Adelaide, Australia Mycophenolic acid (MPA) has been reported to be mainly glucuronidated by UGT1A9, but the enzyme(s) responsible for MPA glucosidation, which occurs in humans in vivo, remains unknown. Recent data from this laboratory has shown that UGT2B7 catalyzes both the glucuronidation and glucosidation of morphine. By anology with morphine, we hypothesised that UDPglucuronosyltransferase (UGT) enzymes catalyse MPA glucuronidation and glucosidation. Thus, the aim of the project was to characterize the kinetics of MPA glucosidation and glucuronidation by human liver microsomes (HLMs) and to identify the human UGT enzyme(s) capable of glucosidating MPA. The formation of MPA phenolic glucuronide (MPAGlcUA), MPA phenolic glucoside (MPAGlc), MPA acyl glucuronide (AcMPAGlcUA) and MPA acyl glucoside (AcMPAGlc) from incubations with microsomes from four livers and recombinant UGT enzymes (expressed in HEK293 cells) supplemented with the appropriate cofactor, UDP-glucuronic acid (UDP-GlcUA) or UDP-glucose (UDP-Glc), was quantified by HPLC with UV detection. MPAGlcUA, AcMPAGlcUA and AcMPAGlc formation by HLM followed Michaelis-Menten kinetics with average intrinsic clearances (CLint) of 45.5 9.8 mL/min/mg, 8.4 1.3 mL/min/mg and 0.73 0.08 mL/min/mg, respectively. MPAGlc formation exhibited either Michaelis-Menten kinetics or weak substrate inhibition, with an average CLint of 5.0 2.4 mL/min/ mg. MPAGlcUA was formed only by UGT1A family enzymes. In comparison, both UGT1A family enzymes and UGT2B7 formed MPAGlc although highest activity was observed for UGT1A9. UGT1A10 and UGT2B7 were the main enzymes responsible for the formation of AcMPAGlcUA. UGT2B7 was the only enzyme found to convert MPA to AcMPAGlc. MPAGlcUA and MPAGlc formation by recombinant UGT1A9 and AcMPAGlcUA formation by recombinant UGT2B7 followed Michaelis-Menten kinetics with CLint values of 56 12.6 mL/min/mg, 4.8 1.8 mL/min/mg and 0.27 0.03 mL/min/mg, respectively. Overall, the major hepatic enzyme involved in the glucosidation of MPA is UGT1A9. Like morphine, the glucosidation and glucuronidation of MPA occur as parallel metabolic pathways although these reactions were catalysed by several enzymes from the UGT1A family.

P306. CHARACTERIZATION OF THE BIOTRANSFORMATION OF THE HIV NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR RILPIVIRINE Julie M. Lade and Namandje´ N. Bumpus Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD


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Rilpivirine, 4-[[4-[[4-[(1E)-2-cyanoethenyl]-2,6-dimethylphenyl]amino]-2-pyrimidinyl]amino]benzonitrile, is a non-nucleoside reverse transcriptase inhibitor used to treat HIV-1; however, the biotransformation of rilpivirine has yet to be reported. Using human liver microsomes, two mono- and two di-hydroxylated metabolites were identified by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Fragment ions of these metabolites suggested oxygenation of the 2,6dimethylphenyl ring and methyl groups. The identities of these metabolites were confirmed using a rilpivirine analogue deuterated at both methyl groups. cDNA-expressed cytochromes P450 (CYPs) and chemical inhibition studies indicated that CYP3A4 and CYP3A5 were responsible for forming the oxygenated metabolites. Additionally, 24h rilpivirine treatment of primary human hepatocytes resulted in a maximal 28.4-fold increase of CYP3A4 mRNA. UDP-glucuronosyltransferases (UGTs), UGT1A1 and UGT1A4, contributed to O-linked glucuronidation of the more abundant monohydroxymethyl metabolite and N-linked glucuronidation of rilpvirine on the pyrimidine ring, respectively. All hydroxylated and glucuronide metabolites were detected in plasma and urine from patients receiving rilpivirine as part of an antiretroviral drug regimen. UPLC-MS-based targeted metabolomics screening of these clinical samples indicated that arginine levels may be decreased while citrate levels may be increased in plasma of rilpivirine-treated subjects. In addition, increased citrate and adenosine urine levels were observed in these patients. Metabolism assays were also performed using liver microsomes isolated from a range of mammals. Of these, only Golden Syrian hamster liver microsomes formed metabolites in equal or greater abundance to human liver microsomes. The modes of rilpivirine metabolism have been determined in this present study, and these data are expected to inform clinical use of rilpivirine. Further, these studies provide novel insight into the potential impact of rilpivirine therapy on the endogenous metabolome.

P307. BIOTRANSFORMATION OF BISPHENOL S BY HUMAN AND RAT SUBCELLULAR FRACTIONS: CHARACTERIZATION OF THE CYP450 ISOZYMES INVOLVED Elisabeth F. Perdu1, Sandrine Bruel2 and Daniel Zalko2 1 TOXALIM (Research Center in Food Toxicology), INRA (French National Institute for Agricultural Research), Toulouse, France, 2Toxalim, INRA (French National Institute for Agricultural Research), Toulouse, France Bisphenols, which are widely used for the manufacture of polycarbonates and epoxy resins, can be released into the environment and contaminate foodstuffs1. Some have been demonstrated to generate disorders in animals and are suspected endocrine disruptors in human. Bisphenol S [4,40 -Sulfonyldiphenol] (BPS) is used in fast-drying epoxy glues and as an anticorrosive. BPS shares estrogen-mimicking properties with bisphenol A (BPA), and is increasingly used as an alternative to BPA in plastic consumer products and thermal papers2. Recent works have demonstrated that BPS is already present in the environment, in paper products and in the human body3. Thermal receipt papers have been suggested to be the major sources of human exposure to BPS4. In the present work we investigated the in vitro biotransformation of radio-labelled BPS using rat and human liver subcellular fractions (microsomes and S9 fractions) as well as a range of human recombinant CYP450 isozymes. BPS metabolites were separated and quantified by high-performance liquid chromatography coupled to radioactivity detection (R-HPLC), and kinetic studies were carried out. Metabolites were further isolated by HPLC, and analysed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Many of these metabolites were characterized for the first time in rodents and in humans. BPS was metabolised into the corresponding glucuronide (liver S9 fractions). In addition to this phase II biotransformation product, hydroxylated metabolites were formed. Phase I metabolic pathways predominated in incubations performed using liver microsomes but were also involved in incubations performed with liver S9 fractions. The formation of the main metabolite, namely hydroxylated BPS, was P450 dependent. The role of individual human liver microsomal P450 enzymes in the oxidative metabolism of BPS was further investigated using bactosomes containing recombinant human cytochromes P450 enzymes (1A1, 1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 4A11) coexpressed with NADPH-cytochrome P450 reductase. P450 2C19 was the most effective regarding BPS oxidation. P450 3A4, 2C18, 2C8 and 2C9 also catalyzed BPS, but at a lesser extent. P450 2C19 was previously shown to be involved in the oxidation of others bisphenols (BPA, BPF). Additional studies are underway to explore the biological activity of BPS metabolites.

References 1. Liao C., Kannan K. (2013). Concentrations and profiles of bisphenol A and other bisphenol analogues in foodstuffs from the United States and their implications for human exposure. J Agric Food Chem. 61(19):4655–62 2. Liao C., Liu F., Kannan K. (2012). Bisphenol S, a new bisphenol analogue, in paper products and currency bills and its association with bisphenol A residues. Environ Sci Technol. 46(12):6515–22. 3. Liao C., Liu F., Alomirah H., Loi VD., Mohd MA., Moon HB., Nakata H., Kannan K. (2012). Bisphenol S in urine from the United States and seven Asian countries: occurence and human exposures. Environ Sci Technol. 46(12):6860–66. 4. Konkel L. (2013). Thermal reaction: the spread ob bisphenol S via paper products. Environ Health Persp. 121(3): A76.

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P308. C-10068: A NOVEL SIGMA AGONIST MORPHINAN WITH A SUPERIOR METABOLISM AND PHARMACOKINETIC PROFILE


Vinita Uttamsingh, Sophia Nguyen, Gary Bridson, Adam Morgan and Phil Graham Concert Pharmaceuticals, Inc., Lexington, MA C-10068 is a selectively deuterium-modified analog of dextroETHorphan which is closely related to dextromethorphan (DM), an anti-tussive agent and active ingredient in NuedextaÕ . C-10068 has complex pharmacological actions at several neurologically relevant receptors which appear generally similar to those of DM, although C-10068 is a more potent sigma agonist. C-10068 has shown anticonvulsant properties and evidence of neuroprotective, anti-nociceptive and anti-depressant effects which suggest potential as a therapeutic agent for the treatment of pain, depression, seizures, and traumatic brain injury. DM is rapidly metabolized by CYP2D6 resulting in low and highly variable oral bioavailability and formation of the psychotropic O-desmethyl metabolite, dextrorphan (DX). DX has been associated with undesirable effects which may be due to antagonism at the NMDA receptor. The purpose of this investigation was to characterize the in vitro metabolism and nonclinical pharmacokinetic profile of C-10068. The comparison of the amounts of DX formed from DM, dextroethorphan and C-10068 (which forms partially deuterated DX) was of particular interest. The metabolic stability of C-10068 was determined in human liver microsomes (HLM) and CYP2D6 supersomes and compared with that of dextroethorphan and DM. The in vitro half-life for C-10068 was 2-fold longer than that for dextroethorphan and DM in both HLM and CYP2D6 supersomes. The CYP450 profile of all three compounds was determined in HLM using CYP450-selective chemical inhibitors. The CYP450 profile of C-10068 was similar to that of dextroethorphan and DM in that CYP2D6 formed DX from all three compounds. However, the amount of DX formed from C-10068 was substantially lower than that formed from dextroethorphan and DM. The kinetic parameters, Km and Vmax, for the formation of DX from DM, dextroethorphan, and C-10068 were determined in HLM. The intrinsic clearance, Vmax/Km, for DX formed from C-10068 was about 2-fold lower than that formed from DM. The pharmacokinetic profile of all three compounds was investigated in male Sprague-Dawley rats following administration of a 30 mg/kg oral dose. The oral exposure, Cmax and AUC, of C-10068 was 3-fold greater than that of dextroethorphan and DM in rats. In conclusion, C-10068 is a deuterated new chemical entity in the morphinan class and has superior metabolism and pharmacokinetic properties compared to dextroethorphan and the approved drug DM.

P309. DEUTERATED ISOTOPOLOGS OF IVACAFTOR HAVE IMPROVED METABOLISM AND PHARMACOKINETIC PROPERTIES Sophia Nguyen, Gary Bridson, Adam Morgan, Richard Gallegos, Scott Harbeson, Lijun Wu and Vinita Uttamsingh Concert Pharmaceuticals, Inc., Lexington, MA Ivacaftor (KalydecoÕ ) is an approved therapeutic agent for the treatment of cystic fibrosis (CF) in patients with the G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Ivacaftor is extensively metabolized by CYP3A4 in humans and hence is administered at a dose of 150 mg twice daily (300 mg/day) to patients with CF. The primary site of metabolism of ivacaftor is on the tert-butyl adjacent to the hydroxyl group of the phenol leading to the formation of hydroxymethyl ivacaftor (M1) and ivacaftor carboxylate (M6). In humans, the plasma levels of M1 and M6 are substantially greater than that of the parent drug. Deuterium isotope effects are often unpredictable; therefore, Concert prepared and tested the pre-clinical metabolism and pharmacokinetic (PK) properties of multiple deuterium-modified isotopologs of ivacaftor. The isotopologs were deuterated at one (D9 or D90 ) or both (D18) of the tert-butyl groups present on ivacaftor. The metabolic stability of ivacaftor and the three deuterated isotopologs were compared in human CYP3A4 supersomes. The in vitro half-life (t1/2) of the compounds deuterated at the primary site of metabolism (D9 and D18) was approximately 50% longer than that of ivacaftor while the t1/2 of the D90 isotopolog was similar to that of ivacaftor. The metabolite profile of ivacaftor, D9- and D18-ivacaftor was examined in human hepatocytes. Semi-quantitative HPLC-UV analysis allowed detection of M1 in the supernatants from the human hepatocyte incubations. The relative amount of M1 formed from the two deuterated ivacaftor isotopologs was approximately half the amount formed from ivacaftor. The in vivo PK profile of ivacaftor, D9- and D18-ivacaftor was assessed and compared in the beagle dog. Male, beagle dogs were administered a 3 mg/kg oral, discrete dose of each compound in a cross-over study. The exposure parameters, Cmax, AUC0–24hr and C24hr for the deuterated compounds were enhanced by about 30–60%, 40–70%, and 40–80%, respectively, compared to ivacaftor. Surprisingly, the exposure of D9-ivacaftor was higher than that of D18-ivacaftor in the dogs despite similar stabilization of the two deuterated compounds in human CYP3A4 supersomes. The metabolite profile in dog plasma showed a reduction in the relative amount of M1 when the dogs were administered both the deuterated compounds compared to ivacaftor. In summary, deuteration of ivacaftor at the major site of biotransformation stabilized it to metabolism in human in vitro systems and greatly improved the PK properties of the deuterated isotopologs in dogs. This enhanced metabolism and pharmacokinetic profile of deuterated ivacaftor may ultimately allow for a more convenient or more effective CFTR potentiator.

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P310. HAPLOTYPE VARIATIONS INFLUENCE HUMAN GSTZ1 GENE EXPRESSION AND THE KINETICS OF THE ANTI-TUMOR DRUG DICHLOROACETATE (DCA)


Taimour Langaee1, Caitrin W. McDonough1, Albert L. Shroads2, Peter W. Stacpoole3 and Margaret O. James4 1 Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL, 2Department of Medicine, University of Florida, Gainesville, FL, 3Medicine, University of Florida College of Medicine, Gainesville, FL, 4Medicinal Chemistry, University of Florida, Gainesville, FL The zeta-1 family isoform of GST biotransforms DCA and certain other halogenated xenobiotics. As maleylacetoacetate isomerase (MAAI), this bi-functional enzyme also catalyzes the penultimate step in tyrosine metabolism. The most common human GSTz1/MAAI haplotypes EGT, KGT, EGM, and KRT, are inferred from the following 3 SNPs rs7975 (E32K), rs7972 (G42R), and rs1046428 (T82M). EGT is the most frequent haplotype (50%), followed by KGT (30%), EGM (15%), KRT (5%) and KGM (0.4%). The influence of GSTZ1/MAAI haplotype on DCA kinetics was recently examined in healthy adult subjects (1). Carriers of at least one EGT allele generally showed faster DCA plasma clearance and build-up of certain tyrosine intermediates, compared to individuals not carrying this haplotype. We tested the hypothesis that the presence of a lysine (K) amino acid in the KGT haplotype reduces the expression of GSTZ1. The influence of KGT and EGT haplotypes on GSTZ1/ MAAI expression was determined by analyzing 44 liver samples from subjects older than age of seven of various racial/ethnic backgrounds (2). Expression data were analyzed based on the presence or absence of K. We found that the difference in GSTZ1/ MAAI expression between KGT and EGT carriers was significant (p ¼ 0.017) and was even more striking when samples obtained from African-Americans were excluded from the analysis (p ¼ 0.005). When analyzed alone, there was no difference in GSTZ1/MAAI expression between KGT and EGT carriers in the African-American group. We attribute this difference in GSTZ1/MAAI expression among KGT haplotype carriers in Caucasians, Asians and Hispanics to the linkage disequilibrium (LD) between the K or A allele from the G4A SNP (rs7975) with the promoter G4A -1002 SNP (rs7160195) A allele. The A allele of this promoter SNP was shown to cause a lower transcription level of GSTZ1/MAAI gene (3). There is no LD between these two polymorphisms in African-Americans. We conclude that the lower expression of GSTZ1/MAAI in Caucasians, Hispanic and Asians who possess the KGT haplotype results in lower enzymatic activity and slower metabolism of DCA compared to those who possess the EGT haplotype. The data also suggest that tyrosine metabolism may be slower in non-EGT individuals, leading to accumulation of potentially toxic tyrosine intermediates.

References 1. Shroads AL, Langaee T, Coats BS, Kurtz TL, Bullock JR, Weithorn D, Gong Y, Wagner DA, Ostrov DA, Johnson JA, Stacpoole PW. Human polymorphisms in the glutathione transferase zeta 1/maleylacetoacetate isomerase gene influence the toxicokinetics of dichloroacetate. J Clin Pharmacol. 2012 Jun;52(6):837–49. 2. Li W, Gu Y, James MO, Hines RN, Simpson P, Langaee T, Stacpoole PW. Prenatal and postnatal expression of glutathione transferase 1 in human liver and the roles of haplotype and subject age in determining activity with dichloroacetate. Drug Metab Dispos. 2012 Feb;40(2):232–9. 3. Fang YY, Kashkarov U, Anders MW, Board PG. Polymorphisms in the human glutathione transferase zeta promoter. Pharmacogenet Genomics. 2006 May;16(5):307–13.

P311. IN VIVO RAT METABOLISM OF AN OBESITY DRUG BELORANIB Heasook Kim-Kang1, Yijun Yi2, Philip Inskeep3 and James Vath4 1 Drug Metabolism and Biotransformation, XenoBiotic Laboratories, Inc., Plainsboro, NJ, 2XenoBiotic Laboratories, Inc., Plainsboro, NJ, 3Inskeep DMPK, East Lyme, CT, 4Zafgen, Inc., Cambridge, MA Beloranib, an analog of the natural chemical compound fumagillin, is an experimental drug candidate for the treatment of obesity. Sprague Dawley rats, both intact and bile duct-cannulated (BDC), were administered a single IV dose of 4 mg/kg (200 mCi/kg) of [14C]beloranib. Blood, plasma, urine, feces, and bile were collected for mass balance, metabolite profiling, and identification. Within 24 hours, approximately 82% and 87% of the dosed radioactivity was recovered in intact male and female rats, respectively. Total recoveries were 92.2% and 95.6%, respectively. From intact male and female rats, 67% and 60% of the dosed radioactivity was excreted in the feces, with less found in urine (19% and 30%), respectively. In BDC rats; ca. 60% of the dosed radioactivity was found in bile within 72 hrs. Metabolite profiles were determined by radio-HPLC and LC/FT-MS was used for identification of metabolites. Unchanged beloranib was detected at negligible levels in both bile and urine, accounting for 51% of the administered dose. Glutathione conjugates and subsequent degradation products accounted for the majority of radioactivity in bile with 34% (males) and 35% (females) of the dose derived from this pathway. Fumagillol-related metabolites accounted for 8% and 7% of the dose in males and females, respectively. A small amount of the N-oxide of beloranib was also observed in bile. Very little radioactivity derived from the GSH pathway was recovered in urine (3–4% of the dose). The major metabolites in urine were fumagillol-related metabolites, accounting for 7% of the dose in males and 13% of the dose in females, respectively. Beloranib was a prominent circulating radioactive component in rat plasma, accounting for ca.10% of the total

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radioactivity in 0–48-hr rat plasma samples. The highest proportion of radioactivity in plasma of rats (ca. 30%) was fumagillolrelated metabolites. The metabolic fate of dimethylaminoethanoxyl cinnamic acid, the non-radiolabeled portion of the hydrolysis products of beloranib, was evaluated and the results indicated that it and each of its metabolites account for510% of dose in bile or urine. Overall, beloranib cleared mainly by metabolism and biliary excretion of metabolites in rats. The major metabolic pathways include glutathione conjugation-derived metabolites and fumagillol-derived metabolites. No single major metabolite represented 410% of the dose except for one (10%) detected in feces. Some non-extractable radioactivity was observed in later time-point (448-hr post-dose) liver, however, it accounted for less than 0.15% of the administered dose.

P312. IN VITRO AND IN VIVO INHIBITION OF N-OXIDATION AND ITS ASSOCIATION WITH HEPATOTOXICITY IN DOG


Anthony J. Lee1, Jill Wetter1, Kennan Marsh1, Lise Loberg2 and William Bracken2 1 Drug Metabolism and Pharmacokinetics, AbbVie, North Chicago, IL, 2Preclinical Safety, AbbVie, North Chicago, IL ABT-288, a cyclic tertiary amine-containing compound, was metabolized primarily to its N-oxide in dog liver microsomal preparations. Assays using recombinant human Cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) isoforms showed that N-oxidation of ABT-288 was mediated by FMO1 and FMO3. In dogs in vivo, N-oxide/ABT-288 ratios in Cmax and AUC were over 10 following an oral administration of ABT-288, indicating N-oxidation is a major metabolism pathway in vivo. Elevated levels of liver specific transaminase enzymes such as ALT and AST were observed following administration of ABT288. The objective of current study was to investigate if the liver enzyme changes were caused by ABT-288 or its N-oxide. Experimentally, N-Oxide metabolite was administered in dog and N-oxide/parent amine ratios were determined. In addition, ABT-288 was incubated in dog liver microsomes in the presence or absence of methimazole, a non-specific FMO inhibitor and 1-aminobenzotriazole, a non-specific CYP inhibitor. To inhibit in vivo N-oxidation, methimazole was co-administered with ABT-288 and pharmacokinetic parameters and liver specific enzyme levels were determined in dogs. When N-oxide metabolite was administered in dog, N-oxide/parent amine ratios in Cmax and AUC were similar to the ratios observed following administration of ABT-288, suggesting reduction of N-oxide back to parent drug occurs in vivo. Following incubations of ABT288 in dog liver microsomes in the presence or absence of methimazole and/or 1-aminobenzotriazole, N-oxidation of ABT-288 was inhibited by methimazole in dose dependent manner, suggesting FMOs mediate the N-oxidation of parent compound in dog. Following oral administration of ABT-288 at 2 mg/kg with methimazole (20 mg/kg BID), AUC of parent drug was greater than that observed following ABT-288 at 20 mg/kg dose without methimazole, while AUC of N-oxide was 5% of 20 mg/kg dose or 21 25% of 2 mg/kg dose without methimazole. Only minimal changes in ALT and AST values were noted following oral administration of ABT-288 at 2 mg/kg with methimazole (20 mg/kg BID). Taken together, the results of current study suggest that the changes in liver transaminases following oral administration of ABT-288 in dogs are not due to the concentrations of parent drug, but are likely due to the concentrations of the N-oxide metabolite.

P313. DISPOSITION AND METABOLISM OF [14C]DABRAFENIB, A RAF KINASE INHIBITOR, IN MICE, RATS, DOGS AND HUMANS Lauren E. Richards-Peterson1, Thomas C. Wilde1, Donna B. Mamaril-Fishman1, David A. Bershas1, Molly Z. Karlinsey1, Ernest M. Schubert1, Stephen Castellino2, Daniele Ouellet3 and Peter D. Gorycki1 1 PTS Dmpk, GlaxoSmithKline, King of Prussia, PA, 2PTS Dmpk, GlaxoSmithKline, Research Triangle Park, NC, 3Cpms, GlaxoSmithKline, Research Triangle Park, NC Dabrafenib (TAFINLARÔ, GSK2118436, N-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzene sulfonamide) is an orally bioavailable, potent and selective RAF kinase inhibitor. The RAS/RAF/ MEK/ERK pathway is a critical proliferation pathway in many human cancers and BRAF mutations have been identified in approximately 40 to 60% of melanoma. The objective of these studies was to characterize the metabolism and disposition of [14C]dabrafenib following a single oral administration to mice, rats, dogs and humans. Blood, urine (rats, dogs and humans only), bile (rat only), and feces (rats, dogs and humans only) were collected for at least 96 hours post dose. Liquid scintillation counting was used to measure radioactivity in plasma, urine, bile and feces. Radio-HPLC was used to profile and quantify metabolites. The structures of metabolites were elucidated by LC/MS/MS. These studies demonstrated that the biotransformation of dabrafenib occurs mainly via stepwise oxidation of the tert-butyl moiety to give hydroxy-dabrafenib, then carboxydabrafenib, followed by decarboxylation to generate the isopropyl derivative, desmethyl-dabrafenib. At 2 hours following a single dose of 14C-dabrafenib, carboxy-dabrafenib and hydroxy-dabrafenib were the major components in mouse and rat plasma, respectively, while dabrafenib was the main component in dog and human plasma. By 6 hours post dose, dabrafenib and its 3 main metabolites were detectable in plasma from all 4 species, with the exception that desmethyl-dabrafenib was not detected in dog plasma. By 10 hours post dose, carboxy-dabrafenib was the major drug-related component in human plasma. Dabrafenib was eliminated mainly in feces, primarily as intact dabrafenib in rat and dog and as oxidative metabolites in humans. On average, intact dabrafenib (in 3 patients) or desmethyl-dabrafenib (in one patient) was the main drug-related fecal component in humans

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and carboxy-dabrafenib was the main drug-related component in human urine. A comparison of the respective amounts of carboxy- and desmethyl-dabrafenib in bile from BDC male rats (8.3% and 2% dose) to their respective amounts in the feces of intact male rats (2% and 13.4% dose) suggests that carboxy-dabrafenib is also converted to desmethyl-dabrafenib in the gut following biliary secretion. In all species, following oral administration of dabrafenib, it is absorbed into the bloodstream and is metabolized to hydroxy-dabrafenib in the liver. Hydroxy-dabrafenib circulates in blood and is metabolized to carboxy-dabrafenib in liver (alternatively, hydroxy-dabrafenib is glucuronidated). Carboxy-dabrafenib circulates (at varied concentrations across species) until it is eliminated slowly via the urine or bile. Following biliary secretion, carboxy-dabrafenib may be decarboxylated in the gut to form desmethyl-dabrafenib, which is either excreted in feces or reabsorbed back into the bloodstream before elimination.

P314. METABOLISM AND DISPOSITION OF [14C]VELUSETRAG, A NOVEL AND HIGHLY SELECTIVE 5-HT4 RECEPTOR AGONIST, IN HUMANS


Jayaprakasam Bolleddula1, William Fitch2, Carla Washington1, Pierre-Jean Colson1, Vijay Sabesan1, Philip Worboys1 and David L. Bourdet1 1 Theravance, Inc, South San Francisco, CA, 2Stanford University, Stanford, CA Velusetrag (TD-5108), N-((1R,3R,5S)-8-((R)-2-hydroxy-3-(N-methylmethylsulfonamido)propyl)-8-azabicyclo[3.2.1]octan-3yl)-1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxamide, is a highly selective 5-HT4 agonist currently in development for the treatment of GI disorders such as chronic idiopathic constipation and gastroparesis. The mass balance and metabolism of [14C]velusetrag was studied in six healthy human volunteers after a single oral administration of [14C]velusetrag (15 mg, 100 mCi). A mean of 76.7% of the administered dose was recovered in feces and 15.9% recovered in urine. Unchanged velusetrag was the major component of drug related material identified in both the feces and urine and its excretion accounted for approximately 58% (feces) and 12% (urine) of the administered dose. The major metabolite recovered in feces and urine was an N-demethylated metabolite (M1) and represented approximately 16% (feces) and 3% (urine) of the administered dose. Unchanged velusetrag was the major circulating component of drug-related material in human plasma and represented approximately 45% of the total circulating radioactivity. The major circulating metabolite in human plasma was identified as a direct O-glucuronide (M8) of velusetrag and represented approximately 31.5% of the total circulating radioactivity. Interestingly, the metabolite profiles in plasma from this study included the presence of a significant radio-impurity (I1) which accounted for approximately 51% of the total plasma radioactivity. Despite its high abundance in plasma, the radioimpurity was not identified in human excreta consistent with the low total dose of the impurity (0.1%). Assignment of the radio-impurity was based upon the difference in the 14C/12C ratio observed for this peak relative to the ratio observed for velusetrag and all velusetrag related metabolites in the plasma from animal studies. The high plasma abundance of this low level radio-impurity can be explained by its high bioavailability, low volume of distribution, and low systemic plasma clearance as observed in animal studies. The radioactivity associated with this peak was thus excluded from the final assessment of the plasma metabolite profile in humans. Overall, the major metabolic pathways of velusetrag in humans arise from N-demethylation (M1) and direct Oglucuronidation (M8).

P315. VALPROIC ACID AND PRIMARY METABOLITES OF VALPROIC ACID ARE NOVEL ACTIVATORS OF AMP-ACTIVATED PROTEIN KINASE Lindsay B. Avery and Namandje´ N. Bumpus Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis. Valproic acid (VPA), a widely prescribed anti-epilepsy drug, has been demonstrated to modulate hepatic lipid/carbohydrate metabolism; however, the mechanism underlying this is unknown. In the present study, treatment of primary human hepatocytes with 800mM VPA, which is a concentration reported to be achieved in plasma in humans receiving VPA therapeutically, resulted in phosphorylation (activation) of AMPK. This effect was conserved in mice in that stimulated phosphorylation of AMPK that was detectable after a 1 hour incubation of primary hepatocytes isolated from 10–12 week old C57Bl/6 mice with 800mM VPA (1.9 0.4 fold change, p ¼ 0.0065). In addition, phosphorylation of acetyl-coA carboxylase (ACC), a substrate of AMPK, was observed 2 hours following VPA treatment (2.2 0.6 fold change, p ¼ 0.0056) and this effect was abrogated in the presence of the AMPK inhibitor 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine. Pre-incubation with STO-609, which is an inhibitor of an upstream kinase of AMPK (CamKK2), decreased phosphorylation of AMPK in response to VPA. Treatment with 20mM of VPA metabolites (4-ene-VPA formed by the cytochromes P450; 2-ene-VPA, 3-OH-VPA, and 3-keto-VPA produced via b-oxidation) also resulted in phosphorylation of AMPK and ACC. Pre-incubation with the Cytochrome P450 inhibitor aminobenzotriazole, decreased the VPA-mediated phosphorylation of AMPK and ACC indicating a role for metabolites of VPA in the activation of AMPK. Collectively, these results suggest that VPA and metabolites of VPA are able to

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activate the AMPK/ACC signaling pathway. As such, these compounds may have potential therapeutic benefit in the treatment of metabolic disorders.

P316. METABOLIC STABILITY ANALYSIS: NOVEL METHOD FOR CORRECTION OF MICROSOMAL BINDING


Hinnerk Boriss and Kathleen Boehme Sovicell GmbH, Leipzig, Germany Measurements of intrinsic clearance rates by microsomal incubations are frequently prone to underestimation of the clearance rate due to microsomal binding. In the present study we present validation data for a novel assay for assessing microsomal binding. The TRANSIL Microsomal Binding assay assesses the affinity of drugs to microsomal membranes. The free fraction in the microsomal incubation is calculated from the microsome concentration in the incubations. For a set of 30 compounds spanning a broad range of chemical structures and microsome binding we find a correlation with an r2 of 0.95 with data obtained by dialysis which indicates that the methods yield comparable results. Advantages of the TRANSIL method are its short incubation time of only 12 minutes, the ability to easily multiplex and rapidly analyze samples, and the ability to use one measurement for all microsomal species.

P317. A CLINICAL MICRODOSE STUDY WITH 14C-ELACYTARABINE IN HEALTHY MALE SUBJECTS TO GENERATE MASS BALANCE DATA TO SUPPORT REGULATORY SUBMISSION: AN INNOVATIVE MICROTRACER APPLICATION Iain Shaw1, Lloyd Stevens1, Suzanne Sweet1, Stuart Mair1, Finn Myhren2, Athos Gianella-Borradori2, Petter-Arnt Hals2, Tone Veiteberg2, Stephen Dueker3 and Hubert Dirven2 1 Quotient Clinical, Nottingham, United Kingdom, 2Clavis Pharma, Oslo, Norway, 3Eckert & Ziegler Vitalea Science, Davis, CA Background: Obtaining human mass balance data for a cytotoxic drug product has always been regarded as a challenge. While it is not feasible to perform a conventional mass balance study at clinical doses in healthy volunteers, it is difficult to subject severely ill patients to the rigours of prolonged residency in a clinical unit and to the comprehensive excreta collection and blood sampling that is required. Notwithstanding these challenges, regulatory authorities do ask pharmaceutical companies to describe the routes and rates of elimination for cytotoxic drug molecules as a minimum description of drug metabolism in the registration packages for new drug products. Elacytarabine is a novel, lipid derivative of the anti-cancer drug cytarabine commonly used in the treatment of acute myeloid leukemia. The drug is in the final stages of a phase 3 study in relapsed or refractory AML. Elacytarabine is a stable ester of elaidic acid and cytarabine. Aims: This paper describes an open label, single-dose study to assess the mass balance recovery of an intravenous microdose of 14C-elacytarabine in healthy male subjects. The study requirement was driven by a specific request from the US Food and Drug Administration (FDA) to provide supporting information on the routes of elimination of total radioactivity and total recovery. The study synopsis was submitted for FDA for review before implementation. Methods: The program of work from the radiosynthesis of the 14C API through to the final study report was delivered as a synthesis-to-clinic package. The clinical study design comprised a single intravenous administration of 50 mg 14C-elacytarabine containing not more than 270nCi of 14C to a single cohort of 6 healthy male subjects. The subjects remained resident in the clinical unit for 7 days post dose and returned for 24 hour visits on Days 10 and day 14. Blood, urine and faecal samples were collected through the residential periods and samples were submitted for total 14C analysis by accelerator mass spectrometry. Results: A mean of 90% of the administered dose of radioactivity was recovered in urine and faeces over the course of the study. A mean of 89.6% of total radioactivity was recovered from the urine, indicating that renal clearance was the main pathway of elimination. A single IV administration of [14C]-elacytarabine was well tolerated in this study. There were no AEs reported during the study, and no clinically significant findings in any clinical laboratory evaluations, vital signs assessments or ECG recordings. Summary/Conclusion: This study demonstrated the validity of a microdose approach to generate mass balance data to support regulatory submission for a drug where obtaining the data by traditional methods was not possible.

P318. IN VITRO DRUG METABOLISM BY HUMAN CARBOXYLESTERASE 1 WITH FOCUS ON ANGIOTENSIN-CONVERTING ENZYME INHIBITORS Ragnar Thomsen1, Henrik B. Rasmussen2 and Kristian Linnet1 1 Dept. of Forensic Medicine, University of Copenhagen, Copenhagen, Denmark, 2Mental Health Centre Sct. Hans, Copenhagen University Hospital, Denmark Introduction: Carboxylesterase 1 (CES1) is the predominant hydrolase in human liver. The enzyme is involved in the metabolism of several therapeutic agents and drugs of abuse as well as endogenous compounds. CES1 activates several ester-containing

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prodrugs, including oseltamivir, dabigatran etexilate, mycophenolate mofetil and some angiotensin-converting enzyme (ACE)inhibitors including trandolapril, imidapril, benazepril and quinapril. However, the role of human CES1 in the activation of the commonly prescribed ACE-inhibitors, enalapril and ramipril, has not been studied. The enzyme also inactivates a multitude of drugs, including methylphenidate, pethidine, clopidogrel, rufinamide and oxybutynin. Methods: The hydrolytic activation of ester prodrugs enalapril and ramipril was studied using recombinant human CES1 and CES2 and compared to trandolapril. Michaelis-Menten kinetic parameters were determined using the recombinant enzyme. Additionally, a panel of therapeutic drugs and drugs of abuse was screened for inhibition of p-nitrophenyl acetate hydrolase activity in recombinant CES1 and HLM. Results: Enalapril, ramipril and trandolapril were readily hydrolyzed by CES1, but not by CES2. Km for the three compounds were roughly equal, but Vmax was 20-fold higher for trandolapril and ramipril compared to enalapril. The screening assay confirmed several known inhibitors of CES1 and identified two previously unreported inhibitors, namely the calcium antagonists isradipine and nimodipine. Conclusions: The clinically important ACE-inhibitors enalapril and ramipril were identified as novel substrates of CES1. In addition, isradipine and nimodipine were found to moderately inhibit CES1. Due to CES1’s involvement in the metabolism of several drugs used in the treatment of common conditions such as hypertension, congestive heart failure and diabetes mellitus there exists a potential for clinically relevant drug interactions. The findings in the present study may contribute to the prediction of such interactions in humans.

P319. ALDO-KETO REDUCTASE 1C3 INDUCES ANTHRACYCLINE RESISTANCE IN CANCER CELLS BY THE REDUCTION OF DAUNORUBICIN AND IDARUBICIN Jakub Hofman, Beata Malcekova, Adam Skarka, Eva Novotna and Vladimir Wsol Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic Drug resistance belongs to the main obstacles emerging during the cancer chemotherapy. By the conversion of drugs into corresponding less active metabolites, some biotransformation enzymes significantly contribute to this phenomenon. Aldo-keto reductase 1C3 (AKR1C3) is an NADPH-dependent enzyme frequently overexpressed in various cancers. In this study, we investigate possible role of AKR1C3 in the resistance of tumour cells to anthracyclines. Reducing activity of AKR1C3 toward anthracyclines was first tested using incubations with purified recombinant enzyme prepared in E. Coli. AKR1C3 was shown to most efficiently catalyse the formation of daunorubicinol, followed by idarubicinol and doxorubicinol. We further examined the reduction of daunorubicin and idarubicin employing transfection of A549, HeLa, MCF7 and HCT 116 cancer cells with AKR1C3 encoding vector. Production of daunorubicinol and idarubicinol was markedly accelerated in transfected cells; this acceleration was significantly blocked by 20 -hydroxyflavanone, a recognised AKR1C3 inhibitor. To demonstrate the participation of AKR1C3 on anthracycline resistance, we conducted MTT proliferation assays employing transfected HeLa, MCF7 and HCT 116 cells. Introduction of AKR1C3 into cells significantly reduced the antiproliferative effect of both daunorubicin and idarubicin in all three tested cell lines. In another experiment, we combined daunorubicin and idarubicin with 20 -hydroxyflavanone in non-transfected A549 cells, which endogenously express considerable amounts of AKR1C3. As a result, we observed that 20 -hydroxyflavanone efficiently sensitised A549 cells to anthracycline treatment. In conclusion, our data suggest substantial impact of AKR1C3 on the pharmacokinetic behaviour of daunorubicin and idarubicin, affecting their distribution as well as elimination. In addition, we demonstrate that the reduction of daunorubicin and idarubicin catalysed by AKR1C3 contributes to the resistance of cancer cells to anthracycline chemotherapy. Concomitant administration of anthracyclines with specific AKR1C3 inhibitor could be an efficient strategy for the successful treatment of AKR1C3 overexpressing tumours. This work is co-financed by the European Social Fund and the state budget of the Czech Republic. Project no. CZ.1.07/2.3.00/30.0061, CZ.1.07/2.3.00/30.0022 and CZ.1.07/2.3.00/20.0235.

P320. THE HEPARG CELL LINE: A VALUABLE CELLULAR MODEL FOR BIOTRANSFORMATION AND METABOLOMIC STUDIES Nicolas J. Cabaton, Maria-Helena Meireles, Cecile Canlet, Marie Tremblay-Franco, Emilien L. Jamin, Laurence Dolo and Daniel Zalko TOXALIM (Research Centre in Food Toxicology), INRA (French National Institute for Agricultural Research), Toulouse, France Humans are daily exposed to a variety of man-made chemicals entering the food-web and/or present in the environment. Many are known to behave as endocrine disruptors, and this currently raises new challenges in the field of toxicology. Exposure can occur not only to parent molecules, but also to their metabolites. These biotransformation products are sometimes involved in toxic mechanisms. The entering in force of specific regulations such as the REACH directive and the need to reduce the number of animals used for toxicity studies, require the development of reliable in vitro tools, enabling to examine the potential effects of a variety of chemicals. Such tools should be as close as possible to human in vivo models, and should be suited for the investigation of low doses of contaminants as well, especially when dealing with active molecules such as endocrine disruptors.


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Ideally, in vitro models should express the largest possible metabolic capabilities, both regarding general metabolism and xenobiotics metabolism, when attempting to accurately decipher the effects of the tested molecules. Novel approaches in the field of metabolomics are being developed to highlight metabolic changes in organisms or biological systems exposed to nutritional, pharmacological, or toxic stimuli. The use of metabolomics to examine metabolic shifts induced in vitro by endocrine disruptors is expected to contribute to a better understanding of their capability to disrupt metabolic pathways, with possible extrapolation to human. In that context, the HepaRG cell line is a valuable model that allows to perform both biotransformation and metabolomic studies. This particular cell line expresses high metabolic capabilities, like human primary hepatocytes, with the advantage of having the indefinite growth capacity of hepatic cell lines. We performed metabolic profiling studies to examine the metabolic pathways and biotransformation rates of a variety of model food and environmental contaminants such as bisphenols (BPA, BPF), phthtalate (DEHP), and polycyclic aromatic hydrocarbons (Fluoranthene, Benz[a]pyrene). Concentration ranges were optimized for each contaminant and metabolic profiling was performed using HPLC after a 24-h exposure. Metabolomic approaches were also investigated on HepaRG cell extracts, in the context of low concentration exposure (BPA, as low as 10-12 M). Sample extracts were submitted to 1H NMR spectroscopy and NMR data were analyzed by multivariate statistical methods. We were able to discriminate between the control group and all BPA-exposed groups. Discriminant metabolites identification suggests a disruption in energetic and lipid metabolism pathways. This study confirms that metabonomic approach using 1H NMR spectroscopy is a promising tool for the characterization of shifts in the global metabolism after an exposure to very low concentrations of xenobiotics. Taken together, our data demonstrate that the HepaRG cell line is a valuable model for toxicological studies, taking into account phase I as well as phase and II biotransformations, but also highly suitable for the study of subtle metabolic shifts, highlighted by metabolomic approaches.

P321. CHARACTERIZATION OF THE METABOLISM AND ABSORPTION ON NOVEL SYNTHETIC BIOACTIVE PEPTIDES Chunyuan Zhang1, Bin Ma2, Dan Yang1 and Ge Lin2 1 Department of Chemistry, The University of Hong Kong, Hong Kong, Hong Kong, 2School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong Ion channel dysfunction can cause many sever human diseases, such as cystic fibrosis, epilepsy, myotonia and hypertension. Thus, research and development referring to regulating ion transport dysfunction to correct ion channel dysfunction, have drawn an increasing attention. Previously, several peptide candidates for treatment of diseases associated with chloride channel dysfunction were synthesized. However, these peptides exhibited poor metabolic stability and absorption profiles, which decreased their potential for therapeutic utility. Recently, a novel class of bioactive peptides has been synthesized by our group as promising preclinical candidates. The aim of the present study was to explore the metabolic stability and absorbability of four (Y1-Y4, Fig.1) of this novel class peptides, in order to ranking the best candidate with good metabolic stability and absorbability for further development. These four peptides share a common core structure with differences in the linker part and side chain. Firstly, the stability and metabolism of these four novel synthetic peptides in gastrointestinal tract and hepatic sub-cellular fractions were investigated. Gastrointestinal stability study revealed that both peptides Y1 and Y2 were unstable in GI tract with more than 41% and 47% degradation in simulated gastric fluid and simulated intestinal fluid. Metabolism study also showed that Y1 and Y2 were extensively metabolized for more than 31% and 45% in rat liver S9 and microsomes, respectively. However, the stability of Y3 and Y4 was significantly better with less than 5.6% degradation in both fluids. For peptide Y3, only 4.41% was metabolized by rat liver S9 and 5.57% by microsomes, respectively, while no metabolism of Y4 occurred. Subsequently the two peptides with good metabolic stability were chosen for the follow-up absorption study using Caco-2 monolayer cell model. The absorption study results showed that peptide Y3 [Papp(AB): (0.376 0.018)x106 cm/s, Papp(BA): (0.661 0.013)x106 cm/s] was poorly absorbed, while Y4 had a good absorbability [Papp(AB): (1.61 0.326)x106 cm/s, Papp(BA): (1.15 0.021)x106 cm/s]. In conclusion, according to the results obtained from metabolism and absorption study, peptide Y4 is the best candidate with both good metabolic stability and absorbability for further development. [Supported by Hong Kong Research Grants Council Collaborative Research Fund (HKU 2/06C and HKU8CRF10)]

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P322. BIOTRANSFORMATION FROM PYRROLIZIDINE ALKALOID N-OXIDES TO PYRROLIZIDINE ALKALOIDS IN LIVER AND GASTROINTESTINAL TRACT


Mengbi Yang, Jianqing Ruan and Ge Lin School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong The pyrrolizidine alkaloids (PAs) and their N-oxides are a group of toxic alkaloids that widely exist in plants all over the world. Content of PA N-oxides in plants are often equal or even higher than that of PAs. However, PAN-oxides intoxication is largely unknown. In our previous clinical study, HSOS (hepatic sinusoidal obstruction syndrome) had been found in patients who consumed PA N-oxide-containing herbs, demonstrating that PA N-oxide also exhibited hepatotoxicity similar to their parent PAs. PAs exert hepatotoxicity via cytochrome P450 (CYPs)-mediated metabolic activation to pyrrole-protein adducts. We propose that PA N-oxides may be metabolically converted to the corresponding PAs followed by the same metabolic activation, leading to hepatotoxicity. The present study aims to unveil the in vitro metabolic conversion of PA N-oxides to the corresponding PAs in both liver and gastrointestinal tract. After PA N-oxides were incubated with rat liver and intestine S9 preparations for 1 hour aerobically, the NADPH-dependent reduction of PA N-oxides to PAs occurred in both liver (metabolic rate 17.9%–33.1%) and intestine (metabolic rate 10.7%– 17.6%), and the same pyrrole-protein adducts, which were also generated by PAs, were detected for all four PA N-oxides tested. When different hepatic subcellular fractions were investigated, higher conversion rate was found in hepatic microsome, while cytosol contributed to a less extent. Moreover, anaerobic condition was found to be preferable to the reduction, while aerobic condition for metabolic activation and pyrrole-protein adducts formation. The nonselective CYPs inhibitors including SKF525A and n-octylamine significantly inhibited microsomal formations of both parent PAs and protein adducts, indicating that CYPs were the enzyme mediating the reduction of PA N-oxides. The anaerobic incubation of a representative PA N-oxide (riddelliine N-oxide) with pooled rat intestinal microbiota also demonstrated significant conversion (58.7%) of the N-oxide within 6 hour, suggesting a pivotal role of intestinal microbiota in the conversion of PA N-oxides to the PAs. This study firstly unveiled that the conversion of PA N-oxides to the PAs catalyzed by enzymes such as CYPs occurred in the liver and also in gastrointestinal tract mediated by intestinal microbiota. Out findings demonstrated that hepatotoxicity of PA N-oxides might be due to such biotransformation to their parent PAs followed by the same metabolic activation and protein adducts formation. [Supported by Research Grant Council of Hong Kong (GRF Grants no. 471310 and 469712) and CUHK (Direct Grants no. 2041744 and 4054047)].

P323. METABOLISM AND EXCRETION OF [14C]EVOGLIPTIN, A DPP-4 INHIBITOR, IN RATS Kyung-Jin Park and Hyun Joo Shim Drug Discovery Team IV, Dong-A ST Research Institute, Kyunggi-do, South Korea Evogliptin, a novel, orally active, highly specific and potent inhibitor of dipeptidyl peptidase-4 (DPP-4) that is currently in clinical development for the treatment of T2DM. The objective of this study was to characterize the mass balance and metabolic profiling and metabolite identification. For mass balance and metabolite profiling studies, SD male rats were administered a single oral dose of 30 mg/kg of [14C]evogliptin. The preliminary route of elimination in rats was in feces which accounted for 66.5% of the administered radioactivity, and urine accounted for 29.7%. Full recovery of the administered radioactvity was achieved in all animals with approximately 96% in the urine, feces, and expired air up to 168 h after administration. Metabolite profiles of plasma and excreta samples were generated by HPLC and online radiometric detection and identification of metabolites by mass spectrometric analysis. In plasma, urine, and feces, the unchanged evogliptin was accounted for 31.7%, 54.1%, and 27.4% of total peak area in radio-chromatogram, respectively. The major metabolite was hydrolyzed metabolite in rat plasma, urine, and feces. The metabolites of evogliptin in rats were generated via oxidation, dealkylation, hydrolysis, sulfation, and glucuronidation.

P324. COMPARATIVE BIOTRANSFORMATION AND MASS-BALANCE OF [14C]BMS-754807, A POTENT AND REVERSIBLE ANTAGONIST OF IGF1R, AFTER ORAL ADMINISTRATION TO RATS, DOGS AND HUMANS Peggy Liu-Kreyche1, Nirmala Raghavan1, Xiaomei Gu1, L. J. Christopher1, Pamela. L. Clemens1, Richard Burrell2, Jinping Gan1 and Rama Iyer1 1 Department of Biotransformation-PCO, Bristol-Myers Squibb, Princeton, NJ, 2Bristol-Myers Squibb Company, Wallingford, CT BMS-754807,{(2S)-1-{4-[(5-cyclopropyl-1H-pyrazole-3-yl)amino]pyrrolo[2,1-f][1,2,4]triazin-2-yl}-N-(6-floropyridin-3-yl)-2methylpyrrolidine-2-carboxamide}, is an antagonist of the tyrosine kinase activity of the type 1 insulin-like growth factor


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receptor (IGF-1R). It is under development as an oral treatment for a variety of cancer types. A series of studies were conducted to determine the routes of excretion and biotransformation of [14C]BMS-754807 following oral administration to intact and bile-duct cannulated (BDC) rats, dogs, and humans. Plasma, urine, bile and feces samples were collected at various time intervals and measured for radioactivity by liquid scintillation counting. Metabolites of [14C]BMS-754807 were profiled with HPLC-MS and HPLC-radioactivity detection. BMS-754807-derived radioactivity was eliminated mainly via fecal excretion in rats, dogs and humans. The percent recovery of the radioactive dose in feces was 83.4% in rats, 65.1% in dogs, and 84.6% in humans. The percent recovery of the radioactive dose in urine was 4.1% in rats, 5.2% in dogs, and 10.4% in humans. In BDC rats and dogs, 84% (0–24 h) and 41% (0–48 h) of the dose was recovered in the bile. Biotransformation profiles of plasma and feces from rats, dogs and humans were qualitatively similar. Unchanged BMS-754807 and M5, a mono-hydroxylated metabolite, were the most prominent drug-related components in the plasma and feces from all species. Glucuronide metabolites were present as major metabolites in bile for all species. Except for M2 in rat feces, the other glucuronide metabolites were not present in feces, indicating hydrolysis of these metabolites back to aglycone occurred during transit in GI tract. Urine profiles varied between the species. Rat urine contained high levels of a glucuronide conjugate of parent (M2) and multiple oxidation products (M5, M62 etc.). Dog urine contained mainly unchanged parent drug. Human urine contained predominantly M2. The extent of in vivo metabolism was similiar in animals compared to humans. The parent compound comprised 518.8% of the excreted dose (urine þ feces) indicating that BMS-754807 was highly metabolized in all species. The major metabolic pathways for metabolism of BMS-754807 were oxidation and glucuronidation. The metabolites included mono- and di- oxygenated metabolites and glucuronide conjugates of parent and various oxidation products. All metabolites found in humans were present in at least one of the animal species.

P325. METABOLISM OF AND ITS IMPACT ON SYSTEMIC EXPOSURE TO TANSHINOL, A VASOACTIVE COMPOUND FROM SALVIA MILTIORRHIZA ROOTS (DANSHEN), IN RATS Junling Yang1, Dandan Tian1, Xinwei Liu1, Junhua Liu1, Weiwei Jia1, Li Li1, Yuxing Huang1, Feifei Du1, Fang Xu1, Fengqing Wang1, Lihong Hu1, Zhu Yan2 and Chuan Li1 1 Laboratory for DMPK Research of Herbal Medicines, Shanghai Institute of Meteria Medica, Chinese Academy of Sciences, Shanghai, China, 2Tianjin University of Traditional Chinese Medicine, Tianjin, China Background and Aim: Tanshinol, a main medicinal ingredient present in Salvia miltiorrhiza roots (Danshen), has vasodilating and antioxidant properties. The major elimination pathways of this phenolic acid include renal excretion and the hepatic metabolism. This study aimed to understand the metabolism of tanshinol. Experimental: LC-MS-based metabolic profiling of tanshinol in rats was enhanced by knowledge-based metabolic prediction. The major metabolites were chemically synthesized for NMR-based structure elucidation. Rat or human enzymes mediating formation of tanshinol’s major metabolites were characterized using specific enzyme inhibitors or recombinant individual enzymes, while transporters mediating the metabolites’elimination were also identified with transfected cells. Vasodilating activities of the metabolites were compared with those of tanshinol. The influences of the tanshinol’s major metabolic process on its systemic exposure levels were further investigated in rats with or without chemically diminished metabolism. Key Results: Catechol-O-methyltransferase- and sulfotransferase-mediated biotransformations represented the major metabolic pathways of tanshinol in rats and 3-O-sulfated tanshinol (M2) and 3-O-methyltanshinol (M11) were the major plasma metabolites measured. The formation of these metabolites could also be mediated by human catechol-O-methyltransferase and sulfotransferases (SULT1A3, SULT1A1, and SULT1B1). M2 and M11 were eliminated via rapid renal excretion (involving human OAT1/rat Oat1-mediated tubular secretion). M2 and M11 exhibited weak and negligible vasodilating activities. Despite their important roles in tanshinol elimination, diminishment of the methylation did not significantly affect the systemic exposure levels of the phenolic acid, while it appeared to be difficult to find an in vivo inhibitor to diminish the sulfation. Conclusions: Methylation and sulfation represent the major metabolic pathways of tanshinol, which lead to the inactivation of its vasodilating activity.

References Lu T., Yang J-L., Gao X-M., Chen P., Du F-F., Sun Y., Wang F-Q., Xu F., Shang H-C., Huang Y-H., Wang Y., Wan R-Z., Liu CX., Zang B-L., Li C (2008) Plasma and urinary tanshinol from Salvia miltiorrhiza (Danshen), can be used as pharmacokinetic markers for cardiotonic pills, a cardiovascular herbal medicine. Drug Metab. Dispos. 36: 1578–1586. J-L. Yang and D-D. Tian contributed equally to this work

P326. METABOLIC ACTIVATION OF DAPAGLIFLOZIN AND CANAGLIFLOZIN IN VITRO IN LIVER MICROSOMES AND HEPATOCYTES Mark Grillo1, Thuy Tran2 and Smriti Khera3 1 Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, CA, 2Pharmacokinetics and Drug Metabolism, Amgen, Inc, South San Francisco, CA, 3Life Sciences Group, Agilent Technologies Inc, Santa Clara, CA


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Dapagliflozin and canagliflozin are C-aryl glycoside-containing drugs that are potent sodium-glucose cotransporter type-II (SGLT2) inhibitors developed for the treatment of type 2 diabetes. Dapagliflozin was not approved by the US Food and Drug Administration (FDA) in 2012 due to concerns of increased, albeit modest (1.47% versus 1.35%; dapagliflozin versus control groups), incidences of breast and bladder tumors in clinical trials in type 2 diabetics; however dapagliflozin (ForxigaÔ) has received approval in the EU for the treatment of type 2 diabetic patients 18 years of age and older with a dosage of 10 mg once daily. Chemically-reactive metabolites of drugs can be generated by oxidative metabolism (e.g., via phase-I mediated cytochrome P450-mediated oxidative bioactivation) which can lead to covalent binding to protein that in some cases leads to toxicity. Herein, we utilized in vitro methods to investigate the potential for bioactivation of dapagliflozin and canagliflozin to chemically-reactive intermediates. Thus, covalent binding study results showed irreversible binding to protein in rat (RLM) and human (HLM) liver microsomes incubations with [3H]dapagliflozin (10 mM); however no evidence for P450-mediated (i.e., NADPH-dependent) bioactivation was observed. After 1-h of incubation with RLM and HLM, covalent binding was 93-170 and 5-8 pmol equivalents/mg protein, respectively. Covalent binding to protein occurred in liver microsomes across species in a timedependent and NADPH-independent fashion with a rank order of rat &dog4cynomolgus monkey4human. Reactive metabolite trapping studies with semicarbazide in incubations with RLM indicated the reactive metabolite formed to be dapagliflozin-C-6aldehyde, which was confirmed by LC-MS/MS and by 1H-NMR analyses of the HPLC-purified dapagliflozin-semicarbazideadduct. Time-dependent and linear covalent binding of [3H]dapagliflozin (10 mM) to protein in incubations with rat and human hepatocytes was observed and led to 218 and 20 pmol/mg protein after 4-h of incubation, respectively. Coincubation of rat and human liver microsomes or cytosol with NADþ or with 4-methylpyrazole had no effect on covalent binding to microsomal or cytosolic proteins, indicating no alcohol dehydrogenase-mediated bioactivation. Corresponding in vitro experiments with canagliflozin also showed the formation of the corresponding chemically reactive C-6-aldehyde and the trapped semicarbazide Schiff-base adduct from incubations with RLM and rat hepatocytes, but not from incubations with HLM or human hepatocytes. Canagliflozin (InvokanaÔ) was approved by the FDA in March, 2013 for the treatment of adults with type 2 diabetes. In summary, in incubations with rat liver microsomes and rat hepatocytes, both dapagliflozin and canagliflozin were shown to undergo bioactivation on the 6-hydroxymethyl-alcohol of the glucose-moiety to a chemically-reactive C-6-aldehyde intermediate. Further studies in our laboratory are ongoing to determine the identity of the enzyme(s) catalyzing the bioactivation of dapagliflozin and canagliflozin to the C-6-aldehyde metabolite.

P327. ONTOGENY OF HEPATIC ENERGY METABOLISM GENES IN MICE Helen J. Renaud1, Julia Yue Cui1, Hong Lu2, Xiao-bo Zhong3 and Curtis Klaassen1 1 Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS, 2Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY, 3Department of Pharmaceutical Sciences, University of Connecticut School of Pharmacy, Storrs, CT The liver plays a central role in metabolic homeostasis by coordinating the synthesis, storage, breakdown, and redistribution of nutrients. Due to different demands for nutrients and energy during growth and development, hepatic energy metabolism is dynamically regulated throughout different life stages. However, the patterns of ontogenic changes of genes important in hepatic energy metabolism throughout development are not well understood. The aim of this study was to perform detailed transcript analysis of energy metabolism genes during various stages of liver development in mice. Livers from male C57BL/6 J mice were collected at twelve ages, including perinatal and postnatal time points (n ¼ 3/age). The mRNA was determined by RNA-Seq, with transcript abundance estimated by Cufflinks. One thousand sixty energy metabolism genes were examined; 794 were above detection, of which 627 were significantly changed during at least one developmental stage compared to adult liver. Two-way hierarchical clustering revealed three major clusters dependent on age: GD17.5 - Day 5 (perinatal-enriched), Day 10 – Day 20 (prepuberty-enriched), and Day 25 – Day 60 (adolescence/adulthood-enriched). Clustering analysis of cumulative mRNA expression values for individual pathways of energy metabolism revealed 3 patterns of enrichment: glycolysis, ketogenesis, and glycogenesis were all perinatally enriched; glycogenolysis was the only pathway enriched in prepuberty; whereas lipid droplet metabolism, cholesterol and bile acid metabolism, gluconeogenesis, and lipid metabolism were all enriched in adolescence/ adulthood. Within these individual pathways, there were several interesting patterns of age-dependent expression. The rate limiting steps of fatty acid beta-oxidation, Acox1 (peroxisomal beta-oxidation), and Cpt1a (mitochondrial beta-oxidation), become divergently expressed as the liver matures into adulthood, with Acox1 expression increasing and Cpt1a expression decreasing. This may indicate an increase for the need of peroxisomal beta-oxidation over that of mitochondrial beta-oxidation during growth into adulthood. Stearoyl-CoA desaturases (Scd) are rate limiting in the process of fatty acid desaturation. Scd1 has been implicated in the metabolic syndrome as it is highly upregulated in rat and mouse models of obesity and type 2 diabetes. Interestingly the present study shows a sharp upregulation of Scd1 after the age of 30 days. Elovl3 regulates the elongation of saturated and mono-unsaturated fatty acids and Elovl3-null mice are resistant to diet-induced obesity. After 25 days of age, the present study shows a stark increase in Elovl3 transcript abundance. What regulates these increases in Scd1 or Elovl3 is unknown. These data shed new light on the ontogeny of homeostatic regulation of hepatic energy metabolism, which could ultimately provide new therapeutic targets for metabolic diseases. [Support: NIH grants DK-081461, ES-019487, and ES009649; CIHR]

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P328. STRUCTURE-ACTIVITY RELATIONSHIPS OF A SERIES OF 17 PARABENS FOR HISTAMINE RELEASE IN RAT PERITONEAL MAST CELLS, SKIN ALLERGIC REACTION IN GUINEA PIGS, AND HYDROLYTIC ACTIVITY IN RAT AND GUINEA PIG SKIN


Naoto Uramaru1, Toshio Inoue2, Yoko Watanabe1, Shigeru Ohta3 and Shigeyuki Kitamura1 1 Biohealth Sciences, Nihon Pharmaceutical University, Saitama, Japan, 2Pharmacotherapy, Nihon Pharmaceutical University, Saitama, Japan, 3Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan Parabens, which are a homologous series of esters of p-hydroxybenzoic acid, have been used as preservatives in cosmetics, medicines and foods based on their antimicrobial activity. However, parabens in cosmetics have the potential to cause allergic contact dermatitis. In this study, we examined paraben-induced histamine release in rat peritoneal mast cells and skin allergenicity in guinea pigs using a series of 17 parabens with different alcohol side chains, ranging from methylparaben to dodecylparaben. Furthermore, to examine the structure-activity relationship, we investigated the effects of different side chains and different phenolic hydroxyl group substituent positions, as well as alkyl benzoate, phenylalkane and alkylphenol derivatives. Among parabens, the highest histamine release activity was observed with octylparaben, and shorter and longer side chain parabens showed weaker activity. However, octyl benzoate or octyl o-hydroxybenzoate caused no significant degranulation of mast cells. In contrast, octyl m-hydroxybenzoate caused concentration-dependent degranulation. Phenyloctane did not induce marked degranulation, but octyl phenol exhibited significant activity. Paraben metabolites, p-hydroxybenzoic acid and alcohols containing methyl to dodecyl straight chain moieties, as well as branched alkyl, phenyl and benzyl moieties, did not show histamine release-inducing activity. When skin allergenicity was examined in guinea pigs, butylparaben induced a typical weak skin reaction and heptylparaben induced a typical strong skin reaction, whereas methylparaben, dodecylparaben and p-hydroxybenzoic acid were inactive. The hydrolytic activity of skin microsomes of rats and guinea pigs toward parabens was also examined. Higher activity was observed towards shorter-side-chain parabens. These results indicate that interaction of parabens with rat mast cells requires phenolic m- or p-hydroxyl group substitution and characteristic lipophilicity of the alkyl side chain. Metabolites of parabens showed less potent effects than the mother compounds in mast cells and did not induce skin erythema in a guinea pig skin reaction test. Therefore, parabens appear to act as direct allergens.

P329. INHIBITORY EFFECT OF HIGH FAT DIET-INDUCED NON-ALCOHOLIC STEATOHEPATITIS WITH FIBROSIS BY PLATYCODON GRANDIFLORUM ROOT-DERIVED SAPONIN IN RATS Jae Ho Choi, Tilak Khanal, Sun Woo Jin and Hye Gwang Jeong College of Pharmacy, Chungnam National University, Daejeon, South Korea Non-alcoholic steatohepatitis (NASH) is characterized by diffuse fatty infiltration, lobular inflammation, ballooning degeneration, and sinusoidal fibrosis in the liver. Simple steatosis in the early stage may gradually develop into fatty hepatitis, and subsequently develop towards hepatic fibrosis and liver cirrhosis. The progression of liver injury leads to fibrosis, a condition characterized by distortion of normal architecture, septae and nodule formation, altered blood flow, portal hypertension, hepatocellular carcinoma, and ultimately, liver failure. This study was performed to the inhibitory effect of Platycodon grandiflorum root-derived saponin, CKS, in an experimental NASH model induced by a high fat diet (HFD). HFD treatment for 8 weeks led to marked NASH as examined by serum biochemistry, histopathological examination, and hepatic malondialdehyde (MDA) and collagen content. CKS is used as a food and in traditional oriental medicine to treat chronic inflammatory diseases. CKS inhibited HFD-induced serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and hepatic content of MDA and collagen. CKS inhibited HFD-induced body and liver weights in rat. Reverse transcription polymerase chain reaction and western blot analyses revealed that CKS inhibited HFD-induced fibrosisrelated mRNA expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and tumor necrosis factor- alpha (TNF- alpha) and protein expression of collagen type I and alpha-smooth muscle actin (alphaSMA). Also, CKS inhibited HFD-induced cyclooxygenase-2 (COX-2) expression and nuclear factor-kappa B (NF-kB) activation. These results suggest that CKS manifests effective hepatocellular protective action and ameliorative effects against chronic liver damage and developing liver fibrosis induced by HFD treatment.

P330. INVESTIGATION OF GLUCURONIDE AND SULFATE METABOLITES FORMATION AFTER ORAL ADMINISTRATION OF PARACETAMOL IN HEALTHY HONG KONG CHINESE SUBJECTS Siu kwan Wo1, Benny Fok2, Brian Tomlinson2, Vincent HL Lee1 and Zhong Zuo1 1 School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, 2Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Aim: Paracetamol (P), a widely used analgesic and antipyretic agent, is mainly metabolized into glucuronide (PG) and sulfate (PS) metabolites mediated by UGT1A1 and SULT1A1, respectively. Toxicity of P usually appears after its overdosing resulting


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in saturation of UGT and SULTs. In clinical practice in China, polymorphisms of UGT and SULT have already been reported to lead to large inter-individual variability and side effects of several clinical extensively used drugs. The present study is to 1) evaluate the formation of PG and PS in Hong Kong Chinese subjects after a single oral dose of 500 mg P and 2) to compare the metabolic ratio of both glucuronides and sulfates in Chinese subjects with that from other ethnic groups. Methodology: Protocol was approved from the local ethics committee prior to the study. Healthy subjects were administered with a single oral dose of 500 mg Panadol caplet after an overnight fast of 10 h. Venous blood samples were collected at pre-dose (0 h), 10 min, 20 min, 30 min, 45 min, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 h post-dose. Simultaneous quantification of P, PG and PS in plasma was conducted using a validated LC/MS/MS method and the calibration standards were prepared by spiking known amount of these three standards in drug-free plasma. Pharmacokinetic parameters (Cmax, Tmax, AUC0–12h, AUC0–inf, T1/2) of P, PG and PS were calculated by non-compartmental model from the plasma concentration vs time profiles. The metabolic ratios of PG and PS (i.e. AUCPG/AUCP, AUCPS/AUCP, AUC(PGþPS)/AUCP) and AUCPG/AUCPS ratio among ethnical groups were obtained and compared. Results: Totally sixteen Hong Kong Chinese male subjects completed the study. The Cmax, AUC0–inf, Tmax and T1/ 2 of P was found to be 7.9 2.9 mg/ml, 28.2 6.5 mg h/ml, 0.64 0.28 h and 2.92 0.30 h, respectively, which are comparable to those reported before on other ethnical group at the same dosage. The Cmax of PG and PS were found to be 3.3 0.9 and 3.9 1.0 mg/ml, respectively. Although the ratio of AUC(PGþPS)/AUCP (1.8 0.45) in Hong Kong Chinese subjects was similar to that from American (2.15), a much lower AUCPG/AUCP (0.98 0.38) and a slightly higher AUCPS/AUCP (0.89 0.23) ratio were observed in Hong Kong Chinese when compared with that from American (1.60 and 0.59, respectively). The results suggest that phenotypic and genetic variations of UGT and SULT may account for the difference in metabolism of P in Chinese subjects from the other ethnic groups. Conclusion: Different metabolic ratio of PG or PS to P was observed in Hong Kong Chinese subjects when compared to American. The effect of phenotyping and geneotyping on the metabolic pathways of paracetamol may warrant further study.

P331. SELECTION OF HUMAN LIVER S9 AND CYTOSOL FRACTIONS FOR EVALUATING CLEARANCE BY ALDEHYDE OXIDASE (AO): THE IMPACT OF LOW VERSUS HIGH AO ACTIVITY LOTS Phyllis Yerino, Clayton J.M. Otwell, Zell Woodworth and David B. Buckley XenoTech, LLC, Lenexa, KS Aldehyde oxidase (AO) is a cytosolic enzyme present in the liver of humans and other mammals that catalyzes various oxidation and reduction reactions. Biotransformation by AO is an important clearance mechanism for numerous drugs and drug candidates and AO metabolism can lead to rapid in vivo clearance. Several publications have described the under-prediction of in vivo human clearance for compounds metabolized by AO based on scaled in vitro clearance data, which are typically obtained from experiments conducted with human liver subcellular fractions such as S9 or cytosol. Zientek and colleagues (2010) described a rank order, or ‘yard-stick’, approach to categorize known AO substrates as low, medium or high clearance compounds based on in vivo data. With this approach, new drug candidates can be evaluated in vitro in S9 or cytosol and the predicted in vivo clearance can be qualitatively ranked from low to high clearance. The subcellular fractions S9 and cytosol are commercially available from multiple sources and in many formats (individual lots and pools of various sizes), which causes variation in AO activity. Consequently, the purpose of this study was to evaluate the impact of low versus high AO activity in human liver S9 and cytosol preparations on the prediction of scaled clearance for AO substrates with the ‘yard-stick’ approach. To evaluate clearance of drugs metabolized by AO in low versus high activity lots, S9 and cytosol fractions were selected from five individuals that spanned 410-fold variation in AO activity. Experiments were conducted to evaluate the disappearance of six drugs (at 1 mM) with varied, known AO clearance, namely carbazeran, deoxypenciclovir, zoniporide, benzylguanine, zaleplon and methotrexate. Incubations were conducted in human liver S9 (2.5 mg/mL) or cytosol (1 mg/mL) from all five individuals for up to 1–4 hours; time points were typically adjusted to account for expected half-life. In S9, half-life and scaled clearance values could be calculated for all five individuals, including the lowest activity sample. In each case the ‘yard-stick’ approach could be applied to all samples tested with reasonable thresholds. In cytosol, half-life and scaled clearance values could be calculated from four of five individuals. For five of six drugs, the half-lives and clearance values could not be calculated for the lowest AO activity sample because of low substrate turnover. Similarly, the ‘yard-stick’ approach could be applied with reasonable thresholds for all but the lowest AO activity sample. Based on available characterization data, most available pooled human liver S9 and cytosol lots have AO activities within the range of S9 and cytosol activities tested here; typically 3- to 5-fold higher than the lowest AO activity sample. These data indicate that, due to the necessity to scale AO clearance with a rank-order approach, most human liver S9 and cytosol lots (individual or pooled) can be utilized to predict in vivo AO clearance once threshold values are determined with appropriate probe drugs.

Reference Zientek et al., In vitro-In vivo Correlation for Intrinsic Clearance Drugs Metabolized by Human Aldehyde Oxidase, Drug Metabol Dispos 38:1322–1327, 2010.

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P332. A COMPARISON OF ALDEHYDE OXIDASE ACTIVITY IN CRYOPRESERVED HEPATOCYTES FROM MULTIPLE ANIMAL SPECIES: CD-1 MOUSE, SD RAT, WISTAR RAT, BEAGLE DOG, CYNOMOLGUS MONKEY, RHESUS MONKEY AND MAN


Utkarsh Doshi and Albert P. Li In Vitro ADMET Laboratories LLC, Columbia, MD During drug development, human liver microsomes (HLM) are routinely used for the selection of new chemical entities (NCE) with appropriate metabolic stability. This practice has led to the elimination of compounds that are rapidly metabolized by microsomal phase I oxidation, especially by P450 pathways. One outcome of this is the elimination of NCE with P450 liability but the accumulation of NCE that are metabolized by non-microsomal drug metabolizing enzyme (DME) pathways. Aldehyde oxidase (AO), a cytosolic phase I DME, has emerged as a key nonP450 DME for metabolic stability, metabolic activation, and detoxification. One key property of AO is the known species differences in its activity. Most of the findings on species differences in AO activities were reported with liver fractions containing cytosolic DMEs (e.g. S9), with only limited number of reports on intact hepatocytes. We hypothesized that findings with intact hepatocytes, especially that concerning species differences, would advance our understanding of the role of this cytosolic phase I DME in hepatic drug metabolism. A key practical application is to use hepatocyte results to guide the selection of an appropriate nonhuman animal species to model human metabolism for compounds metabolized predominantly by AO. We thereby embarked upon a comprehensive examination of AO activities in cryopreserved hepatocytes from multiple animal species: male CD-1 mouse , male SD rat, male Wistar rat, male beagle dog, male and female cynomolgus monkey, male and female Rhesus monkey, and male and female human. Our major objective is to evaluate if major species differences observed in vivo could be observed in the intact hepatocytes. AO activity was quantified via the evaluation of intrinsic hepatic clearance (Clint) of O6-benzylguanine (OBG; 1 uM) upon incubation with hepatocytes in 48-well plates as suspension cultures. The known AO inhibitor, hydralazine (25 uM) was used to confirm the role of AO in the metabolism of OBG. AO activity was readily quantifiable in the hepatocytes from all the species. For the rodent hepatocytes, mouse had the highest activity (Clint of 60 mL/min/kg body weight), followed by Wistar rat (42 mL/min/kg) with SD rat having the lowest activity (19 mL/min/kg). Hepatocytes from 4 human donors yielded similar AO activities (ranged from 40 to 49 mL/min/kg). The hepatocytes from the various primate species yielded unexpected results: Rhesus monkey had the lowest activity (6.5 mL/ min/kg (male) and 10 mL/min/kg (female); cynomolgus monkey had a higher activity of 17 mL/min/kg (male), and 7.2 mL/ min/kg. Of all the species evaluated, male beagle dog hepatocytes had lowest AO activity of 8.6 mL/min/kg. The results therefore suggest that male Wistar rat hepatocytes represent the experimental model most similar to human. Near complete inhibition of OBG metabolism by 25 uM hydralazine was observed in all species, confirming that AO is responsible for the activity. The results with AO activities measured in intact cryopreserved hepatocytes support the use of intact cryopreserved hepatocytes in metabolic stability screening. The presence of P450 activities, cytosolic phase I DME such as AO as reported here, phase II conjugating pathways, as well as other intact cell properties (ICP) including plasma membrane and the associated uptake transporters in the hepatocytes allow the generation metabolic stability results more similar to that in vivo than that with cell free fractions such as liver microsomes.

P333. EVALUATION OF SPECIES AND TISSUE-DEPENDENT DIFFERENCES OBSERVED IN CARBOXYLESTERASE METABOLISM: APPLICATION TO PRO-DRUG STUDIES Katie Fox and Phil Butler Cyprotex, Macclesfield, United Kingdom Pro-drugs are substances which are administered in an inactive form and then metabolised in the body into the active drug. Many pro-drugs are designed to improve oral bioavailability of drugs which exhibit poor absorption from the gastrointestinal tract. A common approach to pro-drug design is using a carboxylic ester linkage to aid passive transport and undergo hydrolysis to active drug on entering systemic circulation1. This hydrolysis is catalysed by esterases such as carboxylesterases (CES), butyrylcholinesterase (BuChE) and paraoxonase (PON). In human, CES are found primarily in liver and intestinal endoplasmic reticulum, however, in rat and mouse, CES are also found in plasma2. There are different forms of CES, most notably CES1 and CES2 which have differing substrate specificity. In human, hCE-1 is located primarily in the liver and hCE-2 in the intestine and kidney. Rat and mouse CES1 and CES2 are expressed differently to human. Knowledge in this area has focused primarily on mRNA expression and activity in plasma and blood. Further investigations of CES1 and CES2 activity in liver and intestinal microsomes are needed. Our aim was to gain an improved understanding of species and tissue differences in CES-mediated hydrolysis by comparing incubations of human, rat and mouse liver microsomes, intestinal microsomes and plasma. The investigation focused on two prodrugs oseltamivir and irinotecan (CPT-11) which are specific CES1 and CES2 substrates, respectively, in human. Both parent depletion and specific hydrolytic metabolite formation were monitored by LC-MS/MS over a 45 minute time course and intrinsic clearance and half-life (t1/2) determined. To confirm enzyme-specific metabolism, we included isoform-specific inhibitors

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27-hydroxycholesterol (CES1) and loperamide (CES2), and the non-specific inhibitor phenylmethanesulfonyl fluoride (PMSF) in the incubations. We confirmed previous findings that no CES activity is detectable in human plasma3 with neither oseltamivir nor irinotecan undergoing metabolism. However in both rat and mouse plasma we did observe hydrolysis of oseltamivir and CPT-11. 7-ethyl10-hydroxycamptothecin (SN-38) formation from CPT-11 was observed in both rat and mouse liver microsomes at a higher rate than in human, suggesting a difference in CES expression. This difference in tissue expression of CES between human and rodents should be carefully considered when performing preclinical work. Awareness of inter-species differences in membrane-bound CES activity between human, mouse and rat is helpful for intelligent study design and subsequent data interpretation especially when using carboxylic ester linkages for pro-drug activation.

References


1. Imai T., (2006), Drug Metab. Pharmacokinetics, 21:173–185 2. Satoh T. et al., (2006), Chemico-Biological Interactions, 162:195–211 3. Li B. et al., (2005) Biochemical Pharmacology, 70:1673–1684

P334. CHARACTERIZATION OF MICROSOMAL ALDEHYDE OXIDASE ACTIVITY Ryan A. Dick1, Caitlin Brown2, Ji Young Lee3, James Driscoll1 and Marc Evanchik1 1 Dmpk, MyoKardia, South San Francisco, CA, 2Celsis In Vitro Technologies, United States, MD, 3Celsis In Vitro Technologies, Baltimore, MD Aldehyde oxidase (AO) is a well-known enzyme capable of oxidizing various azaheterocyle and aromatic aldehyde-containing small molecules without the requirement of a cofactor. While AO is thought to be cytosolic, in vitro studies have identified significant levels of activity of this enzyme in microsomal preparations derived from human and non-human liver. Rat, dog, monkey and human microsomes from three commercial manufacturers (Celsis, BD Biosciences and Xenotech) were assayed for AO activity using probe substrates including carbazeran, zoniporide, O6-benzylguanine, phthalazine, and zaleplon. AO activity was greatest in monkey liver microsomes with carbazeran exhibiting a52 min half-life. Comparison of specific activities among microsomes and cytosol for rat, monkey and human indicates that microsomes have 5-10% of the cytosol AO activity. Further, incubation of each of the probe substrates with monkey, human, and rat liver microsomes generated AO-specific metabolites that were verified by comparison with cytosolic controls. Immunoblotting was used to confirm the presence and relative abundance of AO in microsomes. The feasibility of wash strategies to minimize contamination was also investigated. Together these data confirm the unexpected presence of significant AO activity in commercial microsomes which may lead to inaccuracies in metabolic reaction phenotyping and ultimately have implications on assessment of drug-drug interaction potential.

P335. SPECIES DIFFERENCES IN HEPATIC AND INTESTINAL ESTERASE ACTIVITIES BETWEEN HUMAN, RAT, DOG AND MONKEY Haruka Nishimuta1, J. Brian Houston2 and Aleksandra Galetin2 1 Discovery Pharmacokinetics Group, Preclinical Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan, 2 School of Pharmacy and Pharmaceutical Sciences, Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, United Kingdom Carboxyl esterases (CEs) play an important role in metabolism of prodrugs [1]. However, unlike cytochrome P450, characterisation of CEs, including species differences, are poorly understood. In the current study, species differences in hepatic and intestinal metabolic esterase activities were investigated for 10 compounds. Metabolic stabilities were assessed in pooled human cryopreserved hepatocytes, pooled liver S9 (LS9) and intestinal S9 (IS9) (prepared without esterase inhibitors); the S9 studies were conducted without NADPH and at a single substrate concentration of 0.2 mM. In addition, species differences were assessed by obtaining the CLint values in the same in vitro systems described above but from Sprague-Dawley rats, beagle dogs and cynomolgus monkeys. Compounds investigated are predominantly metabolized by CE1, with the exception of candesartan cilexetil (substrate for CE2) and mycophenolate mofetil (both CE1 and CE2). The scaled human CLint,hepatocytes ranged from 15– 3900 mL/min/kg for perindopril and candesartan cilexetil, respectively. CLint values obtained in monkey hepatocytes were the most comparable to human for the current dataset, with CLint values for 6 compounds within 2-fold; exceptions were benazepril, trandolapril, ramipril and oseltamivir. Hepatocyte CLint,dog / CLint,human was within 2-fold for only 4 compounds; osertamivir and mycophenolate mofetil being the most pronounced outliers. The most pronounced species differences in CLint,hepatocytes were observed between rat and human where for certain drugs (e.g., temocapril) up to 100-fold higher CLint was seen in rat; the only drug with comparable CLint in rat and human hepatocytes was perindopril. The scaled human CLint,LS9 ranged from 8-46000 mL/min/kg for perindopril and mycophenolate mofetil, respectively; the scaled CLint values were up to 2-fold greater

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than CLint,hepatocytes across all species, with the exception of dog where opposite trend was apparent. Although the fold range in CLint,LS9 was analogous to hepatocytes, species differences were more pronounced; no trends in differences in metabolic activity of each CE were evident. In the case of intestine, the metabolic activities for CE1 substrates were not detected in human S9, in contrast to rat and monkey. In the case of CE2 substrates, metabolic activities were greater in human and monkey S9 relative to rat and dog, reflecting species differences in expression of CEs. In conclusion, monkeys showed the most comparable hepatic esterase activity to human and could be a useful animal model to predict human but with the potential limitation of the lack of comparability in intestinal esterase activity.

Reference 1. Imai T, Ohura K. (2010) The role of intestinal carboxylesterase in the oral absorption of prodrugs. Curr Drug Metab 11:793–805.


P336. CHARACTERIZATION OF ESTERASES INVOLVED IN THE METABOLISM OF PARA-NITROPHENYL ACETATE IN HUMAN PLASMA Shin Takusagawa1, Kanako Yajima2, Takafumi Iwatsubo1, Shotaro Uehara2, Aiji Miyashita1 and Takashi Usui1 1 Drug Metabolism Research Laboratories, Astellas Pharma Inc., Osaka, Japan, 2Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., Wakayama, Japan p-Nitrophenyl acetate (p-NPA) is a general substrate widely and routinely used to assay carboxylesterase (CES) activity because the hydrolysis of p-NPA can easily be followed by yellow color development due to the liberation of free p-nitrophenol, but this same substrate is also hydrolyzed by acetylcholinesterase (AChE), butyrylcholinesterase (BChE), albumin, and paraoxonase (PON). It is reported that human plasma contains only fouresterases (BChE, PON, AChE, and albumin), and one of them, AChE, is present in negligible amount.1 To our knowledge, the contribution of each esterase enzyme to the total enzyme-mediated hydrolysis of p-NPA in human plasma has not been reported in details. In order to characterize the esterases involved in the hydrolysis of p-NPA in human plasma, the enzyme kinetics and the inhibition profiles of p-NPA hydrolysis in human plasma were investigated and compared with those in serum albumin, liver and intestinal microsomes, recombinant AChE, and purified BChE solutions. Liver and intestinal microsomes were included as enzyme sources of CES1 and CES2, respectively. EDTA, a specific PON inhibitor, inhibited the p-NPA (75 mM) hydrolysis in human plasma most strongly among the esterase inhibitors tested, and the inhibitory effects were comparable between 0.1 and 1 m MEDTA (by 50%). Stronger inhibition by EDTA (0.1 mM) was observed at higher concentrations of p-NPA (more than 80% inhibition at 1000 mM p-NPA), while weaker inhibition at lower concentrations (30% inhibition at 10 mM p-NPA). Both the p-NPA hydrolysis activity in human plasma in the presence of EDTA (0.1 mM) and that in albumin solution without EDTA exhibited biphasic enzyme kinetics, with a Km1 value (high affinity component) of 20.1 and 51.2 mM, respectively, and a Km2 value (low affinity component) of both42000 mM. Inhibition profiles of the p-NPA hydrolysis activity in human plasma in the presence of EDTA and in albumin solution without EDTA were also very similar. In both reaction mixtures the p-NPA hydrolysis activity at 40 mM p-NPA was inhibited by 5,50 dithiobis (2-nitrobenzoic acid) (DTNB) and bis(4-nitrophenyl) phosphate sodium salt (BNPP), and at 1000 mM p-NPA it was inhibited by DTNB only. Little or no inhibitory effects by the other esterase inhibitors tested were observed, indicating that the remaining p-NPA hydrolysis activity in human plasma after inhibition of PON can be attributed to albumin. In conclusion, enzyme-mediated p-NPA hydrolysis in human plasma was catalyzed by PON and albumin almost exclusively. Albumin contributes equally to or greater than PON at 75 mM p-NPA or below while PON contributes more than albumin at higher concentrations of p-NPA.

Reference 1. Li B, Sedlacek M, Manoharan I, Boopathy R, Duysen EG, Masson P, Lockridge O. (2005). Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma. BiochemPharmacol 70:1673–1684.

P337. THE ROLE OF ESTERASES IN METABOLISM OF PONATINIB Caroline Woodward Dmpk, ARIAD PHARMACEUTICALS, Cambridge, MA The Role of Esterases in Metabolism of Ponatinib Caroline N. Woodward, Scott Wardwell, Emily Ye, Narayana Narasimhan ABSTRACT Ponatinib (IclusigÕ ) is an oral multi-targeted tyrosine kinase inhibitor (TKI) with potent activity against native BCR-ABL and its mutants. Recently, IclusigÕ received accelerated approval from US FDA for the treatment of adult patients with chronic myeloid leukemia or Philadelphia chromosome-positive acute lymphoblastic leukemia who are resistant or intolerant to prior TKI therapy. In a phase I, open-label mass balance study to investigate the absorption, metabolism

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and excretion of [14C]ponatinib in healthy volunteers, AP24600 was identified as a major circulating metabolite (14.9% of total radioactivity [TRA]). This metabolite was also observed as a major metabolite of ponatinib in rat plasma (50% of TRA) and a trace metabolite (51% of TRA) in monkey plasma. AP24600 was formed via hydrolysis of the amide bond. Several in vitro/in vivo studies were conducted to explore the formation of AP24600 in hepatic and pre- hepatic systems in order to understand the physiological site of hydrolysis and to determine the mechanism of AP24600 formation. 1. In incubations with liver subcellular fractions or hepatocytes, ponatinib was not metabolized to AP24600. However, ponatinib was metabolized to AP24600 in intestinal subcellular fractions in the absence of NADPH. The rate of hydrolysis of ponatinib in human, monkey, and rat intestinal S9 fractions was in the order of rat4human4monkey. 2. After oral dosing of ponatinib in rats, AP24600 concentrations were higher in portal vein compared with jugular vein plasma, indicating that the hydrolysis occurred presystemically. 3. Pretreatment of Sprague Dawley rats with bis-nitrophenylphosphate (BNPP), a known serine esterase inhibitor1 prior to ponatinib oral administration resulted in a four-fold decrease in AP24600 exposure compared with the control group. These in vitro and in vivo studies suggest that formation of AP24600 is pre-systemic and that esterases were involved in the hydrolytic cleavage of ponatinib.


Reference 1. Li P, Callery PS, Gan LS, Balani SK. Drug Metab Dispos. Esterase inhibition attribute of grapefruit juice leading to a new drug interaction. 2007;35(7):1203–8. ISSX 2013

P338. ENHANCEMENT OF PARAOXONASE 1 (PON1) DETOXICATION ACTIVITY TOWARD NERVE AGENT SURROGATES BY PHENOXYALKYL PYRIDINIUM OXIMES Janice E. Chambers1, Edward C. Meek2 and Howard W. Chambers3 1 Coll of Vet Medicine, Mississippi State Univ, Mississippi State, MS, 2Coll Vet Medicine, Mississippi State Univ, Mississippi State, MS, 3Biochem, Molec Biol, Entomol, Plant Pathol, Mississippi State Univ, Mississippi State, MS Novel phenoxyalkyl pyridinium oximes (patent pending) are being developed at present as reactivators of acetylcholinesterase that is inhibited by highly relevant surrogates for sarin (nitrophenyl isopropyl methylphosphonate; NIMP) and VX (nitrophenyl ethyl methylphosphonate (1). The goal of the development of this line of oximes was to synthesize acetylcholinesterase reactivators with enhanced lipophilicity that could penetrate the blood-brain barrier, and several have been developed that have shown efficacy as reactivators as well as ability to penetrate the brain in animal models (2). Paraoxonase 1 (PON1) is a calciumdependent hydrolase associated with high density lipoproteins that destroys oxidized lipids and contributes to cardiovascular health. Named for the hydrolysis of paraoxon, PON1 also has limited efficacy in hydrolyzing organophosphates, including some nerve agents. Because oximes are nucleophiles, the hypothesis for the current project is that these more lipophilic oximes could serve as better nucleophiles than water in the PON1 active site to enhance nerve agent detoxication. Commercially obtained human serum was used as a source of PON1. A direct assay using a high concentration (0.3 mM) of NIMP and NEMP monitored 4-nitrophenol spectrophotometrically as an index of surrogate detoxication. Oximes were tested at 0.1 mM. An indirect assay using low concentrations of NIMP (120 nM) and NEMP (30 nM) used the residual inhibition of exogenous acetylcholinesterase following oxime (0.1 mM) plus surrogate incubation as an index of surrogate detoxication; acetylcholinesterase activity was measured spectrophotometrically. The indirect assay allows more realistic concentrations of organophosphates to be assayed. Some substituted 6-phenoxyhexylpyridinium 4-oximes have shown enhancement of surrogate detoxication. Three oximes have resulted in 30–40% enhancement of NEMP detoxication and 25–30% enhancement of NIMP detoxication in the direct assay, while there are four oximes providing up to 10% enhancement with NEMP but minimal to no enhancement with NIMP. Preliminary results indicate that the three most effective oximes in the direct assay also provide appreciable enhancement of NIMP detoxication in the indirect assay. These novel chemistries, which are being further expanded, have promise as future drugs that could enhance the normally inefficient PON1-mediated detoxication of nerve agents by PON1, and therefore have potential as antidotes to nerve agent toxicity. (Supported by Defense Threat Reduction Agency HDTRA1-12-1-0043).

References 1. Meek, E., H. Chambers, A. Coban, K. Funck, R. Pringle, M. Ross and J. Chambers. Synthesis and in vitro and in vivo inhibition potencies of highly relevant nerve agent surrogates. Toxicol. Sci. 126(2):525–33. 2012. 2. Chambers, J., H. Chambers, E. Meek and R. Pringle. Testing of novel brain-penetrating oxime reactivators of acetylcholinesterase inhibited by nerve agent surrogates. Chemico-Biol. Interact. 203:135–8. 2013.

P339. N-HYDROXYLATION OF 4-AMINOBIPHENYL BY CYP2E1 AND ASSOCIATED OXIDATIVE STRESS MAY INFLUENCE ITS TUMORIGENICITY IN MOUSE LIVER Shuang Wang, Kim S. Sugamori and Denis M. Grant Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada


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Oxidative stress may play an important role in human liver cancer since all of the major risk factors for this disease, including viral hepatitis, alcohol, obesity and chemical carcinogen exposure, have been associated with oxidative stress. In a previous study from our laboratory, exposure of mice to two doses of the arylamine liver carcinogen 4-aminobiphenyl (ABP) on postnatal days 8 and 15 resulted in the detection of liver tumors at 1 year of age in male but not in female mice. Male mice deficient in the arylamine N-acetyltransferases NAT1 and NAT2 were also significantly protected against liver tumor growth. However, acute genotoxic effects of ABP did not correlate with the sex and strain differences in tumor incidence. Thus our overall goal is to determine whether ABP also has non-genotoxic effects, such as the induction of oxidative stress, and how these non-genotoxic effects may impact liver tumorigenesis. To determine whether oxidative stress may be involved in ABP-mediated tumorigenesis in mouse liver, we first assessed the ability of ABP to generate oxidative stress in the mouse Hepa1c1c7 hepatoma cell line. ABP did not produce reactive oxygen species (ROS) or oxidative DNA damage in Hepa1c1c7 cells. However, N-hydroxy-ABP (HOABP), an in vivo metabolite of ABP, was a potent inducer of both ROS and oxidative DNA damage. We characterized the kinetics of ABP N-hydroxylation by mouse liver microsomes and found that more than one cytochrome P450 (CYP) appears to be involved in the reaction. As predicted by the traditional model of ABP bioactivation, CYP1A2 is the high-affinity ABP N-hydroxylating enzyme, while at least one additional low-affinity CYP is responsible for significant ABP N-hydroxylation at the concentrations of ABP produced from the doses used in our tumor study. In vitro chemical inhibition studies revealed that ABP and the CYP2E1-selective substrate p-aminophenol mutually inhibited each other’s oxidation. ABP was also able to generate ROS in Hepac1c1c7 cells transiently expressing CYP2E1 but not CYP1A2. Thus our data suggest that CYP2E1 is the low-affinity ABP N-hydroxylating enzyme, and that it is capable of inducing oxidative stress from this reaction. In preliminary in vivo studies, we detected increased expression of the oxidative stress-inducible genes Nqo1 and Hmox1 in mouse liver following postnatal exposure to tumorigenic doses of ABP, supporting the involvement of oxidative stress in our tumor model. Future studies will focus on measuring sex and strain differences in oxidative stress and antioxidant levels in mouse liver following tumorigenic doses of ABP, and correlating these changes to our tumor results.

P340. DEVELOPMENT OF A NOVEL LUMINESCENT ASSAY FOR MEASURING H2O2 IN CULTURED CELLS James J. Cali1, John Shultz2, Sarah Duellman2, Gediminis Vidugiris3, Hui Wang4, Wenhui Zhou5 and Poncho Meisenheimer5 1 Promega Corporation, Madison, WI, 2R&D, Promega Corporation, Madison, WI, 3Engineering, Promega Corporation, Madison, WI, 4R&D, Promega Bioscience, SAn Luis Obispo, CA, 5R&D, Promega Bioscience, San Luis Obispo, CA The induction of reactive oxygen species (ROS) is a common mechanism of cytotoxicity that can be studied in cultured cells. Inducible ROS include H2O2, superoxide, hydroxyl radical, and singlet oxygen. H2O2 is convenient to assay because of its relatively long half-life and it is useful as a general marker of oxidative stress because various ROS are converted to H2O2 within cells. Common H2O2 detection reagents include fluorogenic probes that react directly with H2O2 or through catalysis by an enzyme such as horseradish peroxidase (HRP). The direct probes are convenient for imaging ROS induction in cells, but tend to be non-selective among the various ROS and they have limited utility in plate based assay formats because of high background signals that result in small assay windows. While HRP-dependent probes are H2O2 selective and quite sensitive, they are prone to high false hit rates in compound screening scenarios and they perform poorly in cell based assays. To overcome these shortcomings we turned to a luminescent approach that would employ firefly luciferase and a pro-substrate. The pro-substrate would be selectively activated for luciferase activity by H2O2. Based on the wellknown characteristics of luciferase assays we expected low intrinsic background signals, a wide dynamic range, and low false hit rates in compound screens. For the H2O2 probe we synthesized a cyanobenzothiazole derivative containing a borate ester and self-cleaving linker (2-ethynyl-6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)benzo[d]thiazole). The borate ester reacted directly and selectively with H2O2 to expose the linker that spontaneously eliminated a free cyanobenzothiazole. The latter moiety is a luciferin precursor that reacts rapidly with D-cysteine to form luciferin, which in turn generates light with luciferase. This probe formed the basis for a homogenous assay in which cells in a multi-well plate are first exposed to test articles (e.g. ROS inducers), then to the probe, and then to a luciferase formulation containing D-cysteine. The luciferase produces light in proportion to the H2O2 concentration of the sample and the light output is read with a luminometer. The method detected the effects of prototypical ROS inducers with sensitivity and a wide dynamic range. To test the performance of the assay in a small molecule library screen, we screened the 1280 compound LOPAC library against HepG2 cells. Cells in medium were compared to medium alone to select H2O2 modulators and to discriminate between cell-based and abiotic effects. Verification of positive compounds revealed a very low false hit rate (e.g. due to luciferase inhibition; 51%). These results indicate that our goal of developing an H2O2 assay that would obviate the limitations of fluorescent methods described above was realized. This novel cell based H2O2 assay should prove effective and practical for small scale and high throughput applications.

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P341. IRON PROCHELATOR BSIH PROTECTS CARDIAC CELLS AGAINST CATECHOLAMINEINDUCED OXIDATIVE INJURY


Pavlina Haskova1, Hana Jansova1, Jan Bures2, Miloslav Machacek1, Petra Kovarikova2 and Tomas Simunek1 1 Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic, 2Department of Pharmaceutical Chemistry and Drug Control, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic Oxidative damage plays a role in the process of catecholamine-induced cardiotoxicity due to the ability of catecholamines to undergo iron-promoted oxidation. That results in formation of unstable toxic oxidation products capable of redox cycling and reactive oxygen species (ROS) production that may be responsible for the cardiotoxicity of catecholamines. Free iron ions are significantly involved in all redox reactions leading to catecholamine oxidation and ROS formation. Iron chelation has been shown as a suitable tool of cardioprotection, which has considerable potential to protect heart cells against catecholamine cardiotoxicity (1, 2). Iron chelation offers efficient protection against harmful effects of oxidative stress on cells; however, prolonged exposure of cells to classical chelators may interfere with physiological iron homeostasis. Prochelators represent novel approach to decrease oxidative injury by activating a chelating agent only under conditions of oxidative stress when they are needed. BSIH (isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene]hydrazide is an iron prochelator, which is converted by H2O2 into chelating agent SIH (salicylaldehyde isonicotinoyl hydrazone) that efficiently inhibits ironcatalysed hydroxyl radical generation (3). In this in vitro study, we have investigated the process of prochelator BSIH activation in buffered solution by oxidation products of catecholamines epinephrine and isoprenaline into active chelating agent using calcein assay and HPLC analysis. Cellular experiments on H9c2 rat embryonic cardiomyblasts and neutral red uptake assay were used for examinations of its toxicity and the degree of protection of cardiac cells against catecholamine-induced oxidative injury. Furthermore, cellular experiments evaluated by 20 ,70 -dichlorodihydrofluorescein diacetate assay demonstrated inhibition of intracellular ROS formation. Finally, we compared BSIH with known information about SIH. Our results demonstrate ability of prochelator BSIH to efficiently protect cardiac cells against oxidative injury of catecholamines. It is able to significantly preserve cellular viability during oxidative impact of catecholamines and their oxidation products with almost equal efficiency as SIH, but with significantly reduced own toxicity in contrast with SIH. Hence, while retaining all the useful antioxidant properties of SIH, BSIH could offer solution of typical adverse effects of iron chelators related to cellular iron deficiency. This study was co-financed by the European Social Fund and the state budget of the Czech Republic. Project no. CZ.1.07/2.3.00/30.0061.

References (1) Haskova P, Kovarikova P, Koubkova L, Vavrova A, Mackova E, Simunek T: Iron chelation with salicylaldehyde isonicotinoyl hydrazone (SIH) protects against catecholamine autooxidation and cardiotoxicity. Free Radical Biology and Medicine.50(4):537–549; 2011. (2) Haskova P, Koubkova L, Vavrova A, Mackova E, Hruskova K, Kovarikova P, Vavrova K, Simunek T: Comparison of various iron chelators used in clinical practice as protecting agents against catecholamine-induced oxidative injury and cardiotoxicity. Toxicology. 289(2–3):122–131; 2011. (3) Charkoudian LK, Dentchev T, Lukinova N, Wolkow N, Dunaief JL, Franz KJ: Iron prochelator BSIH protects retinal pigment epithelial cells against cell death induced by hydrogen peroxide. Journal of Inorganic Biochemistry. 102(12):21302135; 2008.

P342. DIETARY PROTECTION BY GARLIC EXTRACT AGAINST LEAD INDUCED OXIDATIVE STRESS AND GENETIC BIRTH DEFECTS Omotade I. Oloyede1 and Oladimeji S. Tugbobo2 1 Biochemistry, Ekiti State University, Ado-Ekiti, Nigeria, 2Science Technology (Biochemistry unit), Federal Polytechnic, AdoEkiti, Nigeria The antioxidant potentials of Allium sativum extract was studied in bone marrow cells of albino rats using micronucleus assay with the use of 100 mg/ml crude garlic extract as dietary supplement via oral gavage. The rats were divided into three groups; A [distilled water], B [lead acetate], C [garlic extract þ lead acetate]. After the short-term exposure, rats were sacrificed by cervical dislocation and chromosomal preparations were made from bone marrow according to colchicine hypotonic-fixation airdrying Giemsa schedule. The cytogenic end points observed were chromosomal aberrations and tissue damage. The chromosomal aberration induced by lead was reduced significantly in animals fed with the extract in group C (2%) with [0.025 0.053]mean frequency of polychromatic erythrocytes while lead acetate administered to animals in group B was highly mutagenic with (25%) chromosomal aberration and [0.013 0.988]mean frequency of polychromatic erythrocytes respectively. Besides, the antioxidant properties of garlic was further demonstrated in the in vitro experiment where it provided significant protection to thiobarbituric reactive substances (TBARS)levels, reduced glutathione (GSH) and oxidized glutathione (GSSG)

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contents in liver tissues, suggesting their ability to act as free-radical scavengers and in protecting the cell against oxidative stress for normal cellular functions. The results thus, suggest the involvement of reactive oxygen species [ROS] in lead toxicity and also revealed the beneficial role of garlic therapeutic efficacy supporting the hypothesis that cellular redox status may be significantly reversed by utilizing a thiol-containing antioxidant compound. This research further indicates the antimutagenic and antioxidant potentials of garlic against oxidative stress and mutation.

P343. COMPARISON OF IRON CHELATOR SIH AND OXIDATIVE STRESS-ACTIVATED PROCHELATOR BSIH AGAINST OXIDATIVE STRESS-INDUCED CARDIOMYOCYTE DAMAGE


Hana Jansova1, Pavlina Haskova1, Jan Bures2, Eliska Mackova1, Petra Kovarikova2 and Tomas Simunek1 1 Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic, 2Department of Pharmaceutical Chemistry and Drug Control, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic Oxidative stress is a common denominator in various cardiovascular diseases. Free cellular iron plays a crucial catalytic role in the formation of highly toxic hydroxyl radicals, and thereby it may aggravate the cellular oxidative injury. Therefore, chelation of Fe may be an effective therapeutic approach. Salicylaldehyde isonicotinoyl hydrazone (SIH) is able to quickly penetrate through cellular membranes and efficiently chelate labile intracellular iron and protect cardiac cells against oxidative stress. However, main drawbacks of SIH are related to its rapid hydrolysis due to the labile hydrazone bond as well as due to its potential toxicity caused by iron deficiency in states without systemic iron overload. BSIH (boronyl salicylaldehyde isonicotinoyl hydrazone), is novel oxidative stress-activated pro-chelator of SIH, that has shown potential to protect cells against various oxidative stressinducing agents [1]. H9c2 rat embryonal cardiomyoblast-derived cell line was used in our experiments. Neutral Red Uptake Assay and xCELLigence System were used for assessment of cellular viability. Calcein Assay and Calcein-AM Assay were used to determine efficiency of prochelator activation by H2O2 to effective chelator in buffered solution and inside the cells. Epifluorescence microscopy was used for photodocumentation of mitochondrial inner membrane potential by JC-1 probe and lysosomal integrity by LysoTracker Blue. IC50 and EC50 were calculated using the CalcuSyn software. HPLC-UV method was used for determination of SIH and BSIH stability. Whereas SIH showed dose-dependent decrease of cellular viability, BSIH did not display any own toxicity when incubated with the cells for up to 72 hours and concentrations up to its solubility limit of 600 mM. SIH significantly protected cells against injury caused by H2O2 (200 mM) at concentrations 10 mM, whereas BSIH significantly protected H9c2 cells at concentration 60 mM and at its higher concentrations its protective effect was even more pronounced than the parent chelator SIH. We determined almost comparable chelating activity of BSIH following its activation by H2O2 to SIH in solution and in cells its activity reached even 70% of chelating activity of SIH. We also confirmed higher stability of BSIH in buffer, medium and in cells as compared to SIH. In conclusion, our results demonstrate very good properties of pro-chelator BSIH. It shows higher stability and does not display almost any own toxicity while retaining the useful protective properties of parent chelator SIH. Therefore BSIH could be applicable in prevention and/or in treatment of cardiovascular disorders with a known (or presumed) role of oxidative stress without a risk of toxicity due to the iron deficiency, which is typical adverse effect of Fe chelators. This study was supported by the Charles University grant No. 367911.

References [1] Charkoudian LK, Pham DM, Franz KJ. J Am Chem Soc. 2006 Sep 27; 128(38):12424–5. A pro-chelator triggered by hydrogen peroxide inhibits iron-promoted hydroxyl radical formation.

P344 - CYP2D6 GENE LOCUS CHARACTERIZATION BY NEXT GENERATION SEQUENCE (NGS) ANALYSIS Andrea Gaedigk1, Greyson P. Twist2, Neil A. Miller2, Darrell Dinwiddie2, Laurel K. Willig2, Emily G. Farrow2, J. Steven Leeder1 and Stephen F. Kingsmore2 1 Division of Clinical Pharmacology & Therapeutic Innovation, Children’s Mercy Hospital & Clinics, Kansas City, MO, 2 Center for Pediatric Genomic Medicine, Children’s Mercy Hospital & Clinics, Kansas City, MO Background: The human CYP2D gene locus comprises three genes, CYP2D6 encoding a major drug metabolizing enzyme and the CYP2D7 and CYP2D8 pseudogenes. Over 100 CYP2D6 allelic variants and subvariants have been described. Many carry SNPs, small indels, CYP2D7-derived conversions or a combination thereof. In addition, copy number variations (CNV) and hybrid genes composed of CYP2D6 and 2D7 are also observed in a variety of arrangements. The complexity of this gene locus poses a particular challenge for NGS platforms, specifically the mapping of the relatively short NGS sequences to the highly homologous genes. This investigation aimed to evaluate state of the art NGS technology and tools to determine their suitability for clinical application. Methods: In silico-modeling was applied to determine optimal DNA library preparation and read lengths


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that would allow accurate sequence alignment to the Hg19 and GRCh37 references. Subsequently, NGS data were generated for 19 patients and 3 Coriell samples on an Illumina HiSeq 2500 utilizing standard TruSeq DNA PCR-Free preparation kits. Sequence reads were aligned and analyzed for variants with CASAVA 1.8.2 with gapped ELAND (Illumina), patient samples were additionally analyzed with GSNAP (Genentech) and GATK 1.4 (Broad Institute). NGS data were compared to Sanger sequencing, genotype information, and quantitative CNV analysis. Results: In silico modeling predicted excessive multiple alignments across all exons and introns for sequence fragments shorter than single 100 base pair reads, whereas single reads 4250 bases produced partially interpretable data (ambiguous alignments of CYP2D6 and CYP2D7 reads were most prominent in intron 1 and exons 2 , 7 and 9). The model predicted acceptable accuracy for 2 100 paired end reads with 250 base inserts, and a decrease of incorrect alignments with an increase in size of the insert; read length was predicted to contribute to accuracy to a lesser degree. Clinical sample allele calls were accurate and concordant between NGS and Sanger. Alleles represented and accurately identified include CYP2D6*1, *2, *3, *4, *17, *33, *35, *41, *45 and *59. The CYP2D6*68 þ *4 tandem hybrid gene detected in 4 subjects by XL-PCR and CNV analysis was indirectly inferred by NGS via a characteristic misalignment pattern. Conclusions: This is the first study analyzing high-quality clinical NGS data. Using the most advanced NGS technology and an innovative combination of analysis tools we were able to accurately align and call CYP2D6 genotypes in all but one sample that carried a CYP2D6*5 gene deletion. Algorithms and software to detect copy number variations, such asCYP2D6*5, gene duplications/multiplications and gene rearrangements involving CYP2D6/7 and CYP2D7/6 fusion genes are currently being developed to ultimately determine a patient’s CYP2D6 genetic make-up in the clinical setting at 100% accuracy.

P345. INTERINDIVIDUAL VARIABILITY IN TPMT ENZYME ACTIVITY: TEN YEARS OF EXPERIENCE WITH THIOPURINE PHARMACOGENETICS AND THERAPEUTIC DRUG MONITORING Laurent Chouchana1, Ce´line Narjoz2, Denis Roche2, Brigitte Pineau3, Gilles Chatellier4, Philippe Beaune1 and Marie-Anne Loriot1 1 Inserm Umr-S775, Universite´ Paris Descartes, Sorbonne Paris Cite´, Paris, France, 2Biochimie, pharmacogeńe´tique et oncologie molećulaire, Hopital Europeén Georges Pompidou, Assistance Publique-Hopitaux de Paris, Paris, France, 3De´partement d’informatique hospitalie`re, Hopital Europeén Georges Pompidou, Assistance Publique-Hopitaux de Paris, Paris, France, 4Unite´ de recherche clinique, Hopital Europeén Georges Pompidou, Assistance Publique-Hopitaux de Paris, Paris, France Background: Interindividual variability in clinical response to thiopurines, including azathioprine (AZA), is related to differences in TPMT enzyme activity, which governs the production of active metabolites (6-thioguanine nucleotides [6-TGN] and 6methylmercaptopurine nucleotides [6-MMPN]). Both genetic and nongenetic determinants can influence TPMT activity. Methods: A retrospective review of the laboratory database of a French university hospital identified 7360 patients referred for TPMT phenotype and genotype determination, and for 6-TGN and 6-MMPN monitoring. We assessed associations linking demographic and clinical variables to TPMT activity, and we evaluated the impact of TPMT variability on metabolite levels. Results: TPMT activity (n ¼ 4911) was slightly higher in males, children younger than 5 years, and cystic fibrosis patients. Four TPMT phenotypes were identified: low, intermediate, normal/high, and very high activity. Wild-type patients displayed 1.7- and 4.5-fold higher mean TPMT activity than did heterozygotes and mutant homozygotes, respectively. Overall genotype-phenotype concordance was 93.4%, with 27% discordance for heterozygotes. Based on 6775 assays, mean 6-TGN concentrations were 1.6fold higher in TPMT-deficient patients compared to TPMT-normal patients. An unfavorable 6-MMPN:6-TGN ratio ( 20) (17.4% of patients) was associated with higher mean TPMT activity. 6-MMPN and, consequently, 6-MMPN:6-TGN were 1.6and 2.2-fold higher in females than males, despite similar mean 6-TGN levels and AZA doses. Conclusion: TPMT activity and sex strongly influences the thiopurine metabolite production. Our results highlight the usefulness of therapeutic drug monitoring to increase the proportion of patients within the 6-TGN therapeutic range and to identify patients at high risk for toxicity or therapeutic resistance.

P346. A PHARMACOGENOMICS STUDY OF SEVERE TOXICITY IN METASTATIC COLORECTAL CANCER PATIENTS TREATED WITH 5-FLUOROURACIL/IRINOTECAN (FOLFIRI)-BASED REGIMENS: RESULTS OF A PROSPECTIVE STUDY Sylvia Chen1, Mario Harvey1, E´ric Le´vesque2, Isabelle Laverdiere1, Felix Couture2, Derek J. Jonker3 and Chantal Guillemette1 1 Center Hospitalier de l’Universite Laval Research Center and Faculty of Pharmacy, Laval University, Quebec, QC, Canada, 2 Center Hospitalier de l’Universite Laval Research Center and Faculty of Medicine, Laval University, Quebec, QC, Canada, 3 Hematology-Oncology Department, Ottawa Hospital Cancer Center and Ottawa University, Ottawa, ON, Canada Current pharmacogenomic studies on irinotecan have repeatedly established a predictive UGT1A1*28 variant associated with severe neutropenia while the contribution of additional pathways is still unresolved. Our goal was to assess association of germline variations in a series of genes involved in irinotecan metabolism, transport and action, and their influence on drug response and toxicity. We studied a cohort of 167 advanced colorectal cancer (CRC) patients receiving FOLFIRI-based regimens. A haplotype-tagging Single Nucleotide Polymorphism (htSNPs) strategy was used to maximize coverage of 38

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candidate genes with 390 htSNPs selected from the International HapMap Project. Genotyping was performed by SequenomÕ iPLEX matrix-assisted laser desorption/ionization–time-of-flight mass spectroscopy. We report novel markers namely within the common region of the UGT1A locus associated with a lower risk of grade 3–4 neutropenia (ORs ranging from 0.18–0.35; p50.04). Haplotype analyses allowed the identification of 3-marker UGT1A haplotypes either protective or conferring a higher risk of severe neutropenia (ORs of 0.21; p ¼ 0.04 and 8.95; p ¼ 0.004). Furthermore, several variants in genes coding for ABC transporters (ABCG1, ABCC1, ABCG2), carboxylesterases (CES1 and CES2) and DNA repair proteins (PARP1, RECQL5) were associated with an altered risk of grade 3–4 neutropenia. A replication study is ongoing in a larger cohort of FOLFIRI-treated CRC patients. Ultimately, these findings may help improve therapeutic strategies in managing irinotecan-based treatments and predicting toxicity. This work was supported by CIHR and The Canada Research Chair program.


P347. MRP2/ABCC2 GENETIC VARIABILITY INFLUENCES BREAST CANCER SURVIVAL Isa Cavaco1, Hege Edvardsen2, Magnus Ingelman-sundberg1, Vessela Kristensen3 and Sarah Sim1 1 Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 2Department of Genetics, Institute for Cancer Research, OUS Radiumhospitalet, Oslo, Norway, 3The K.G. Jebsen Center for Breast Cancer Research, University of Oslo, Oslo, Norway Multidrug resistance-associated protein 2 (MRP2) or ATP-binding cassette sub-family C member 2 (ABCC2) is a protein encoded by the ABCC2 gene and it is involved in multi-drug resistance. In vitro studies have shown that MRP2 transports a variety of anticancer drugs, including methotrexate, cisplatin, irinotecan and paclitaxel (reviewed [1]). Furthemore, MRP2 was shown to be overexpressed in tamoxifen (TAM) resistant MCF-7 cell line [2] and a polymorphism in the ABCC2 gene (rs3740065) was associated with clinical outcome in a Japanese cohort and suggested to predict the prognosis of patients treated with TAM [3]. These results motivated a large study in a Caucasian cohort and here we determined 37 SNPs in the ABCC2 gene in 1568 Norwegian breast cancer patients using the Illumina 660 K SNP array. Part of these patients was also genotyped for the rs3740065 SNP using a commercially available TaqManÕ assay (C_22271640_10, Life Technologies). A number of SNPs included in the array were not present in the patients analyzed, e.g. rs17216156, rs17222674, rs7080681, rs17216198, rs17222547, rs3740070, rs8187699, rs7899457, rs17216296, rs4148383, rs4148387, rs4148393, rs3740072 and rs4148402. From the remaining SNPs, rs8187706 and rs8187709 were present at very low frequencies (51%), whereas the rest of the SNPs were present at higher frequencies (from 5 up to 80%). Using Haploview [4] we performed analysis of the ABCC2 haplotypes and linkage disequilibrium (LD) between the different SNPs included in the array and found that only one SNP was nonsynonymous whereas all others were present in the 50 or intronic regions. When performing univariate analysis of ABCC2 genotype and adjuvant therapy outcome, significant correlations for poor breast cancer specific survival were found for several SNPs (e.g. rs717620, rs2804400 and rs2756114) Univariate analysis of subjects receiving cyclophosphamide, methotrexate and 5-fluorouracil (CMF) similarly showed that some of the ABCC2 SNPs (e.g. rs2756107) correlate with poor breast cancer specific survival. This study indicates an association between ABCC2 polymorphism and breast cancer treatment outcome (both for adjuvant and chemotherapy) among Caucasians. Further functional analyses are required in order to identify the basis for the observed associations.

References 1. 2. 3. 4.

Chen ZS, Tiwari AK. 2011. FEBS J. 278: 3226–45. Choi HK, Yang JW, Roh SH, Han CY, Kang KW. 2007. Endocr Relat Cancer 14: 293–303. Kiyotani K, Mushiroda T, Imamura CK, Hosono N, Tsunoda T, et al. 2010. J Clin Oncol 28: 1287–93. Barrett JC, Fry B, Maller J, Daly MJ. 2005. Bioinformatics 21: 263–5.

P348. EFFECTS OF UGTS AND DRUG TRANSPORTERS POLYMORPHISMS ON PHARMACOKINETICS OF TELMISARTAN IN KOREAN HEALTHY VOLUNTEERS Jae-Eun Kim1, Ho-Jung Shin1, Su Jeong Lim1, Ji Eun Seo2, Min Hye Kim1, Kyung Hoon Oh1, Ho Sook Kim1, Dong-Hyun Kim1 and Jae Gook Shin1 1 Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Busan, South Korea, 2Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Busan, South Korea Telmisartan, an angiotensin II receptor blocker, is widely used for the treatment of hypertension and cardiovascular risk reduction. It is mainly metabolized by UDP-glucuronyl transferases (UGTs) and UGT1A3 is predominantly involved in the glucuronidation of telmisartan. Clinical studies showed the large inter-individual variation in the exposure of telmisartan when orally administered. The purpose of present study was to examine whether telmisartan exposure could be affected by genetic polymorphisms of UGTs and drug transporters. The blood samples were collected from 45 healthy volunteers who participated in a study of telmisartan bioequivalence study (80 mg) and genotyping were conducted in drug transporters (ABCC1, SLC22A1,

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SLCO1B3 and SLCO2B1) and UGTs (UGT1A1 and UGT1A3). All SNPs in the hepatic uptake of telmisartan (ABCC1 2168G4A, SLC22A1 14676C4T, SLCO1B3 -5035A4G,47088G4A, 42808T4G, 77022A4G and SLCO2B1 21151G4A) had no effects on the area under the plasma concentration-time curve (AUC) of telmisartan. Unexpectedly, the AUC0–48 in volunteers carrying one or two UGT1A1*28 alleles was significantly lower than that with UGT1A1*1/*1 participants (wild, 2813 1158; hetero, 1414 490; homo, 1130 247, p ¼ 0.0004). A similar phenomena was observed in UGT1A1*60 variant (wild, 3220 1021; hetero, 2407 1174; homo, 1720 468, p ¼ 0.0002). Heterozygotes of UGT1A3*2a also showed significantly lower AUC0–48 (wild, 3172 1044; hetero, 1201 214, p50.0001). Furthermore, Linkage disequilibrium analyses demonstrated a strong linkage betweenUGT1A3 haplotypes and UGT1A1 variants; UGT1A3*2a and UGT1A1*28 (D’ ¼ 0.945, r2 ¼ 0.705), and UGT1A3*2a and UGT1A1*60 (D’ ¼ 1, r2 ¼ 0.371). Our results collectively suggested that the effects of UGT1A1*28 and UGT1A1*60alleles on the pharmacokinetics of telmisartan were attributed to their strong linkage with UGT1A3*2a which enhances UGT1A3 expression.

P349. FREQUENCIES AND METABOLIC ACTIVITIES OF THE CYP2D6 GENOTYPES IN KOREANS


So-Young Park, Hye-In Lee, Mi-Jung Kim, Ji-Yeong Byeon, Chang-Ik Choi, Se Hyung Kim and Seok-Yong Lee School of Pharmacy, Sungkyunkwan University, Suwon, South Korea More than 100 different human CYP2D6 variant and subvariant alleles have been identified. Among them, the CYP2D6*2, *5, and *10 alleles, along with CYP2D6 gene duplication, are the most clinically important and widely distributed polymorphisms in East Asians, including Koreans. We determined the genotypes of 1901 Korean subjects and compared the metabolic activity of CYP2D6 in different CYP2D6 genotypes, by measuring the metabolic capacity of metoprolol, a CYP2D6 substrate. In Koreans, most frequently observed genotypes were CYP2D6*wt/*wt (24.7%, 95% CI 22.7–26.7), *wt/*10 (43.3%, 95% CI 41.0–45.6) and *10/*10 (25.0%, 95% CI 23.0–27.0). In addition, the frequency of CYP2D6*wt/*5 and *5/*10 was 3.1% (95% CI 2.3–3.9) and 1.9% (95% CI 1.3–2.5), respectively. Thirty-seven healthy Korean subjects with six different CYP2D6 genotypes (CYP2D6*wt/ *wt, *wt/*10, *wt/*5, *10/*10, *5/*10, and *5/*5) received a single oral dose of metoprolol 100 mg, and plasma concentrations of metoprolol were determined up to 24 hours by using high-performance liquid chromatography with fluorescence detection. Oral clearance (CL/F) of metoprolol in CYP2D6*wt/*10, *wt/*5, *10/*10, *5/*10 and *5/*5 group was 66%, 53%, 22%, 13% and 8% of that in CYP2D6*wt/*wt genotype group, respectively. Area under the plasma concentration-time curve (AUCinf) of metoprolol in each genotype group was 1.4-, 1.7-, 4.3-, 7.0- and 11.4-fold higher than CYP2D6*wt/*wt group, respectively. In conclusion, the enzymatic activity in CYP2D6*wt/*10, referred as a heterozygous extensive metabolizer (EM) genotype, was close to that in CYP2D6 intermediate metabolizer (IM) rather than EM, and CYP2D6*10/*10, referred as homozygous IM, was close to that in CYP2D6 poor metabolizer (PM).

P350. EFFECTS OF CYP3A5 GENETIC POLYMORPHISMS ON THE PHARMACOKINETICS OF CILOSTAZOL AND ITS ACTIVE METABOLITES Se Hyung Kim, Hye-In Lee, Mi-Jung Kim, So-Young Park, Joo-Yeon Lee, Jung-Eun Lee, Ji-Yeong Byeon, Choon-Gon Jang and Seok-Yong Lee School of Pharmacy, Sungkyunkwan University, Suwon, South Korea Cilostazol inhibits platelet aggregation by inhibition of phosphodiesterase type III. The metabolic pathway of cilostazol is very complex and CYP3A4, 2C19 and 3A5 are involved in the biotransformation of cilostazol. Of 11 known metabolites of cilostazol, OPC-13015 and OPC-13213 are considered to be pharmacologically active. In this study, the effects of CYP3A5 genetic polymorphism on the pharmacokinetics of cilostazol and its two active metabolites were determined. Fourteen CYP3A5 expressors and nineteen CYP3A5 non-expressors were selected for this study. After overnight fasting, each subject received a single 100 mg oral dose of cilostazol. Blood samples were collected up to 48 hours after drug intake and plasma concentrations of cilostazol and its two active metabolites were determined by using LC-MS/MS analytical system. Cmax, CL/F, and AUCinf of cilostazol were not different between two CYP3A5 genotype groups (CYP3A5 expressors and CYP3A5 non-expressors). Cmax and AUCinf of OPC-13015 and OPC-13213 were not different in two genotype groups. The potency-adjusted total active moiety of cilostazol was also not different in two genotype groups. These results suggest that the CYP3A5 genetic polymorphisms may not affect therapeutic effects of cilostazol.

P351. SIGNIFICANTLY HIGHER EXPOSURE OF ACTIVE MOIETY OF CILOSTAZOL IN CYP2C19PMCYP3A5 NON-EXPRESSOR Jung-Eun Lee, Hye-In Lee, So-Young Park, Mi-Jung Kim, Se Hyung Kim, Joo-Yeon Lee, Ji-Yeong Byeon, Choon-Gon Jang and Seok-Yong Lee School of Pharmacy, Sungkyunkwan University, Suwon, South Korea

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Cilostazol is prescribed to inhibit the platelet aggregation. It shows a considerable interindividual variation in bioavailability and biotransformation and it is metabolized mainly by CYP3A4, 2C19 and 3A5. Of 11 known metabolites of cilostazol, OPC-13015 and OPC-13213 are considered to be pharmacologically active. We investigated the effects of CYP2C19 and CYP3A5 genetic polymorphisms on the pharmacokinetics of cilostazol and its two active metabolites. Thirty-three healthy Korean volunteers were administered a single 100-mg oral dose of cilostazol. Blood samples were collected up to 48 hours after drug intake and plasma concentrations of cilostazol and its two active metabolites were determined by using LC-MS/MS analytical system. In CYP2C19EM-CYP3A5 non-expressor group, the pharmacokinetic parameters of cilostazol, OPC-13015 and OPC-13213 were not different with those in CYP2C19EM-CYP3A5 expressor group. In CYP2C19PM-CYP3A5 expressor group, AUCinf of OPC-13015 was significantly higher than in CYP2C19PM-CYP3A5 non-expressor group, but those of cilostazol and OPC-13213 were not. In CYP2C19PM-CYP3A5 expressor group, Cmax and AUCinf of OPC-13015 were significantly higher and those of OPC-13213 were significantly lower than in any other three haplotype groups. The potency-adjusted total active moiety of cilostazol was the greatest in subjects with CYP2C19PM-CYP3A5 non-expressor group. These results suggest that CYP2C19 and CYP3A5 genetic polymorphisms significantly affect the therapeutic effects of cilostazol total active moiety and CYP2C19 plays a crucial role in the biotransformation of cilostazol.

P352. ASSOCIATION OF BREAST CANCER WITH CYTOCHROME P450 2C19 GENETICS – RISK AND RESPONSE TO ADJUVANT THERAPY Isa Cavaco1, Hege Edvardsen2, Magnus Ingelman-Sundberg1, Vessela Kristensen3 and Sarah Sim1 1 Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 2Department of Genetics, Institute for Cancer Research, OUS Radiumhospitalet, Oslo, Norway, 3The K.G. Jebsen Center for Breast Cancer Research, University of Oslo, Oslo, Norway CYP2C19 polymorphism has been associated with reduced breast cancer risk (CYP2C19*17; (1, 2)). In terms of the effect of CYP2C19 genotype on clinical response to adjuvant tamoxifen therapy, the studies performed have been inconclusive and beneficiary roles for both the defective CYP2C19*2 allele (3, 4) and the increased-activity CYP2C19*17 allele (5) have been suggested, whereas others have shown no association (6–8). We aimed in this study to analyze the effect of CYP2C19 alleles on the overall risk for breast cancer as well as on tamoxifen treatment outcome using a large collection of Norwegian DNA samples from healthy donors and from breast cancer (BC) patients. Genotyping analysis for CYP2C19*2 and CYP2C19*17 was performed in 1828 controls and 2154 breast cancer cases. In a subgroup of the BC patients (n ¼ 667), the association betweenCYP2C19 alleles and clinical outcome (time to any relapse and time to death by BC) was evaluated. CYP2C19*3 and CYP2C19*4 variants were also analyzed in a subgroup of the samples. The results confirmed the rarity of the 2C19*3 and 2C19*4 alleles in the Nordic populations and due to their rarity were excluded in the statistical analysis. CYP2C19*2 and 2C19*17 were present in both cases and control samples at expected allelic frequencies (14.7% for CYP2C19*2 and 19.8% for CYP2C19*17 in controls and 15.6% for *2 and 20.3% for *17 in cases). The risk of developing BC was not associated with CYP2C19 alleles, neither when the analyses were stratified for age at diagnosis, ER, HER2, nor postmenopausal status. Univariate analysis for breast cancer survival or recurrence and CYP2C19 genotype did not show any statistically significant associations. Cox analysis using node status and systemic treatment as co-variates did not result in any significant hazard ratio for the different CYP2C19 genotypes. The results obtained in this large study indicate no association betweenCYP2C19 genotype and the outcome of adjuvant tamoxifen therapy in breast cancer.

References 1. 2. 3. 4. 5. 6. 7. 8.

Justenhoven C, Hamann U, Pierl CB, Baisch C, Harth V, et al. 2009. Breast Cancer Res Treat 115: 391–6. Justenhoven C, Obazee O, Winter S, Couch FJ, Olson JE, et al. 2012. Breast Cancer Res Treat 131: 347–50. Ruiter R, Bijl MJ, van Schaik RH, Berns EM, Hofman A, et al. 2010. Pharmacogenomics 11: 1367–75. van Schaik RH, Kok M, Sweep FC, van Vliet M, van Fessem M, et al. 2011. Pharmacogenomics 12: 1137–46. Schroth W, Antoniadou L, Fritz P, Schwab M, Muerdter T, et al. 2007. J Clin Oncol 25: 5187–93. Moyer AM, Suman VJ, Weinshilboum RM, Avula R, Black JL, et al. 2011. Pharmacogenomics 12: 1535–43. Serrano D, Lazzeroni M, Zambon CF, Macis D, Maisonneuve P, et al. 2011. Pharmacogenomics J 11: 100–7. Lim JS, Chen XA, Singh O, Yap YS, Ng RC, et al. 2011. Br J Clin Pharmacol 71: 737–50.

P353. CYP2D6*10 ALLELE SIGNIFICANTLY INCREASES THE EXPOSURE OF TOLPERISONE Ji-Yeong Byeon, Jung-Eun Lee, Joo-Yeon Lee, Se Hyung Kim and Seok-Yong Lee School of Pharmacy, Sungkyunkwan University, Suwon, South Korea Tolperisone, a centrally acting muscle relaxant, is used for relieving spasticity of neurological origin and muscle spasm associated with painful locomotor diseases. The biotransformation of tolperisone is mainly mediated by CYP2D6, and to a lesser

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extent, CYP2C19, CYP1A2, and CYP2B6. We investigated the effects of CYP2D6*10 allele on the pharmacokinetics of tolperisone. Thirty-three subjects with CYP2D6*wt/*wt (*wt ¼ *1 or *2), fifty-four subjects withCYP2D6*wt/*10 and forty-one subjects with CYP2D6*10/*10 were selected for this study. After overnight fasting, each subject received a single 150 mg oral dose of tolperisone. Blood samples were collected up to 12 hours after drug intake, and plasma concentrations of tolperisone were determined by using LC-MS/MS analytical system. Cmax and AUCinf of tolperisone in CYP2D6*10/*10 genotype group were significantly higher than those in CYP2D6*wt/*wt group (p50.001). Apparent oral clearance (CL/F) of tolperisone in CYP2D6*wt/*10 and CYP2D6*10/*10 group was 47% and 76% lower than that in CYP2D6*wt/*wt group, respectively (p50.001). Tolperisone is mainly metabolized by CYP2D6 and CYP2D6*10 allele significantly decreases the metabolism of tolperisone.

P354. EFFECTS OF CYP2D6-CYP2C19 GENOTYPES ON THE PHARMACOKINETICS OF TOLPERISONE


Jung-Eun Lee, Hye-In Lee, Ji-Yeong Byeon, Joo-Yeon Lee and Seok-Yong Lee School of Pharmacy, Sungkyunkwan University, Suwon, South Korea In the previous study, inter-individual variabilities in the pharmacokinetics of oral tolperisone have shown in healthy Korean volunteers. Tolperisone is mainly metabolized by cytochrome P450 (CYP) 2D6 and to a minor extent, metabolized by CYP2C19 and CYP1A2. In this study, the effects of each CYP’s genetic polymorphism on the pharmacokinetics of tolperisone were evaluated. CYP2D6 and CYP2C19 genotyping were performed in over 1,900 healthy Korean volunteers by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and long-PCR. 128 healthy volunteers were recruited and all subjects were given a 150-mg single oral dose of tolperisone. Plasma concentration of tolperisone was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In all genotypes of CYP2C19 (EM, IM and PM), Cmax and AUCinf of tolperisone in CYP2D6*10/*10 group were significantly higher than those in CYP2D6*wt/*wt and *wt/*10 groups, but there are no difference between CYP2D6*wt/*wt and *wt/*10. Similarly, in all the genotypes of CYP2D6 (*wt/*wt, *wt/*10 and *10/*10), Cmax and AUCinf of tolperisone inCYP2C19PM group were significantly higher than those in CYP2C19EM and IM groups, but there are no difference between CYP2C19EM and IM. These results suggest that CYP2D6 and 2C19 are important role in tolperisone metabolism and the PM of both enzymes have markedly increased plasma concentration of tolperisone.

P355. THE EXPOSURE OF TOLPERISONE WAS SIGNIFICANTLY INCREASED IN CYP2C19 POOR METABOLIZERS Jung-Eun Lee, Joo-Yeon Lee, Se Hyung Kim, Ji-Yeong Byeon and Seok-Yong Lee School of Pharmacy, Sungkyunkwan University, Suwon, South Korea Tolperisone is a centrally acting muscle relaxant indicated in the treatment of acute muscle spasms in back pain and spasticity in neurological diseases. Although tolperison is known that it is mainly metabolized by CYP2D6, CYP2C19, a highly polymorphic enzyme, is also involved in the biotransformation of tolperisone. We investigated the effects of CYP2C19 genetic polymorphism on the pharmacokinetics of tolperisone. 128 healthy Korean subjects were selected and divided into three different groups according to CYP2C19 genotype, CYP2C19EM (CYP2C19*1/*1, n ¼ 51), CYP2C19IM (CYP2C19*1/*2 or *1/*3, n ¼ 52), and CYP2C19PM (CYP2C19*2/*2, *2/*3 or *3/*3, n ¼ 25). After overnight fasting, each subject received a single 150 mg oral dose of tolperisone. Blood samples were collected up to 12 hours after drug intake, and plasma concentrations of tolperisone were measured by using LC-MS/MS analytical system. Cmax of tolperison in CYP2C19PM group was significantly higher that in CYP2C19IM and CYP2C19EM (p50.001 for all). AUCinf of tolperisone in CYP2C19PM was also significantly higher than that in CYP2C19IM andCYP2C19PM group (p50.001 for all). Cmax and AUCinf of tolperisone in CYP2C19EM and CYP2C19IM groups were not different. Apparent oral clearance (CL/F) of tolperisone in CYP2C19PM group was 65% lower than that inCYP2C19IM group. The pharmacokinetics of tolperisone is significantly affected by CYP2C19 genotypes. Particularly, CYP2C19PM subjects had markedly increased plasma concentration of tolperisone compared with CYP2C19EM orCYP2C19IM subjects.

P356. NAT2 GENE POLYMORPHISMS IN MEXICAN MESTIZO POPULATION AND THEIR RELATION TO ANTI-TUBERCULOSIS THERAPY Raul Salazar-Gonza´lez1, Diana Patricia Portales-Perez1, Silvia Romano-Moreno1, Rocio Gomez-Ortega2, Martin MaganãAquino3, Susanna Medellıń-Garibay1, Aleida Nun˜ez-Ruiz1, Christian Rodriguez-Pinal1 and Rosa del Carmen Milan-Segovia1 1 Facultad de Ciencias Quı´micas, UASLP, SLP, Mexico, 2Cinvestav, Laboratorio de Genomica Poblacional, 3Departamento de Epidemiologıá, Hospital Central ‘‘Dr. Ignacio Morones Prieto’’


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Tuberculosis (TB) is a common disease in developing countries like Mexico. The anti-tuberculosis therapy includes isoniazid as one of the principal drugs. Isoniazid is metabolized by the second phase enzyme arylamine N-acetyltransferase 2 (NAT2). This enzyme presents numerous variations in its sequence.1 The presence of NAT2 Single Nucleotide Polymorphism (SNP) is responsible for the enzyme decreased efficiency. The high interethnic and intraethnic variations in the sequence of this enzyme makes it interesting to study its distribution in different populations.2 Hypothesis: The presence of NAT2 SNPs will permit to assign the acetylation phenotype in order to establish the response to the TB therapy in Mexican population. The aim of the present work is to determine the distribution and frequency of 282C4T, 341T4C, 481C4T, 590G4A, 803A4G, 857G4A, NAT2 SNPs that permit to assign an acetylation phenotype in Mexican population.3,4 Blood samples were obtained from 40 TB diagnosed unrelated patients and 121 unrelated healthy volunteers that signed an informed consent. We performed genotyping assays by real-time PCR using Taqman reagents. The results showed a 39.6% of slow acetylators, 45.5% of intermediate and 14.9% were rapid acetylators in the group of healthy volunteers. The most common haplotypes present in this group were *4, *5B, *6A. In contrast, for the TB patient group, the results showed a 35% of rapid acetylators, 32.5% of intermediate and 32.5% of slow acetylators. The most common haplotypes present in the TB groups were *4 and *5B. The high prevalence of the slow acetylator haplotypes determined in the group of study demonstrates a higher number of slow and intermediate acetylatorphenotypes. The population with the slow acetylators phenotype must be monitored during the anti-tuberculosis therapy due to the adverse effects that Isoniazid might produce, especially in the liver. In conclusion, the presence of slow and intermediate acetylator phenotype in Mexican population is critical in the anti-tuberculosis therapy.

References 1. Kinzig-Schippers et al. Should We Use N-Acetyltransferase Type 2 Genotyping To Personalize Isoniazid Doses? ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2005, p. 1733–1738. 2. Butcher et al. Pharmacogenetics of the arylamine N-acetyltransferases. THE PHARMACOGENOMICS JOURNAL. 2002, 2, 30–42 3. Hein et al. Changes in consensus arylamine N-acetyltransferase (NAT) gene nomenclature. PHARMACOGENETICS & GENOMICS. 2008; 18(4): 367–368. 4. http://louisville.edu/medschool/pharmacology/consensus-human-arylamine-n-acetyltransferase-gene-nomenclature/

P357. EFFECTS OF CYP2C19 GENETIC POLYMORPHISMS ON THE PHARMACOKINETICS OF CILOSTAZOL AND ITS ACTIVE METABOLITES Mi-Jung Kim, Hye-In Lee, Se Hyung Kim, So-Young Park, Joo-Yeon Lee, Jung-Eun Lee, Ji-Yeong Byeon, Choon-Gon Jang and Seok-Yong Lee School of Pharmacy, Sungkyunkwan University, Suwon, South Korea Cilostazol is a cyclic nucleotide phosphodiesterase type III inhibitor leading to inhibition of platelet aggregation. The metabolic pathway of cilostazol is very complex and many cytochrome P450 (CYP) isozymes are involved in the biotransformation of cilostazol. Of 11 known metabolites of cilostazol, OPC-13015 and OPC-13213 are considered to be pharmacologically active. In this study, the effects of CYP2C19 genetic polymorphism on the pharmacokinetics of cilostazol and its two active metabolites were determined. Seventeen CYP2C19 extensive metabolizers (EM) and sixteen CYP2C19 poor metabolizers (PM) were selected for this study. After overnight fasting, each subject received a single 100 mg oral dose of cilostazol. Blood samples were collected up to 48 hours after drug intake, and plasma concentrations of cilostazol and its two active metabolites were determined by using LC-MS/MS analytical system. Although the pharmacokinetic parameters of cilostazol were similar in different CYP2C19 genotypes, CYP2C19PM subjects showed significantly higher AUC0–1 for OPC-13015 and lower AUC0–1 for OPC-13213 compared to those inCYP2C19EM subjects (p50.01 and p50.001, respectively). The potency-adjusted total active moiety of cilostazol was significantly higher in CYP2C19PM subjects than in CYP2C19EM subjects. These results suggest that theCYP2C19 genetic polymorphisms may affect the therapeutic effects of cilostazol.

A AND POR 831 -35C > T SIGNIFICANTLY INCREASE WARFARIN MAINTENANCE DOSES",5,2,1,0,150mm,150mm,1mm,100mm>P358. NR1I2 7635G4A AND POR 831 -35C4T SIGNIFICANTLY INCREASE WARFARIN MAINTENANCE DOSES Li-zi Zhao1, Xiuyu Leng2, Qishan Zheng1, Hanjing Cen1 and Min Huang1 1 Sun Yat-sen University, Institute of Clinical Pharmacology, Guangzhou, China, 2the First Affiliated Hospital of Sun Yat-sen University, Department of Cardiology, Guangzhou, China Objective: Genetic factors play an important role in the inter-individual variability of warfarin maintenance doses. CYP2C9*3, VKORC1 -1639G4A, CYP4F2 V433M significantly contributed to dose variability in our previous study. However, with

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approximately 50% of the dose variance unexplained, the current dosing algorithms are far from clinical guide. New factors need further exploration. NR1I2 encodes PXR, which regulates the expression of CYPs. PXR was reported to be the shared regulating elements of CYP3A4 and CYP2C9. NR1I2 -25385C4T, 7635G4A which were associated with changed CYP3A4 induction worth considering their impact on warfarin dose variability. Besides, POR, the only electron supplier of CYPs, was reported to account for altered CYPs activity. It was reported that POR A503V affected most CYPs’ activity, while POR 831 -35C4T specially led to increased CYP2C9 activity. Their influence on warfarin dose variability worth exploring as well. In sum, this study aimed to investigate the impact of PXR, POR genetic polymorphisms on warfarin dose variability. Methods: A total of 188 Han Chinese patients with mechanical heart valve replacement were recruited. NR1I2 -25385C4T, 7635G4A and POR A503V were detected by direct sequencing while POR 831 -35C4T,CYP2C9*3 and VKORC1 -1639G4A by PCR-RFLP. Person correlation, t-test and ANOVA were performed to analyze the impact on warfarin maintenance dose. Results: NR1I2 7635G4A significantly increased warfarin maintenance doses. A-allele carriers took higher doses (3.0 mg/d in GA, 2.9 mg/d in AA) than wildtype carriers (2.5 mg/d in GG). POR 831 -35C4T was associated with increased dose as well, with C-allele carriers requiring higher doses (2.9 mg/d in CT, 3.1 mg/d in TT) than wildtype carriers (2.6 mg/d in CC). No significant statistical difference was found among genotypes ofNR1I2 -25385C4T, POR A503V respectively. Considering the obvious influence of CYP2C9*3 and VKORC1 -1639G4A on dose variability, the effect of NR1I2 -25385C4T, 7635G4A, POR A503V and POR 831 -35C4T was further examined in subgroup where the genotypes of CYP2C9*3 and VKORC1 -1639G4A were fixed. In CYP2C9*1*1/VKORC1 -1639GA group (n ¼ 34), no significant statistical difference was found among genotypes of any SNPs. In CYP2C9*1*1/VKORC1 -1639AA (n ¼ 134), the mutated allelic carriers of NR1I2 7635G4A and POR 831 -35C4T still took significantly higher doses than wildtype carriers. Conclusion: NR1I2 7635G4A and POR 831 -35C4T are associated with increased warfarin maintenance doses.

P359. THE EFFECT OF HYPERLIPIDEMIA ON METFORMIN PHARMACOKINETICS IN RATS Raniah Q. Gabr and Dion R Brocks Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada In humans, the renal clearance (CLr) of metformin in obese patients is apparently lower than in lean subjects. We hypothesized that this is due to hyperlipidemia (HL), which is prevalent in obese patients. Giving that metformin excretion is transporter-dependent, HL might have a suppressive effect on the expression of SLC kidney transporters responsible for renal clearance such as organic cation transporters (OCT) 1 and 2, and multidrug and toxin extrusion protein (MATE) 1. The purpose of this study was to investigate the effect of HL on metformin pharmacokinetic parameters and m-RNA expression of the excretory transporters in rats. In the pharmacokinetic experiment 2 days prior to study, male Sprague Dawley rats (525 g) were administered poloxamer 407 (P407) 1 g/kg as intraperitoneal (ip) injection to induce HL. The day before the study, cannulae were implanted into the right jugular veins of P407 treated and control (saline ip) rats. Rats were given 30 mg/kg of metformin intravenously on the study day, followed by collection of blood samples and urine for up to 24 hours after dosing. Plasma and urine samples were assayed for metformin using a validated HPLC method. Real time PCR was used to measure m-RNA of kidney transporters. At 36 and 72 hours after P407 treatment, kidneys were harvested, homogenized and mRNA was isolated and measured. Complementary DNA was synthesized and by the aid of PCR. Quantitative measurements were performed for the required mRNA transporters and the fold of induction was calculated. The following pharmacokinetic results were obtained for metformin (mean SD): Group Control (n ¼ 5) HL (n ¼ 4)

CLr (mL/h/kg)

Vdss (L/kg)

fr (%)

t (h)

1587 472 1266 416

1.06 0.86 4.7 5.3

87.3 9.7 86.2 16.2

5.18 5.19 21.5 22.8

P-407 pretreatment caused no significant change in metformin pharmacokinetics. On the other hand, HL significantly induced m-RNA expression for OCT-1 protein in kidney of rats 72 h after P407 (67% increase). No changes were seen for OCT-2 and MATE-1. In conclusion, HL did not affect the pharmacokinetics of metformin after IV dosing, although it induced the m-RNA expression of OCT-1 in rat kidney.

P360. CYP2E1 IN HUMAN LIVER: A GENE-DOSAGE INSENSITIVE GENE Roman Tremmel, Kathrin Klein, Stefan Winter, Matthias Schwab and Ulrich M. Zanger Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany CYP2E1 IN HUMAN LIVER: A GENE-DOSAGE INSENSITIVE GENE Roman Tremmel1, Kathrin Klein1, Stefan Winter1, Matthias Schwab1,2, Ulrich M. Zanger1 1Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany 2Department of Clinical Pharmacology, University Hospital, University of Tuebingen, Tuebingen, Germany Cytochrome P450


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(CYP) 2E1, is mainly responsible for the oxidation of low-molecular-weight lipophilic compounds, including ethanol, acetone and drugs like chlorzoxazone and acetaminophen. Furthermore, CYP2E1 is an effective generator of reactive oxygen species (ROS) and implicated as a causative player of several liver diseases and cancers. So far, only few genetic variations are known and functional impact could be detected only for three alleles (*1D,*5,*6). The database of structural variants (http://projects.tcag.ca/variation/) describes both gain and loss of gene copy number as a new type of variation (CNV) within the CYP2E1 locus at 10q26.3 (hg18: 135,080-135,250 kb). The goal of this project is to investigate the effect of CNVs on hepatic CYP2E1 expression and activity. We used a collection of 150 Caucasian human liver samples with comprehensive clinical data for quantification of CYP2E1 mRNA (TaqMan), microsomal protein content (Western blot analysis) and activity (chlorozoxazone 6-hydroxylation). Illumina genome-wide SNP genotyping signal intensity values were used for CNV computation with the PennCNV software, based on a hidden Markov algorithm1. Generated CNV calls in the CYP2E1 gene region were validated and confirmed with two specific CNV TaqMan assays (Applied Biosystems), binding within the promoter region and within CYP2E1, using RNase P as reference gene. Expression of mRNA, protein and enzyme activity of CYP2E1 were not normally distributed. Compared to the modest variability of mRNA and activity (30 to 40-fold), protein levels were found to vary more extensively (up to 168-fold). The correlation among the phenotypes was significant, with the protein-activity association being the strongest (rs ¼ 0.48). We identified one liver sample with a homozygous duplication (CN ¼ 4), three samples with heterozygous duplications (CN ¼ 3), and one carrier of a heterozygous deletion of CYP2E1 (CN ¼ 1). The mRNA and protein expression levels and the enzyme activity of CYP2E1 were not associated with the gene copy number, even when other polymorphisms were included in the statistical analysis. Therefore we conclude that hepatic CYP2E1 belongs to the gene dosage-insensitive genes. This is in contrast to the wellknown structural variations within the CYP2 family, i.e. CYP2A6 and CYP2D6 deletions and duplications, which result in lower or higher expression and activity, respectively. This indicates that expression of CYP2E1 in CNV-carriers might be regulated by dosage compensation mechanisms, e.g. negative feedback loops or a differential methylation status. The reason for this type of regulation might be related to CYP2E1’s dangerous role in ROS production and toxicity in human liver. References 1. Wang, K. et al. PennCNV: an integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data. Genome Res. 17, 1665–1674 (2007). Supported by German Federal Ministry of Education and Research (Virtual Liver grant 0315755) and the Robert-Bosch Foundation, Stuttgart, Germany.

P361. FUNCTIONAL SIGNIFICANCE OF SNPS ON CYP3A4 EXPRESSION AND ACTIVITY IN CAUCASIAN LIVERS Amarjit S. Chaudhry1, Xia Yang2, Su-Jun Lee3, Gilbert J. Burckart4, Yiping Fan5, F. Peter Guengerich6, Stephen C. Strom7, Kenneth Thummel8 and Erin G. Schuetz1 1 Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, TN, 2Dept. Integrative Biology and Physiology, University of California, Los Angeles, CA, 3Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, South Korea, 4Office of Clinical Pharmacology, US Food and Drug Administration, Silver Spring, MD, 5Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN, 6Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, 7Department of Laboratory Medicine, Karolinska Institutet and Hospital, Stockholm, Sweden,8Pharmaceutics, University of Washington, Seattle, WA Data on the functional consequence of CYP3A4*1B, CYP3A4*1G, CYP3A4*22 and a PPARa SNP (rs4253728) on CYP3A4 expression or activity have yielded contradictory results and a systematic study correlating these genotypes with CYP3A4 mRNA and activity in a single large human liver cohort is lacking. We carried out the present study to examine the association of these genotypes with CYP3A4 expression/activity in 250 Caucasian livers, and with tacrolimus dosing in adult lung transplant patients in vivo. CYP3A4*1G and CYP3A4*1B were in very high linkage disequilibrium to the CYP3A5*1 expresser allele. Hence, if the population is not first stratified based on CYP3A5 genotype, CYP3A4*1B and CYP3A4*1G genotypes may simply be associated with a phenotype that is mainly dictated by CYP3A5*1. CYP3A4*1B, CYP3A4*1G, CYP3A4*22 and rs4253728 (PPAR) genotypes were not associated with either CYP3A4 mRNA or activity in the human livers. CYP3A4*1G also did not associate with either CYP3A4 mRNA or protein in Korean livers. CYP3A5*3 significantly associated with tacrolimus dose requirements at 1, 3 and 6 months after lung transplantation but CYP3A4*1G was not associated after controlling for CYP3A5*3. Surprisingly, in CYP3A5 non-expressers, CYP3A4*22 was associated with a trend towards a higher tacrolimus dose requirement, that even reached statistical significance at the 3 and 9 month time points. The PPARa rs4253728 did not associate with tacrolimus dose requirements. In conclusion, CYP3A4*1G and CYP3A4*1B are in very high LD to CYP3A5*1 in Caucasians, necessitating stratification by CYP3A5 genotype before comparing the effects of these genotypes on CYP3A activity. Although the CYP3A5*1 expresser genotype was associated with a higher tacrolimus dose requirement, surprisingly, the CYP3A4*22 genotype (which has been linked to a lower CYP3A4 expresser phenotype in some studies), was also associated with a higher tacrolimus dose requirement.

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P362. THE INFLUENCE OF OATP1B1 AND BCRP GENOTYPES ON THE LIPID LOWERING EFFECT AFTER ADMINISTRATION OF ROSUVASTATIN IN HYPERLIPIDEMIA PATIENTS


Li Young Ahn1, Dongseong Shin1, Howard Lee1, Jae-Yong Chung2, Kyoung Soo Lim1, Kyung-Sang Yu1, In-Jin Jang1 and JooYoun Cho1 1 Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, Seoul, South Korea, 2Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Bundang Hospital, Gyeonggi-do, South Korea Rosuvastatin, a HMG-CoA reductase inhibitor, is used to prevent cardiovascular disease by lowering the concentration of blood low-density lipoprotein cholesterol (LDL-C). Rosuvastatin enters the liver by organic anion-transporting peptide (OATP)1B1 and excreted to bile by breast cancer resistance protein (BCRP). This study identified the influence of OATP1B1 (SLCO1B1*15 or *17) and BCRP (ABCG2 421C4A) genotypes on pharmacokinetics, lipid lowering effect and safety of rosuvastatin in hyperlipidemia patients. A total of 17 subjects with LDL cholesterol level more than 130 mg/dL received rosuvastatin 20 mg once daily for 8 weeks. Blood samples for pharmacokinetic and lipid lowering effect analysis was collected on Day 1, 15, 29, 43, and 57 and the blood sampling was conducted before dosing except on Day 57. Plasma concentration was analyzed by chromatography-tandem mass spectrometry (LC-MS/MS). Safety profiles were evaluated by monitoring adverse events (AEs) and clinical evaluations. Subjects were divided by 3 genotype groups, wild type (SLCO1B1 *1/*1, ABCG2 CC), OATP1B1 variant (SLCO1B1 *1/*15, *1/*17, or *17/*17, ABCG2 CA or AA), and BCRP variant (SLCO1B1 *1/*1, ABCG2 CA or AA). OATP1B1 variant and BCRP variant showed higher trough concentration of rosuvastatin by 3.1–5.3 folds and 1.4–2.0 folds, respectively, when compared to the wild type group. The lipid lowering effect was evaluated by the concentrations of LDL-C, total cholesterol, triglyceride, and high-density lipoprotein cholesterol (HDL-C) and the decrease of HDL on Day 15 (p ¼ 0.046), total cholesterol and LDL on Day 29 (p ¼ 0.042 and p ¼ 0.025, respectively) and Day 57 (p ¼ 0.018 and p ¼ 0.018, respectively) from the baseline was significantly different between genotype groups. BCRP variant showed the highest decrease from the baseline. All of the AEs were mild and there were no significant differences of the number of AEs between genotype groups. In conclusion, rosuvastatin was generally well tolerated in hyperlipidemia patients and the genetic variants of OATP1B1 and BCRP influenced on the pharmacokinetics and lipid lowering effect of rosuvastatin.

T/C.3435C > T HAPLOTYPE INCREASES THE EARLY-PHASE ORAL ABSORPTION OF LOSARTAN",5,2,1,0,150mm,150mm,1mm,100mm>P363. ABCB1 C.2677G4T/C.3435C4T HAPLOTYPE INCREASES THE EARLY-PHASE ORAL ABSORPTION OF LOSARTAN Joo-Yeon Lee, Hye-In Lee, Mi-Jung Kim, So-Young Park, Jung-Eun Lee, Se Hyung Kim, Ji-Yeong Byeon, Chang-Ik Choi and Seok-Yong Lee School of Pharmacy, Sungkyunkwan University, Suwon, South Korea Losartan has been shown to be a substrate for the drug-efflux transporter MDR1, encoded by ABCB1 gene. ABCB1 c.2677G4T and c.3435C4T variants are known to be associated with reduced expression and function of P-glycoprotein (P-gp). We investigated the effects of ABCB1 haplotype on the pharmacokinetics of losartan. Thirty-eight healthy Korean volunteers with different ABCB1 haplotypes (c.2677G4T and c.3435C4T; 13, 12 and 13 carriers of GG/CC, GT/CT and TT/TT haplotype) were recruited and administered with a single 50 mg oral dose of losartan potassium. Losartan and its active metabolite E-3174 samples in plasma and urine were collected up to 10 and 8 hours after drug administration, and the concentrations of both samples were determined by HPLC method. Significant differences were observed in Cmax of losartan and losartan plus E-3174 (Lo þ E) among the three haplotype groups (both p50.01). The tmax of losartan and E-3174 in three haplotype groups were also significantly different (both p50.01). The AUC values of Lo þ E were significantly different among the three haplotype groups until 8 hours after losartan administration (p50.01), but the AUCinf was not different among the three haplotype groups. Urinary excretion of Lo þ E until 4 hours after losartan administration in TT/TT group was significantly higher than in GG/CC group (p50.01). Genetic variants of ABCB1 (c.2677G4T/c.3435C4T) may significantly increase the early-phase absorption of losartan, but not the total absorption of losartan.

P364. ASSOCIATION OF MRP2/ABCC2 GENETIC VARIABILITY WITH ARTEMETHER-LUMEFANTRINE TREATMENT EXPOSURE Karin Vos1, Joel Ta¨rning2, Andreas Ma˚rtensson3, Jose´ Pedro Gil4 and Isa Cavaco5 1 Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 2Mahidol-Oxford Research Unit, Bangkok, 3Karolinska Institute, 4CBME/IBB, University of Algarve; Binghamton University; Karolinska Institute,5Centro de Biomedicina Molecular e Estructural, IBB, University of Algarve, Faro, Portugal


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Malaria is a global infectious disease responsible for around 1 million deaths every year. The main worldwide anti-malarial strategy is the artemisinin based combination therapy (ACT). The concept of ACT is based on the fast action of artemisinins (steep decrease in the patient parasite load) and a long half-life partner (kills the remaining parasites). The combination artemether-lumefantrine (ATM-LUM) is one of the most used ACTs in malaria scenario. The metabolism and elimination of anti-malarial drugs in the human host is not completely understood. Drug transporters in the liver, kidney and intestine play an important role in the elimination of drugs. One important drug transporter in the elimination process of many drugs is the Multidrug Resistance Protein 2 (MRP2/ABCC2). The aim of this study was to investigate if genetic variation in MRP2 influences the pharmacokinetic (PK) of ATM, dihydro-artemisinin (DHA, main ATM metabolite) and LUM. We analyzed retrospectively two ATM-LUM clinical trials performed in Tanzania (Hietala et al., 2010; Ngasala et al., 2011) for twononsynonymous SNPs in MRP2, the 1249G4A (V417I) and the 4544G4A (C1515Y) SNPs. For the 1249G4A SNP no significant associations were found between ATM, DHA or LUM pharmacokinetic data and the genotype. In the case of 4544G4A we observed a significant association of the genotype with LUM drug concentrations at day 7 (PK data from the study Ngasala et al., 2011) but not in the first 72 h (PK data from the studyHietala et al., 2010). The patients carrying the AA genotype showed to have significant higher concentrations of LUM when compared with the GG carriers. When analyzing the ATM and DHA PK data combined with 4544G4A MRP2 genotyping we observe an association between 4544G4A SNP and the DHA area under the curve (AUC) 0-72 h, with the AA carriers having higher AUCs that the GG carriers. The results obtained for MRP2/ABCC2 describes for the first time the presence of 1249G4A and 4544G4A SNPs in African populations. Even though ACTs are used globally as first line treatment against uncomplicated malaria this is the first study which showed an association of pharmacokinetic parameters with human ABC transporters in the in vivo context of ACTs malaria treatment. A cost-benefit of pharmacogenetic analysis based personal medicine for malaria is still not available. Although, the knowledge of the pharmacogenetic characteristics of particular populations and ethnic groups can support evidence baseddecisions on the therapies to be adopted, leading to further optimized usage of ACTs.

References Hietala, S.F., et al. (2010). Antimicrob Agents Chemother. 54(11): p. 4780–8. Ngasala, B.E., et al. (2011). Malar J. 10: p. 64.

P365. GENETIC VARIATIONS IN UDP-GLUCURONOSYLTRANSFERASE 2B15 IN A KOREAN POPULATION Mi-Sun Hwang, Su-Jun Lee, Woo Young Kim, Hye-Eun Jeong, Ho Sook Kim and Jae Gook Shin Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, South Korea UDP-glucuronosyltransferase 2B15 (UGT2B15) is involved in the glucoronidation of steroid hormones as well as many drugs. Genetic variations in UGT2B15 have been shown to affect enzyme function and suggested to have a role in human diseases, such as breast and prostate cancers. In the present study, we sequenced genomic DNA from 50 normal Korean subjects to identify single nucleotide polymorphisms (SNPs) in UGT2B15. A total of thirteen genetic variations were found: two in exons, two in introns, seven in the 50 -untranslated region (UTR), and two in the 30 -UTR. The order and frequency distribution of UGT2B15 variations was: -1139T4C (rs9994887), -508G4A (rs1120265), -506T4A (rs1580083), 253T4G (rs1902023) (42%), 23687A4T (rs4148271) (31%), 2635A4T (rs2045100) (28%), -378C4T (14%), 23669C4T (12%), and 23476A4C (rs4148269) (11%), as well as other minor alleles with a frequency of 510%. Thirteen variations were used to characterize linkage disequilibrium structures at the UGT2B15 locus. Five tag SNPs were identified, and the observed allelic frequencies were compared to those of other ethnic populations. This information describing genetic polymorphisms in UGT2B15 could serve as an important resource for studying individual variations in drug and hormone metabolism in Korean as well as other ethnic populations.

P366. PHARMACOKINETICS, TISSUE DISTRIBUTION AND METABOLISM OF FN˜-MANGOSTIN FROM GARCINIA MANGOSTANA IN MICE Young Hee Choi, Young-Won Chin, Seong Yon Han, Jin Kyung Bae and You Jin Kim College of Pharmacy, Dongguk University, Goyang, South Korea Mangosteen, Garcinia mangostana L. as a tropical fruit, contains numerous biologically active compounds and a-mangostin (a-MG) is a major xanthone derivative among the biologically active compounds. The present study was undertaken to investigate the pharmacokinetics of a-MG after its intravenous and oral administration in mice. After the intravenous and oral administration of a-MG, the intravenous and oral AUCs of a-MG were dose-independent at doses of 5–20 and 10–100 mg/kg,

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respectively, in mice. The F value, 1.49% of oral dose, of a-MG was due to the poor gastrointestinal absorption and metabolism of a-MG. It was observed that liver and small intestine were involved in the metabolism of a-MG. Thus, the possible metabolites of a-MG via Phase I and Phase II enzymes were identified in in vitro studies. Also, the distribution and affinities of a-MG to liver, intestine, fat and lung were high after intravenous administration while, in case of oral administration, the distribution and affinities of a-MG were high in most tissues except brain. Thus, these findings may be useful to investigate the efficacy and toxicity of a-MG in various preclinical models with specific diseases and/or basic concepts to design the adjunctive therapy using a-MG. This study was supported by GRRC program of Gyeonggi province (GRRC-DONGGUK2013-B03), Biological Applications of New Therapeutic Target Modulators and the grant of National Research Foundation of Korea (NRF) funded by the government of Korea (S-2013-004424, Y. H. Choi).


P367. SIMPLE REVERSED-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF THE ANTIRETROVIRAL AGENT EFAVIRENZ FROM HUMAN PLASMA Benjamin U. Ebeshi1, Oluseye O. Bolaji2, Margaret Oluka3 and Anastasia N. Guantai3 1 Pharmaceutical & Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Bayelsa State, Nigeria, 2Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria, 3Pharmacology and Pharmacognosy, School of Pharmacy, University of Nairobi, Nairobi, Kenya Background: Sequel to the resurgence of TB co-infection in HIV/AIDS patients in sub-Saharan Africa, efavirenz has become an important component of the highly active antiretroviral treatment (HAART). Efavirenz, is metabolized primarily by the polymorphic cytochrome P450 enzyme, CYP2B6. High plasma concentrations of the drug are associated with a single nucleotide substitution at position 516 (516 G4T) of CYP2B6 giving rise to allelic variant with functional or non-functional activities [1–3]. Objectives: The objective of this study is to provide a simple reversed-phase high performance liquid chromatographic (HPLC) method for the determination of efavirenz in human plasma. Methodology: A 500 ml drug-free plasma sample was each placed in six different centrifuge tubes (2 ml) and varying aliquots of the stock solution (100 mg/ml) of efavirenz were spiked and vortexed for 60 sec to give concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/ml for calibration standards and 2.0, 4.0, 8.0 and 16.0 mg/ml for quality control (QC) samples. The off-column sample pretreatment was carried out by protein precipitation using ice-cold acetonitrile. The samples were chromatographed in a phenomenex (C18) 5 mm particle size column with 250 x 4.6 mm I.D and UV detection at 254 nm using a mobile phase, which was made up of a mixture of solutions A and B. Both consisted of acetonitrile, 25 mM ammonium acetate buffer and glacial acetic acid in proportions of 90:10:0.1 and 10:90:0.1 (v/v), respectively. The analytical technique was validated for precision, accuracy and analyte recovery [4]. This method was applied to determine the steady state plasma levels of efavirenz in 106 HIV/AIDS patients. Results: Linear standard curve was obtained over the concentration range of 0.5 to 16.0 mg/ml. The RSD (%) of efavirenz in intraday and interday assays ranged from 0.44 to 0.78%. Accuracy ranged from 92 to 110% and the recovery was 497%. Of the 106 HIV/AIDS patients investigated, 4% had efavirenz plasma levels below 1 mg/L, predicted to be the minimum effective concentration while 72% had plasma concentrations above the predicted maximum safe concentrations of 4 mg/L. Only 24% had plasma levels within the predicted lower and upper limit of the safety margin (i.e. 41 mg/L54 mg/L). The findings suggest that a possible daily dose reduction in patients with high plasma levels could be effected without any negative impact on the therapeutic efficacy [5–7]. Conclusion: This new HPLC method is simple, reproducible and cost-effective and can be used for therapeutic drug monitoring of efavirenz in HIV/AIDS patients on HAART as demonstrated in this study.

References 1. World Health Organization, Scaling up antiretroviral therapy In resource-limited settings: treatment guidelines for a public health approach, 2003. 2. Harrington M. From HIV to tuberculosis and back again: a tale of activism in 2 pandemics. Clin Infect Dis. 2010;50:S260–66. 3. Puthanakit T, Tanpaiboon P, Aurpibul L, Cressey TR, Sirisanthana V. Plasma efavirenz concentrations and the association with CYP2B6-516G4T polymorphism in HIV-infected Thai children. 2009;14(3):315–20. 4. Poirier JM, Robidou P, Jaillon P. Simultaneous determination of the six HIV protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) plus M8 nelfinavir metabolite and the nonnucleoside reverse transcription inhibitor efavirenz in human plasma by solid-phase extraction and column liquid chromatography. Ther Drug Monit. 2002; 24(2): 302–9. 5. Kappelhoff BS, Huitema AD, Yalvaç Z, Prins JM, Mulder JW, Meenhorst PL, Beijnen JH. Population pharmacokinetics of efavirenz in an unselected cohort of HIV-1-infected individuals. Clin Pharmacokinet. 2005;44(8):849–61. 6. Barret J, Joshi AS, Chai M, Ludden TM, Fiske WD, Pieniaszek HJ. Population pharmacokinetic meta-analysis with efavirenz. Int J Clin Pharmacol Ther. 2002;40:507–19. 7. Matimba A, Oluka MN, Ebeshi BU, Sayi J, Bolaji OO, Guantai AN, Masimirembwa CM. Establishment of a biobank and pharmacogenetics database of African populations Eur J Hum Gen. 2008;16(7):780–3.

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P368. PHARMACOKINETICS OF DRONEDARONE IN RAT USING A NEWLY DEVELOPED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC (HPLC) METHOD


Yousef A. Bin Jardan, Raniah Q. Gabr and Dion R. Brocks Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada Dronedarone is a benzofuran derivative of amiodarone that is used for the treatment of cardiac arrhythmias. Although it was originally thought to possess a superior toxicity profile to amiodarone, recent reports have arisen that describe similar types of toxicities. To date, there is little published information regarding the pharmacokinetics of the drug in human, and apparently none in rats. Therefore, we sought to develop a high-performance liquid chromatographic method for dronedarone analysis using small volumes of rat plasma. Dronedarone and internal standard (IS, ethopropazine) were extracted from 100 mL rat plasma using liquid-liquid extraction with 6 mL hexane. The organic layer was then transferred to clean tubes and evaporated in vacuo. The dried residue was reconstituted in mobile phase and 25 to 130 mL were injected into a C18 analytical column (150 mm 4.6 mm, 5 mm particle size).The mobile phase consisted of acetonitrile: [25 mM KH2PO4: 3 mM sulfuric acid: 3.6 mM triethylamine] in combination of 48:52 v/v was pumped at an isocratic flow rate of 1 mL/min. Detection was by UV absorption at 254 nm for IS, and subsequently changed to 290 nm for dronedarone at 7 min. The components eluted within 20 min. Calibration curves were highly linear (40.999) over the range of 25–1000 ng/mL of dronedarone in rat plasma. The intraday and interday CV% were518% and510%, respectively. The mean error value was512% and Mean recovery was480%. The validated lower limit of quantitation was 25 ng/mL based on 100 mL of rat plasma. To assess the utility of this assay a pharmacokinetic study was performed on three rats which were given 70 mg/kg as dronedarone base orally. Serial plasma samples were collected up to 36 h after dosing. Pharmacokinetic parameters were calculated to be 1439 1780 ng/mL, 4.5 3.27 h, 5.33 0.85 h, 33.2 11.4 L/h/kg and 250 77.1 L/kg for Cmax, tmax, half-life, oral clearance and oral steadystate volume of distribution, respectively. In conclusion, a simple sensitive assay was developed to determine dronedarone concentrations in rat plasma. It was successfully applied to determine dronedarone pharmacokinetic parameters after oral doses. Based on this initial study, rat appeared to possess a higher dronedarone oral clearance and volume of distribution than human.

P369. CAPILLARY MICROSAMPLING IN RATS AND MICE Mira Wenker1, Astrid Capello1, John Korsten2, Theo Noij2 and Harry Emmen3 1 ADME & Kinetics, WIL Research, ‘s Hertogenbosch, Netherlands, 2APC, WIL Research, ‘s Hertogenbosch, Netherlands, 3 GIT, WIL Research, ‘s Hertogenbosch, Netherlands Rodents (rats, mice) are often used to evaluate the safety of new chemical entities. One important drawback in the use of rodents is their relative small size and therefore the inability to draw large amounts of blood from these animals. Often composite plasma concentration-versus-time curves are prepared, whereby different animals are used at different time points. However, this means a higher use of animals and a more ambiguous interpretation on pharmacokinetic data. Capillary microsampling (CMS, sampling of small amounts of blood) combined with sensitive LC-MS instruments can overcome these hurdles and allow for a complete profile from one rat, or even one mouse. In this study, we have investigated the pharmacokinetics of a known compound, acetaminophen, in rats and mice by using CMS. Rats and mice were orally dosed with 10 mg/kg acetaminophen and sampled at 6 time points after dosing by tail vein puncture or saphenous vein puncture. Blood samples of 30 microliter were drawn, centrifuged and cut at the cellular interface. An aliquot of 5 mL was taken by an analytical capillary and washed-out with 115 mL of washing solution (milli-Q/ACN/ formic acid/internal standard). 5 mL of sample was injected on the HPLC/MS/MS system. The results showed that capillary blood sampling via the saphenous vein was a workable technique; sampling via the tail vein required some adjustments, like heating of the tail, especially for animals of lower bodyweight. The plasma concentration curves that were obtained were of good quality, and allowed for a proper pharmacokinetic assessment. Interestingly, the obtained Cmax differed in rats depending on whether tail or saphenous vein sampling was used. Another observation was that acetaminophen displayed gender specific kinetics in rats, with females showing a higher exposure (Cmax and AUC) compared to males. In conclusion, capillary microsampling can be used to obtain proper pharmacokinetic data. A single PK profile of one rat or mouse can be obtained, which leads to the use of less animals, less stress and discomfort to the animals and a better interpretation of pharmacokinetic data.

P370. PHARMACOKINETICS, TISSUE DISTRIBUTION, METABOLISM AND PROTEIN BINDING OF SAUCHINONE IN MICE BASED ON COMBINED LC-MS/MS AND MICROSAMPLING TECHNIQUES: APPLICATION TO EVALUATE THE ADME OF NATURAL PRODUCT IN MICE Young Hee Choi, Young-Won Chin, Jin Kyung Bae and You Jin Kim College of Pharmacy, Dongguk University, Goyang, South Korea


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Sauchinone, a biologically active lignan found in Saururus chinensis (Saururacease), has shown a variety of biological activity for the treatment of fever, jaundice, edema, inflammatory disease, hepatic steatosis and oxidative injury. In spite of its diverse application, there is little information about pharmacokinetic and tissue distribution aspects. Thus, the present study was undertaken to investigate the pharmacokinetic studies in mice using combined liquid chromatography tandem mass-spectrometry and microsampling system. On the developed analytical method, the intra- and inter-validation, precision, accuracy, stability, recovery and matrix effects of sauchinone were conducted in mouse plasma. Based on the developed analytical and microsampling system, it was found that the F value of sauchinone was only 15.1% of oral dose in spite of approximately 85.4% of the oral sauchinone absorbed. This might be due to the systemic and presystemic metabolism of sauchinone. Hence, in order to prove this hypothesis, in vitrometabolism, tissue distribution and plasma protein binding studies were conducted. The metabolism of sauchinone in liver, small intestine and lung homogenates was greater than other tissues. Also, the affinities of sauchinone in various tissues were high: liver, stomach and small intestine after intravenous administration and liver, stomach, small intestine, large intestine, kidney, lung and fat after oral administration. The protein binding values of sauchinone to mouse plasma were 53.0%. These pharmacokinetic phenomena of sauchinone provided a meaningful basis for the preclinical and clinical application. Furthermore, these experimental designs might be adjusted to evaluate the absorption, distribution, metabolism and excretion of natural products in mice. This study was supported by GRRC program of Gyeonggi province (GRRC-DONGGUK2013-B03), Biological Applications of New Therapeutic Target Modulators and the grant of National Research Foundation of Korea (NRF) funded by the government of Korea (S-2013-004424, Y. H. Choi).

P371. PRECLINICAL EVALUATION OF THE B ISOFORM-SPARING PI3K INHIBITOR GDC-0032 AND PREDICTION OF ITS HUMAN PHARMACOKINETICS Jodie Pang1, Matthew Baumgardner1, Jonathan Cheong1, Kyle Edgar2, Timothy P. Heffron3, Hoa Le1, Chudi O. Ndubaku3, Alan G. Olivero3, Emile G. Plise1, Steven T. Staben3, Jeff Wallin2, Susan Wong1, Xiaolin Zhang1 and Laurent Salphati1 1 Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA, 2Cancer Signaling, Genentech Inc., South San Francisco, CA, 3Chemistry, Genentech Inc., South San Francisco, CA The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation and its deregulation is associated with the development of many cancers. Among the Class I PI3Ks (p110a, p110b, p110d and p110g), p110a has been identified as the most critical in carcinomas. In recent years, this pathway has emerged as a major target for the investigation of anticancer drugs. GDC-0032 is a potent and selective inhibitor of Class I PI3K a, d, g isoforms, with 30 fold less inhibition of the PI3K b isoform relative to the PI3Ka isoform. This selectivity profile is expected to maximize efficacy while minimizing off-target activity compared to pan-inhibitors. GDC-0032 has also shown preferential activity against PI3Ka-mutant xenografts. Our objective was to evaluate the preclinical disposition of GDC-0032 and to assess our human PK predictions retrospectively with Phase I clinical data. The pharmacokinetic properties of GDC-0032 were assessed in pre-clinical species following intravenous and oral administrations. The permeability, plasma protein binding and metabolism were also examined in vitro. PBPK modeling was done using GastroPlus. GDC-0032 had low plasma clearance (Clp) in mice, rats, and monkeys of 2.73, 1.77, and 4.15 mL/min/kg, respectively, and moderate CL of 10.1 mL/min/kg in dogs. The terminal half-life (t1/2) ranged from 1.87 hr in mice to 4.42 hr in dogs. The steady state volume of distribution (Vss) varied from approximately 41% total body water volume in rat to 4-fold total body water volume in the dogs. Bioavailability ranged from 41.5% to 117%. In vitro, GDC-0032 displayed moderate to high binding (71.7%–97.6%) to plasma proteins and the apparent permeability determined in MDCK cells was moderate. Human PK parameters were predicted using different approaches, such as allometric scaling and PBPK modeling. Predicted human Clp and Vss were approximately 3.1 mL/min/kg and 3.9 L/kg, respectively. Favorable human predictions from preclinical data enabled the advancement of GDC-0032 into clinical trials. Retrospective look at clinical data further confirms the PBPK predictions with GastroPlus.

P372. PRECLINICAL PHARMACOKINETICS AND IN VITRO METABOLISM OF BMS-605339, A NOVEL HCV NS3 PROTEASE INHIBITOR Susan Jenkins1, Paul Scola1, Fiona McPhee1, Jay Knipe1, Christoph Gesenberg2, Vinod Arora1, Michael Sinz1 and Kenneth Santone1 1 Bristol-Myers Squibb Company, Wallingford, CT, 2Bristol-Myers Squibb Company, New Brunswick, NJ BMS-605339 is a potent HCV NS3 protease inhibitor that suppresses Hepatitis C virus replication and has been under investigation as an oral agent for the treatment of this disease. In vitro and in vivo studies were conducted in mouse, rat, dog and Cynomolgus monkeys to characterize the pharmacokinetics and metabolism of this compound. BMS-605339 was rapidly metabolized in microsomes or hepatocytes from the monkey, with lower rates of metabolism in the other species. BMS-605339 was predicted to be a moderate clearance compound in the human, based on human microsomal and hepatocyte data.

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Experiments using recombinant human CYP enzymes and human liver microsomes suggested that CYP3A4 is likely to play a key role in the metabolic clearance of BMS-605339. Generally, nearly all metabolism of BMS-605339 was oxidative and the nature of the metabolites were similar between in vitro (liver microsomes, hepatocytes) and in vivo (plasma, bile and tissue homogenate) samples. Moderate to high Caco-2 permeability was observed for this compound, with the potential for p-glycoprotein involvement. Experiments with mdr1a-knockout mice (P-gp deficient) supported Caco-2 results, suggesting that BMS-605339 is a substrate for p-glycoprotein. The oral bioavailability of BMS-605339 was low to moderate in mice (11%), rats (13%) and dogs (51%). Bioavailability was variable and dose-dependent in these species suggesting low absorption, possibly due to transporter involvement. Low oral bioavailability was also observed in monkeys (51%), possibly due to oral first-pass hepatic oxidative metabolism, as predicted from in vitro microsomal incubation studies. BMS-605339 possesses low intrinsic aqueous solubility and, in both rats and dogs, administration of an aqueous suspension suggested that BMS-605339 absorption is likely solubility-limited. Liver exposure of BMS-605339 was consistently higher than plasma exposure in all species tested (mouse, rat and dog), indicating the potential for active uptake into hepatocytes. The overall preclinical pharmacokinetic profile supported the selection and development of BMS-605339 as a clinical candidate.

P373. EXPERIMENTAL APPROACH TO INVESTIGATE LUNG PHARMACOKINETIC OF NCES IN RATS AIMED TO REDUCE THE NUMBER OF EMPLOYED ANIMALS Alessandro Fioni, Nicola Cesari, Valentina Cenacchi, Roberta Volta, Giandomenico Brogin and Paola Puccini Pharmacokinetic, Biochemistry and Metabolism Department, Chiesi Farmaceutici S.p.A., Parma, Italy Investigation of the lung pharmacokinetics and assessment of the pulmonary absorption are important issues in preclinical evaluation of NCEs designed to be administered by pulmonary route. These compounds are often required to produce local therapeutic effects with no or negligible GI absorption which is responsible for possible side effects. Methods normally employed in preclinical PK studies require a lot of animals and the data obtained from a great number of different animals are sometimes highly variable. In this work, an alternative approach to characterize the lung PK of a new compound using a reduced number of animals is presented. Test compound was administered by intratracheal (IT) route as a dry powder formulation to catheterized rats (n ¼ 4) by PennCenturyÔ device (Penn-Century, Inc.); after administration, at fixed time points, blood samples were withdrawn from the venous catheter and, 24 hours after administration, animals were sacrificed and lung was collected from each animal, in order to quantify the amount of the test compound which remained in this tissue. All the samples were analyzed by HPLC/MS/MS system and data were evaluated using standard non compartmental pharmacokinetic analysis. Experimental data, together with PK parameters obtained after IV administration, allowed the retrospective calculation of the lung concentrations for the tested compound after IT administration. Using plasma values to calculate the fraction of compound absorbed from the lung in each time interval (e.g. AUC0–10 min, AUC10–30 min, AUC30min–1 hr, AUC1–2 hr, AUC2–3 hr, AUC3–4 hr, AUC4–6 hr, AUC6–24 hr), and knowing the amount of compound which remained in the lung at the last time point (24 hours), the compound concentration profile in lung was estimated, assuming that the amount of the test compound systemically available is only due to the lung absorption. The calculated concentration profile in lung was compared with experimental data obtained by a ‘‘traditional’’ PK experiment using 3 animals for each time point, in order to assess the validity of the new approach. In this work, we have evaluated the use of catheterized rats and PK calculation to reduce the number of animals employed in preclinical studies with less variable data obtaining almost all the information obtained with a greater number of animals, which can be useful in a discovery setting.

P374. PLASMA PROTEIN BINDING MEASUREMENT VIA FAST ASSESSMENT OF EQUILIBRIUM BINDING CONSTANTS TO SERUM ALBUMIN AND ALPHA1 ACID GLYCOPROTEIN Hinnerk Boriss1, Oliver von Richter2 and Kathleen Boehme1 1 Sovicell GmbH, Leipzig, Germany, 2Merck Serono, Darmstadt, Germany Plasma protein binding of can seriously affect the free drug concentration. Thus, it is being increasingly recognized that measurement of the fraction unbound (fu) is important in the early stages of drug discovery, because its impact on drug disposition, active transport, and drug-drug interactions. We determined HSA and AGP equilibrium dissociation constants (KD) independently for 26 structurally unrelated drugs and calculated the fu-values based on these KD-values at physiological and pathophysiological concentrations of the two plasma proteins. The fu-values estimated from HSA and AGP/AAG equilibrium constants correlated well with fu-values determined with equilibrium-dialysis/ LC-MS/MS (r2 ¼ 0.91) and fu-values obtained from the literature (r2 ¼ 0.93). The method was superior to conventional equilibrium dialysis in the fu-determination of drugs highly bound to plasma proteins (fu50.01). Furthermore, the separate determination of HSA and AGP equilibrium constants enabled the assessment of changes in plasma protein binding as a function of variable plasma concentrations when compared with fu-values determined with equilibrium-dialysis/ LC-MS/MS in diluted plasma (30% and 10%) making this assay the first assay to provide the data needed for modeling the impact of altered plasma protein binding on drug disposition and drug effects.

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P375. COMPARISON OF PHARMACOKINETICS OF OSELTAMIVIR AND ITS ACTIVE METABOLITES IN CHINESE HEALTHY VOLUNTEERS VERSUS OTHER ETHNIC GROUPS


Siu kwan Wo1, B. Fok2, Lk Ngai3, MT Ngan4, TV Wong4, Brian Tomlinson2, Thomas YK Chan2, Paul KS Chan3, Moses S.S. Chow5, Vincent H.L. Lee1 and Zhong Zuo1 1 School of Pharmacy, The Chinese University of Hong Kong, Hong Kong, Hong Kong, 2Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, 3Department of Microbiology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, 4Hospital Authority of Hong Kong, Chai Wan, Hong Kong, 5Center for Advancement of Drug Research and Evaluation, College of Pharmacy, Western University of Health Science, Pomona, CA Currently oseltamivir (marketed as Tamiflu) is commonly used for the treatment and prophylaxis of influenza in Hong Kong SAR. This study reports the pharmacokinetics of oseltamivir (O) and its active metabolite (OC) in Chinese healthy subjects for the first time. The study was conducted in single-centre, randomized, open-labelled and multiple dose design. Each subject received oseltamivir (75 mg per dose, twice daily for 5 days). Blood and urine samples were collected on Day 5. Concentrations of O and OC were analysed by developed LC/MS/MS method. The realted pharmacokinetics parameters were calculated with Win-Nonlin and compared between Chinese versus other ethnic groups reported in the literature. Totally 14 adults were enrolled. Upon oral administration of O, it was found to be well tolerated and rapidly absorbed and converted to OC. In general, the concentration of OC in plasma was in orders of magnitude (10-fold) higher than that of O, indicating an extensive metabolism of O in-vivo. Plasma concentration of O reached the peak level at 0.5–1 h, which was considerably faster than that of OC (4–5 h). The concentration of O in plasma at pre-dose (0 h) on Day 5 of the treatment session and 12 h post-dose (trough concentration) was much smaller than those of OC, indicating a faster clearance of O than OC, which was evidenced by the larger renal clearance of O (22 L/h) when compared with OC (16 L/h). Both O and OC are in large quantity in urine than in plasma, indicating that both compounds are readily eliminated via renal excretion. The Cmax (67.9 27.1 ng/ml) and AUC0–12h (134.4 41.1 ng.h/ml) of O in healthy Chinese subjects was within the range reported from Caucasian/African American (86.6 35.0 ng/ml, 169 42.1 ng.h/ml) [1], however slightly higher than that from Caucasian (37.1 18.6 ng/ml, 102 22.3 ng.h/ml) and Japanese (40.8 14.2 ng/ml, 118 27.5 ng.h/ml) [2]. In addition, the Cmax (535.6 102.2 ng/ml) and AUC0–12h(4585 1155 ng.h/ml) of OC in healthy Chinese subjects was found to be higher than the previously reported value for Caucasian/African American (382 70.7 ng/ml; 3008 472 ng.h/ml) [1], Caucasian (244 29.2 389 59 ng/ml, 2270 387 3438 439 ng.h/ml) [2,3], and Japanese (297 90.0 ng/ml, 2276 527 ng.h/ml) [2]. Furthermore, the renal clearance of OC was similar between Chinese (16.3 L/h) and Caucasian (16.8 L/h) [3]. Mechanisms for the discrepancy in O and OC plasma concentrations in different ethinics groups warrant further investigations. In summary, the oseltamivir at the recommended dosages is considered to be safe and without significant pharmacokinetic consequences in Chinese healthy subjects. (Funding surport from Hospital Authority of the Government of Hong Kong SAR HOC271-08).)

References 1. Cirrincione-Dall et al. JCP, vol. 52, pp. 1255–1264, 2012. 2. Schentag et al. JCP, vol. 47, pp. 689–696, 2007. 3. Hill et al. DMD, vol. 30, no. 1, pp. 13–19, 2002.

P376. A PHARMACOKINETIC-PHARMACODYNAMIC (PK-PD) MODEL WITH PREDICTIVE BIOMARKER TO SIMULATE COLORECTAL TUMOR GROWTH IN RATS AFTER 5-FLUOROURACIL TREATMENT Shinji Kobuchi, Yukako Ito and Kanji Takada Pharmacokinetics, Kyoto Pharmaceutical University, Kyoto, Japan 5-fluorouracil (5-FU) is a widely used anticancer agent for the management of colorectal cancer (CRC). However, the optimal dosing regimen for this drug remains debatable. Dosing based on body surface area is associated with a large variability in plasma 5-FU concentrations, and fails to achieve optimal therapeutic concentrations in a high percentage of patients. This interand intra-patient pharmacokinetic variability is a major contributor to 5-FU treatment failure. However, the plasma dihydrouracil/uracil (UH2/Ura) ratio is known to be a possible predictive biomarker of hepatic dihydropyrimidine dehydrogenase (DPD) activity, the rate-limiting step in the catabolism of 5-FU. Considering the relationship between plasma concentrations of 5-FU and treatment efficacy, the plasma UH2/Ura ratio, measured prior to 5-FU treatment, may represent an indirect biomarker for predicting the treatment efficacy of 5-FU. Moreover, pharmacokinetic-pharmacodynamic (PK-PD) analysis of 5-FU using the plasma UH2/Ura ratio may predict tumor growth after 5-FU treatment. To investigate this hypothesis, we conducted pharmacokinetic and pharmacodynamic studies of 5-FU and developed a PK-PD model of 5-FU using plasma

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ratio of UH2/Ura, determined prior to 5-FU treatment, to simulate the colorectal tumor growth curves after 5-FU treatment in rats with 1,2-dimethylhydrazine-induced CRC (CRC rats). In the PK-PD model, the elimination rate constant of 5-FU was estimated using plasma UH2/Ura ratio observed before 5-FU treatment for simulating plasma concentration profiles of 5-FU. The PD part of the model was described using the unperturbed and perturbed growth model [1]. This PK-PD model with plasma UH2/Ura ratio captured well the features of the tumor growth curves (r240.99), providing reliable parameter estimates. These results suggest that the PK-PD model of 5-FU with the plasma UH2/Ura ratio determined before 5-FU treatment can be used to predict treatment responses. Furthermore, we investigated the effect of elimination rate constant of 5-FU on tumor growth using the plasma UH2/Ura ratio; the elimination rate constant of 5-FU and plasma UH2/Ura ratio are considered to correspond with tumor growth after 5-FU treatment. Therefore, the elimination rate constant of 5-FU is an important determinant of the anticancer effects of 5-FU, which could be assessed by measuring plasma UH2/Ura ratio. These results suggest that, for colorectal cancer patients, an assessment of the plasma UH2/Ura ratio and PK-PD analysis with this predictive biomarker could be useful for determining an optimal dosing regimen in a personalized medicine approach.


Reference 1. SimeoniM, Magni P, Cammia C, DeNicolao G, Croci V, Pesenti E, Germani M, Poggesi I, Rocchetti M. Predictive pharmacokinetic–pharmacodynamic modeling of tumor growth kinetics in xenograft models after administration of anticancer agents. Cancer Res 64:1094–1101, 2004.

P377. USE OF HIGH RESOLUTION MASS SPECTROMETRY IMAGING (HR-MSI) COMBINED WITH METABOLOMICS STUDY TO EVALUATE DRUG EFFICACY AND IMPACT ONTO BIOLOGICAL ENVIRONMENT Guillaume Hochart, Raphael Legouffe, Gregory Hamm, Alain Heron, David Bonnel and Jonathan Stauber MS Imaging Department, Imabiotech, Loos, France PK/PD are important information to understand drug efficacy and toxicity in drug development. PK data can be provided by Mass Spectrometry imaging which can not only localize drug within tissue, but also determine its concentration. Unfortunately, no PD information is usually generated by MSI. The combination of targeted HR-MSI experiment with untargeted metabolites identification and distribution on the same biological sample might address this issue. This presentation focus on two examples, firstly with a drug related compound against Alzheimer disease targeting the brain and the second example concerns the study of the impact of xenograft tumors on the local endogenous metabolite composition. HR mass spectrometry (SolariX FTICR 7.0 T, Bruker) was used to perform metabolomics and imaging experiment on tissue thanks to two ionization sources, the LESA (Advion) and the MALDI. For the first study, the drug was administrated to mice at 3 mg/Kg during 3 months. Brains were sectioned and conditioned for MALDI experiment. For the second study, the target molecules were endogenous metabolites in brain from wild-type versus mutant (with xenograft) mice. Visualization and Quantitation data was generated by proprietary Quantinetix software (ImaBiotech). Firstly, MSI analysis was performed to study the distribution of a drug but also exogenous and endogenous metabolites in control and dosed brains. The target molecule was detected in specific histological regions of the brain. Moreover, the quantitative data showed a higher concentration at 20 ng/g in medio-ventral hypothalamus part which plays an important role in Alzheimer disease (crossvalidated by LC-MSMS). Secondly, the 2-hydroxyglutarate (2-HG) and the ketoglutarate (two cancer biomarkers) have been targeted in tumors tissues. The quantification and profiling analyses of brain tumor related metabolites were achieved with a combining approach (validated by LC-MSMS). Higher concentration of 2-HG was observed in tumor region compared to control area region. Some other endogenous metabolites were followed in order to obtain potential readouts of tumor in tissue. In conclusion, we present here a new process which combines QMSI and metabolomics to follow drug distribution, concentration and ‘‘read-out’’ information for PK/PD.

P378. SIMULATED PAKT/AKT BIOMARKER RESPONSE BASED UPON THE OBSERVED CLINICAL EXPOSURES OF PF-04691502, A PI3K/MTOR DUAL INHIBITOR FROM A PHASE 1 CLINICAL STUDY Ravi Rahavendran1, Mary Spilker2, Brett Houk2, Kenneth Luu2, Gary Borzillo2, Shubha Bagrodia2 and Robert Millham2 1 Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, San Diego, CA, 2Pfizer Global Research and Development, San Diego, CA The objective of this work is to simulate the potential biomarker response (inhibition of phosphorylation of AKT) by PF-04691502 at clinical oral doses of 2, 4, and 8 mg QD. A hybrid PK/PD (pharmacokinetic/pharmacodynamic) model was implemented such that the PK was defined by the observed human pharmacokinetics of PF-04691502 and the PD was defined by the preclinical U87MG glioma xenograft tumor pAKTS473/AKT response observed in mice following administration of PF-04691502.


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PF-04691502 is a potent and selective orally administered dual inhibitor of both PI3K and mTOR kinase activity and was evaluated in a Phase 1 clinical study in adult patients with advanced solid tumors. Anti-tumor activity of PF-04691502 was evaluated pre-clinically using SKOV3 (ovary) and U87MG (glioblastoma) human xenograft mouse models. Both models demonstrated dose dependent tumor growth inhibition (TGI) with maximum TGI of approximately 70% at the maximum tolerated dose of 10 mg/kg (oral QD). Inhibition of phosphorylation of AKT S473 (pAKT) was used as the quantitative pharmacodynamic endpoint for PI3K/mTOR inhibition. The U87MG model was used as a basis to develop a comprehensive PK/ PD model. In brief, an indirect response model was developed to link the plasma drug exposure to the pAKT/AKT response observed in U87 mouse xenograft tumors. It was assumed that pAKT has a natural turnover rate defined by a zero order production rate and a first order degradation rate. An inhibitory drug effect was placed on the production rate. The maximal drug effect on this rate (Imax ¼ 0.975), as well as the pAKT turnover parameters (kprod ¼ 489%/hr and kdeg ¼ 4.89/hr) were assumed to be equivalent between mouse and humans. The IC50 value was also assumed to be equivalent between the U87 mouse xenograft tumors and human tumors. The IC50 value was only adjusted to reflect the difference in plasma protein binding between mouse (Fu ¼ 0.15) and humans (Fu ¼ 0.275). Therefore, the IC50defined in mice (38.3 nM (total)) is equivalent to 20.9 nM (total) in humans. Two subjects from each dose group (2 mg and 4 mg) and three subjects (8 mg) were selected to provide realistic clinical PK profiles and a range of potential PD responses. Simulations were performed to determine the potential biomarker response at steady state doses of 2, 4 and 8 mg QD. All simulations were performed using the SAAM II software (The Epsilon Group, Charlottesville, VA). The simulations suggest that at steady state following daily oral administration of 2 mg, 4 mg and 8 mg of PF-04691502, the pAKT/AKT response will be maximally reduced 70–75% (2 mg), 85–90% (4 mg) and 80–95% (8 mg) respectively. As PF-04691502 levels decrease during the dosing interval, the pAKT/AKT response begins to recover and rebounds to 50% (15–24 hr) after a 2 mg dose and 60%– 85% throughout the dosing interval (24 hr) after an 8 mg dose. Results from paired tumor biopsies from 5 subjects collected following an 8 mg oral dose shows a 66% – 80% reduction in pAKT/AKT.

P379. PHARMACOKINETICS/TOXICOKINETICS OF AN ANTI-5T4 ANTIBODY-DRUG CONJUGATE IN MICE, RATS AND MONKEYS Mauricio Leal1, JoAnn Wentland1, Nicole Duriga1, Franklin Spriggs1, Eugene Kadar1, Leslie Lorello1, Nicole Earnhardt1, John Marcek2, Michael Giovanelli2 and Jim McNally1 1 Pdm, Pfizer, Inc., Pearl River, NY, 2Dsrd, Pfizer, Inc., Pearl River, NY The pharmacokinetics of an antibody (huA1)-drug (microtubule disruptive agent MMAF) conjugate, targeting 5T4-expressing cells, were obtained in female nu/nu mice and cynomolgus monkeys after a single dose and in S-D rats and cynomolgus monkeys from multi–dose (IV) GLP toxicity studies. Plasma/serum samples were analyzed using an ELISA-based method for antibody and conjugate (ADC) concentrations as well as for the released payload using an LC-MS/MS method. A comparison across doses suggests lower clearance values at the higher doses in mice. The huA1 and conjugate data in rats and monkeys dosed with A1mcMMAF indicates that exposure (AUC) increases with increasing dose in a linear fashion. Systemic exposure (as assessed by Cmax and AUC) of the released payload increased with increasing dose although exposure was very low and its pharmacokinetics appeared to be formation rate limited. This presentation will discuss the cross species comparison, doserelated pharmacokinetics and relationship between the Ab, ADC and released payload exposure after multiple dosing.

P380. NONLINEAR PHARMACOKINETICS: THE ROLE OF CONCENTRATION-DEPENDENT PARTITIONING TO ERYTHROCYTES IN THE RAT Yaping Tu Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, Research Triangle Park, NC Dose-dependent changes in pharmacokinetic (PK) parameters are often observed as the dose level escalates. This nonlinearity may arise as a result of saturation of a process anywhere from absorption to excretion. In an intravenous (IV) administration, the most common nonlinearity occurs in which the total clearance (CL) decreases with increasing dose resulting from saturation of a metabolizing enzyme or efflux transporter. However, during optimization of a chemical scaffold that targets bacterial infections, we observed a different type of nonlinearity. For several structurally similar compounds, it was found that the total CL in the rats increased with increasing doses. Major PK parameters for these analogues resulting from 3 IV doses to the rats are summarized in Table 1. As a representative, compound GSK2676478A showed a CLtotal of 8.6 mL/min/kg at a dose of 0.3 mg/kg, which increased to 21 mL/min/kg at 1 mg/kg and further to 43 mL/min/kg at 3 mg/kg. The renal clearance CLR, which is approximately 15% of the CLtotal, also increased with dose. The whole blood concentration vs time profile fits in a typical two-compartment model. The distribution phase was followed by a slow elimination phase with a terminal halflife of 15 hr. The volume of distribution (Vss) was 9.7, 22, and 29.8 L/kg at the three doses, respectively. The increase in Vss with increasing dose indicates that the compound may have concentration dependence in plasma protein binding as seen with some antibacterial drugs. However, an in vitro study showed that the free fraction of this compound in rat plasma was 0.35 0.02 in a wide range of concentrations. Therefore, we hypothesized that concentration dependent partitioning to the erythrocytes may have led to the

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observed PK properties. To determine the red blood cell (RBC) over plasma concentration ratios (Krbc/p), the compound was incubated in rat whole blood from 10–3000 ng/mL or dosed to the rat intravenously with whole blood and plasma samples taken simultaneously. As shown in Figure 1, the Krbc/p value was found indeed to be concentration dependent. In the lower concentration region (10–30 ng/mL), the concentration in erythrocytes is 30–80-fold higher than that in plasma, and the Krbc/p value decreases rapidly as the concentration increases. Therefore, at a lower dose, the majority of the compound sequestered in RBCs is unavailable to metabolizing enzymes, resulting in lower CL. As the dose level escalates, uptake to the RBC is being saturated and the plasma concentration (available to the enzymes) increases, which leads to higher clearances.

P381. PLASMA PHARMACOKINETICS AND DISPOSITION OF SAPONINS IN RATS AFTER INTRAVENOUS ADMINISTRATION OF SHENMAI INJECTION


Wei Niu, Junling Yang, Li Li, Feifei Du, Fengqing Wang, Fang Xu and Chuan Li Laboratory for DMPK Research of Herbal Medicines, Shanghai Institute of Meteria Medica, Chinese Academy of Sciences, Shanghai, China Background and Aim: ShenMai Injection, a standardized herbal preparation derived from a combination of steamed Panax ginseng roots (Hongshen) and Ophiopogon japonicus roots (Maidong), is used for treatment of coronary heart disease and shock. Saponins are believed to be responsible for the medicinal effects of the herbal injection, which include ginsenosides from Hongshen and ophiopogonins from Maidong. This study aimed to characterize plasma pharmacokinetics and disposition of the major saponins in rats receiving intravenous ShenMai injection. Experimental: ShenMai injection (CFDA ratification no. Z13020887; Shineway Pharmaceutical Co., Ltd., Sanhe, Hebei Prov., China) was available as a sterile, nonpyrogenic parenteral dosage form and 100 g of Hongshen and 100 g of Maidong were used to make 1 L of the injection. Rats received an intravenous dose of ShenMai injection at 10 mL/kg and the rat samples were analyzed using liquid chromatography/mass spectrometry. Key Results: The major Hongshen ginsenosides contained in ShenMai injection included ppd-type ginsenosides Rb1 (0.21 mM), Rb2 (0.15 mM), Rc (0.14 mM), and ppt-type ginsenoside Rg1 (0.09 mM). Meanwhile, the content level of Maidong saponins was quite low or poorly measurable, i.e. 0.001 mM for ophiopogonin D. After dosing, the ppd-type ginsenosides (t1/2, 14–30 h; CLtot,p, 0.004 L/h/kg; VSS, 0.1 L/kg) had different elimination kinetics from ginsenoside Rg1 (0.2 h; 0.7 L/h/kg; 0.2 L/kg), which resulted in significant difference in systemic exposure level, i.e., the AUC0–t values of the former being 200–500 times greater than the latter. The ppd-type ginsenosides had slow biliary (CLB, 0.2 mL/h/kg) and urinary excretion (CLR, 2.0 mL/h/ kg), while ginsenoside Rg1 was rapidly excreted with a CLB of 312 mL/h/kg and a CLR of 54 mL/h/kg. The liver (AUC0–t, 216– 319 mMh) and kidney levels (40–57 mMh) of the ppd-type ginsenosides were significantly lower than the corresponding plasma levels (189–429 mMh). Meanwhile, the liver (1 mMh) and kidney level (13 mMh) of ginsenoside Rg1 was comparable with and higher than, respectively, the corresponding plasma level (1 mMh). In addition to the ginsenosides, ophiopogonin D was also measured in plasma but at very low level. Conclusions: The differences in elimination kinetics significantly affect the relative systemic exposure levels of the ppd-type and ppt-type ginsenosides.

Reference Liu H-F., Yang J-L., Du F-F., Gao X-M., Ma X-T., Huang Y-H., Xu F., Niu W., Wang F-Q., Mao Y., Sun Y., Lu T., Liu C-X., Zhang B-L., and Li C. (2009). Absorption and disposition of ginsenosides after oral administration of Panax notoginseng extract to rats. Drug Metab Dispos 37:2290–2298. Hu Z-Y., Yang J-L., Cheng C., Huang Y-H., Du F-F., Wang F-Q., Niu W., Xu F., Jiang R-R., Gao X-M., and Li C. (2013). Combinatorial metabolism notably affects human systemic exposure to ginsenosides from orally administered extract of Panax notoginseng roots (Sanqi). Drug Metab Dispos (doi:10.1124/dmd.113.051391).

P382. ANTI FREEZE PROTEIN (AFP): PHARMACOKINETIC AND TISSUE DISTRIBUTION STUDIES IN MICE Stuart G. Wood1, Andrew McEwen2, Stephen Harris2, Stephanie Geoffroy2, Sandeep Chugger2 and Claire Henson2 1 Quotient Bioresearch Limited, Rushden, United Kingdom, 2Metabolic Chemistry, Quotient Bioresearch Limited, Rushden, United Kingdom Purpose: Antifreeze proteins (AFPs) also known as ice structuring proteins (ISPs) are polypeptides produced by a wide variety of organisms (vertebrates, plants, fungi and bacteria) and enable their survival in subzero environments. AFP’s bind to small ice crystals, inhibiting their growth and recrystallisation of ice that could be fatal. Commercially, there are countless applications for antifreeze proteins including increasing shelf-life of stored foods, improving the consistency of ice cream and increasing the cold tolerance of crops and fish, thus extending the harvest season in cooler climates. Medical applications include uses in cryosurgery and preservation of tissues for transplantation or tranfusion. Methods: The protein used in the current study was a Type III AFP (MW 6kD) found in Antarctic eelpout. The proteins was labeled using [3H]-N-succinimidyl propionate, purified


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using a size exclusion column and the purity checked by HPLC prior to use. Groups of mice were dosed intravenously with [3H]protein at a dose level of 10 mg/kg. From one group serial blood samples were taken for determination of radioactive concentrations in plasma. Animals from a second group were prepared for quantitative whole body autoradiography (QWBA), whilst tissues from a third group were removed and prepared for analysis using microautoradiography. Plasma radioactivity analysis was performed by spotting plasma onto FTA DMPK cards (Whatman) and radioactive content determined by placing the cards against imaging plates (FujiFilm) for 168 hours. Radioactive standards were prepared to provide a standard curve for quantification. Sections of whole animals and tissues were taken using a Leica CM3600 microtome (Leica Microsystems). Section thickness was 30 mm for QWBA and 10 mm for microautoradiography. QWBA images were obtained using a BioImage Analyser (Fuji BAS 5100), whilst microautoradiographic images were obtained following exposure of sections to photographic emulsion. Results: The antifreeze protein was rapidly cleared from circulation with a plasma half-life around 40 minutes. Radioactivity was distributed throughout the animal with highest concentrations of radioactivity observed in the liver and lung. Analysis of these tissues using microautoradiography showed association of the radioactivity with the bronchioles (lung) and hepatic veins and artery (liver). Conclusions: The pharmacokinetics and tissue distribution of Type III AFP has been determined in the mouse using radioactively labeled protein. The protein was rapidly cleared from the plasma and generally distributed throughout the animal. The data obtained in this study demonstrate that high quality tissue distribution data can be obtained following radiolabelling of proteins with tritium. The use of tritium also enabled the localization of the protein at the cellular level using microautoradiography

P383. ACOTIAMIDE, A NOVEL ORALLY ACTIVE ACETYLCHOLINESTERASE INHIBITOR FOR THE TREATMENT OF FUNCTIONAL DYSPEPSIA, IS SPECIALLY DISTRIBUTED IN STOMACH Yoshihiro Kawabata, Kazuyoshi Yoshii, Ryoko Toda, Masamichi Hirayama, Takashi Yamaguchi, Minami Iikura and Yukinori Mera Central Research Laboratories, Zeria Pharmaceutical Co., Ltd., Saitama, Japan The novel selective acetylcholinesterase (AChE) inhibitor acotiamide hydrochloride (acotiamide) has proven significantly effective in treating functional dyspepsia (FD) in clinical trials, particularly meal-related symptoms, by improving gastrointestinal motility via AChE activity inhibition. To clarify the site of pharmacological action of acotiamide on subcutaneous administration in rats (18.5, 55.4, and 185 mmol/kg), the time course of plasma and stomach tissue concentrations of acotiamide and the corresponding concentration ratios (Kp) were obtained. Early after administration, the plasma and stomach concentrations were similar, but the decrease in plasma concentration became greater with time. T1/2 and MRT values of acotiamide in the stomach were 1.9 h and 0.91 h, respectively, which are longer than those in plasma. Kp values of the stomach tissue was higher than unity at all examined doses and decreased with increasing dosage of acotiamide, indicating that a saturable component was involved in the stomach distribution. Micro-autoradiography (to confirm acotiamide distribution) and Nissl staining (to confirm the localization of nerve cells) in the stomach after subcutaneous administration of acotiamide to rats at a dose of 55.4 mmol/kg showed high levels of radioactivity being distributed in the muscular layer of the gastric body. Acotiamide distribution was homogenous in the stomach, and its stomach concentration in rats and dogs after subcutaneous or intra-duodenal administration was more than 0.75 mM, which exceeds the Ki value for AChE inhibition. AChE inhibition by acotiamide was directly evaluated by observing the disappearance of AChE-activity staining in sections prepared from dogs. AChE staining was slightly disappeared at 1.0 mM acotiamide, but markedly so at 100 mM. Taken together, our findings indicate that acotiamide inhibits AChE in the stomach and consequently enhances gastric emptying and gastro-intestinal motility. Further, these results demonstrate that acotiamide is distributed around nerve cells in the muscular layer where AChE activity is localized and improves FD symptoms by inhibiting AChE activity in the muscular layer of the stomach.

P384. DRUG DISTRIBUTION WITHIN THE GASTRO-INTESTINAL TRACK - IN VIVO AND MODELING & SIMULATION EFFORTS TO GUIDE PKPD FOR IBD TARGETS Jean-François Le´vesque, Line Ste-Marie, Eric Langlois, Marie-Claude Duquet, Chantal Boudreau, Mohamed Diallo and Nathalie Chauret Dmpk, Vertex Pharmaceutical Canada, Laval, QC, Canada Ulcerative colitis and Crohn’s disease are a multifactorial inflammatory bowel diseases (IBD) characterized by chronic inflammation or discontinuous lesions in gastro-intestinal track causing patients abdominal pain, diarrhea and weights loss. There is evidence that host-predisposition factors, dysbiosis in the gastro-intestinal (GI) track combined with chronic inflammation would be key drivers of IBD. In the design of new drug therapies to treat IBD patients, it is crucial to understand the contribution of local gut exposure to the efficacy read-out in order to establish proper PKPD relationship. Unfortunately, limited data are available on PKPD for IBD targets and on drug distribution within the GI track in human and pre-clinical species used in IBD models. The objective of this study was (1) to determine the whole blood and GI concentration of model compounds

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in mice GI track and (2) validate mice SimCYP ADAM model to simulate whole blood and GI time-concentration profiles in mice. GI distribution studies were performed in mice using rifaximin, a non-absorbed, poorly soluble oral antibiotic as a model compound. Following oral gavage (10 mg/kg), animals were sacrificed a different time-point and whole blood and several GI tissues (stomach, duodenum, jejunum ileum and colon) were collected and analyzed by HPLC-MS/MS. Minimal levels of rifaximin were detected systemically, which is consistent with the fact that rifaximin in a BCS classs IV compound. Millimolar concentrations were recovered in the GI tissues with a Tmax of 1 h in the small intestine and Tmax 4 h in the colon. These in vivo GI distribution results were used to optimize the mice SimCYP ADAM absorption model. SimCYP simulations of rifaximin levels in the jejunum, ileum and colon were within 2 fold of in vivo values. In addition, based on solubility assessment in simulated intestinal fluids, SimCYP model enabled the simulation of soluble and undissolved rifaximin levels in the mice GI track. The knowledge gained from the in vivo GI distribution studies and SimCYP validation will contribute to support dose selection for proof-of-principle (POP) experiment in mice PD model and ultimately, establish PKPD relationship in human clinical studies.


P385. PLASMA PHARMACOKINETICS AND METABOLISM OF LW6, A NOVEL HYPOXIA-INDUCIBLE FACTOR -1A (HIF-1A) INHIBITOR IN MICE Eun Ji Kim1, Kiho Lee2, Kyeong Lee3, Jong Soon Kang1, Ji Eun Yoon1, Chang Woo Lee1, Sang Kyum Kim4 and Soo Jin Oh1 1 Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk, South Korea, 2College of Pharmacy, Korea University, Sejong, South Korea, 3College of Pharmacy, Dongguk University, Seoul, South Korea, 4College of Pharmacy, Chungnam National University, Daejeon, South Korea A novel hypoxia-inducible factor-1a (HIF-1a) inhibitor, (aryloxyacetylamino) benzoic acid derivative, LW6, was identified as a small compound that inhibits the accumulation of HIF-1a. The plasma pharmacokinetics and metabolism of LW6 was characterized in mice. LW6 exhibited a low systemic clearance (1.7 0.1 l/hr/kg), a small volume of distribution (0.5 0.1 l/kg) and a short terminal half-life (0.6 0.1 hr). Following intravenous (5 mg/kg) or oral (5 mg/kg) administration, LW6 was rapidly converted to its metabolite, (4-adamantan-1-yl-phenoxy) acetic acid (APA). Following oral dose, LW6 was rapidly absorbed (tmax ¼ 0.3 0.1 hr). This is correlated with moderate permeability of LW6 in Caco-2 cells with an apparent permeability of 2.1 0.4 106 cm/s. However, its oral bioavailability, estimated using AUClast values, was found to be low (1.7 1.8%). LW6 was slowly degraded in mouse liver microsomes (t1/2460 min) and serum (t1/246 h). Although LW6 was slowly converted to APA in in vitro system, about 54% or 44.8% of it dosed was shown to be available systemically as APA in the mouse after a single intravenous or oral administration, respectively. These results suggest that LW6 as anticancer drug candidate is highly likely to work as its active metabolite APA in the body. Therefore, it would be essential to simultaneously consider the active metabolite as well as the parent compound for a successful evaluation during this lead optimization. However, further studies are necessary to elucidate the mechanism involved.

P386. AUTOMATED BLOOD SAMPLING AND TELEMETRY RECORDING IN AWAKE UNRESTRAINED MACAQUES Brad Gien1, David Hopper2, Paul Kruzich2 and Stacey Kurtz2 1 PK/PD, BASi, West Lafayette, IN, 2BASi, Mount Vernon, IN Automated Blood Sampling and Telemetry Recording in Awake Unrestrained Macaques In an effort to reduce the number of animals used in preclinical cardiovascular safety studies and to improve the quality of data collected while reducing animal and technician stress, we combined automated blood sampling with the collection of radiotelemetry data. Preclinical cardiovascular safety studies using large animals are commonly conducted in species such as Cynomolgus macaques (Cynos). A challenge with using Cynos for the collection of telemetry data and blood sampling is the increase in stress to the animals and technicians as well as telemetry signal fluctuations during blood collection procedures. In order to reduce these issues, we combined automated blood sampling with digital radiotelemetry to compare automated verses manual blood collection and the effects it had on the telemetry data and stress hormones in the blood. Four female Cynos were implanted with telemetry devices and carotid artery catheters. After a recovery period they were dosed with 175 mg/kg of moxifloxacin and heart rate, ECG, blood pressure and temperature were recorded. Blood was sampled manually by technicians during the first phase of the study and by an automated blood sampler, Culex-L, for the second phase of the study. Blood was sampled at predetermined timepoints for both the manual and automated phases and processed to plasma for analysis of moxifloxicin concentrations and cortisol levels. During the automated collection phase of the study, room entry was restricted. This study has shown that automated collection of blood and cardiovascular data simultaneously can be completed in awake unrestrained large animals. Using automated blood sampling when collecting physiological measurements can reduce signal fluctuations, reduce stress in the animals and the technicians while saving lab animal staff time and improving the quality of data collected.

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P387. APPLICATION OF A NOVEL COMPUTATIONAL METHOD TO ESTIMATE FEASIBLE SOLUTION SPACES FOR DETERMINING MULTIPLE PARAMETERS IN PHYSIOLOGICALLY-BASED PHARMACOKINETIC (PBPK) MODEL


Kazuya Maeda1, Kenta Yoshida1, Hiroyuki Kusuhara1, Akihiko Konagaya2 and Yuichi Sugiyama3 1 Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan, 2Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Yokohama, Japan, 3Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Research Cluster for Innovation, RIKEN, Yokohama, Japan Physiologically-based pharmacokinetic (PBPK) modeling is one of the powerful tools to simulate the time profiles of plasma and tissue concentrations of drugs in vivo in any special occasions simply by changing the parameters in PBPK model. However, it is difficult to set up many kinetic parameters for transport and metabolism processes in various organs since some parameters cannot be simply determined by in vitro experiments and need to be modified from the original in vitro parameters to best explain the clinical PK data. In the conventional approach, parameters were optimized with the Gauss-Newton method or its modified ones. However, it is not guaranteed whether a single set of optimized parameters is uniquely determined to reproduce the clinical PK profiles since it sometimes largely depends on initial value of each parameter. Thus, we applied a new computational method to estimate feasible solution spaces to the parameter optimization in PBPK model1. In this method, the lower and upper boundaries of each parameter were set and a group of virtual PK profiles was prepared with random sampling of each parameter within the given range. Then, the next group of data was prepared by approximating a projection from a parameter space into target values with a linear matrix, and calculating temporarily optimized parameters with its inverse matrix. Optimized parameters were finally obtained after 10–20 iterations of this process. We applied this method for analyzing the reported clinical drug-drug interaction (DDI) cases. In the case of OATP-mediated DDI between pitavastatin and cyclosporin A, ca. 3000 parameter sets reproducing observed plasma AUC of pitavastatin in the absence or presence of cyclosporin A were obtained. Most of the calculated parameters varied among optimized sets of parameters, but interestingly, the deviation of the inhibition constant of cyclosporin A for the hepatic uptake of pitavastatin was relatively smaller (less than 2-fold) compared with other parameters, suggesting the robustness of this parameter in this DDI model. We also analyzed the DDI case between pravastatin and rifampicin, and found that considering enterohepatic circulation makes the optimized parameters closer to those obtained from our previous in vitro studies. Since we do not need to care much about the initial value of each parameter, our method is expected to be applied to various types of DDI analyses, particularly involving complex mechanisms. In our presentation, several examples to demonstrate the effectiveness of this method on the optimization of parameters and model structure will be presented.

Reference 1. Aoki et al., NII Technical Report, NII-2011-002E (2011)

P388. METABOLISM AND PHARMACOKINETICS OF PICROSIDE AND II ISOLATED FROM PICRORRHIZA SCROPHULARIIFLORA Wen-Bin Hou, Qi Shan, Fu-Jun Zhou, Xiang Zhou and Chang-Xiao Liu State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Reserch, Tianjin, China Picrorrhiza scrophulariiflora Pennell, a member of the genus Picrorhiza Royleex Benth. of family Scrophulariaceae, is a well known traditional Chinese medicinal plant widely used in Asia for treating hepatitis and other deases. Picroside I and II, Iridoid compounds,are isolated from Picrorrhiza scrophulariiflora Pennell. We carried out the pharmacokinetics and identification of metabolic products of picroside I and II by UPLC-Q-TOF-MS technology. After oral administration of picroside I and II to rats, the samples from blood, urine and feces were checked with LC-MS to analyze the possible metabolites in vivo. Both compounds breaked down to iridoid aglycone, the same as the catalpol aglycone, and phenolic acids with cinnamic acid from picroside I and vanillic acid from picroside II. Picroside I was metabolized to dehydration products with g-pyran structure, and some iridoid aglycones were N-heterocyclized to form pyridine products. The dehydration and pyridine products of iridoid aglycones were conjugated with sulfate and glucorunide, while the vanilloyl catalpol aglycone from picroside II could be conjugated with some amine acids, such as glycine and cysteine. Only picroside II could be detected from the blood, urine and feces samples. The concentration-time curves fitted two-compartment kenitics. The pharmacokinetic parameters revealed that picroside II were absorbed quickly with Tmax as (0.333 0.129) h and Cmax as (176 43.0) ng/mL. The t1/2 was 1.02 0.28 h, and AUC0–T was 222 58.9mg h/L.

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P389. MIMICKING HUMAN NICOTINIC ACID EXPOSURE IN PRECLINICAL SPECIES


Justin Q. Ly1, Connie Chan2, Chou Kang-Jye2, Peter S. Dragovich3, Xiaorong Liang1, Emile Plise1, Leslie Wang1, Lulu X. Yang1 and Bianca M. Liederer1 1 Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA, 2Pharmaceutics, Genentech Inc., South San Francisco, CA, 3Discovery Chemistry, Genentech Inc., South San Francisco, CA Nicotinic acid (NA) (also known as 3-pyridine-carboxylic acid or niacin) is a water soluble B-complex vitamin (B3) and is essential for the metabolism of carbohydrates, fats, and many other substances in the body. Enzymes involving in nicotinamide adenine dinucleotide (NAD) metabolism, such as nicotinamide phosphoribosyltransferase (Nampt), have become attractive targets in recent years for the discovery of novel cancer. Nampt catalyzes the rate-limiting step in the salvage pathway that recycles nicotinamide (NAM) to NAD. Inhibition of Nampt leads to the depletion of cellular NAD levels resulting in significant antitumor effects. However, Nampt inhibition may also cause unwanted toxicity due to NAD depletion in non-tumor tissues. One strategy to minimize Nampt inhibitor toxicity is to co-administer such agents with NA. Here our aim was to mimic human NA exposure and pharmacokinetic (PK) profile in preclinical species to support toxicology and efficacy studies in combination with Nampt inhibitors. PK studies were conducted in female NCR nude mice, male Spraque-Dawley rats, male beagle dogs, and male cynomolgus monkeys. In addition, plasma protein binding and blood plasma partitioning were determined for NA in all species that were used in PK studies. Following a single intravenous dose of 5 mg/kg to mice, rat, and dogs, and 10 mg/kg to monkeys, NA exhibited moderate clearance in mice and rats (43.5 and 38.0 ml/min/kg, respectively), and high clearance in dogs and monkeys (119 and 48.5 ml/min/kg, respectively). Following 30, 100, and 300 mg/kg BID PO dose to mice and 10, 30, and 100 mg/kg BID PO dose to rats, observed exposures were found to be greater than dose proportional in both species. AUClast in nude mice were 144, 917, and 6610 hr*mM for 30, 100, and 300 mg/kg BID dose, respectively, and AUClast in rats were 103, 542, and 722 hr*mM for 10, 30, and 100 mg/kg BID dose, respectively. AUClast of 767 hr*mM was observed following 30 mg/kg BID PO dose to dogs, and 1017 hr*mM was observed following 45 mg/kg BID PO dose to monkeys. Efforts were also made to mimic the human exposure of extended-release NA (NiaspanÕ ) in preclinical species. NA PK was conducted in mice using ALZET osmotic pumps, food and water fortified with NA, and continuous infusion. Capsule dosing of extended release NA was used in rat PK studies and NiaspanÕ (extended released NA) tablets were used in dog and monkey PK studies. Results suggested that (1) significant differences in NA exposures were obtained with different forms of NA (e.g., immediate release vs extended release); (2) even with BID dosing, extended exposure of NA in preclinical species was not achieved using immediate release NA; (3) extended exposure can be achieved in mice with ALZET osmotic pumps or NA fortified food/water, in rats with continuous infusion and capsule dosing, and in dogs and monkeys with NiaspanÕ dosing.

P390. PHARMACOKINETICS OF SINGLE DOSE OF SUBCUTANEOUS PEGYLATED RECOMBINANT HUMAN GRANULOCYTE COLONY STIMULATING FACTOR (PEG-RHG-CSF) IN CANCER PATIENTS: COMPARISON WITH DAILY DOSES OF RHG-CSF Yongming Cai1, Wenjin Shen1, Zihui Song1, Zongpeng Zhang1, Mingkai Xu2, Huiwen Zhang2 and Changxiao Liu1 1 State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, China, 2Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China Background: PEG-rhG-CSF is produced by the covalent attachment of a 20-kDa monomethoxy polyethylene glycol molecule to the N-terminal methionyl residue of rhG-CSF, forming a molecule with a relative molecular mass of approximately 39 kDa. There is demonstrated equivalent efficacy of a single dose of PEG-rhG-CSF to daily rhG-CSF in enhancing neutrophil recovery after chemotherapy, while PEG-rhG-CSF has a significantly longer half-life due to decreased renal clearance and masked proteolytic cleavage sites[1]. PEG-rhG-CSF, investigated in this paper, is manufactured by Qilu Pharmaceutical Co., Ltd., China. The study aims to characterize the pharmacokinetics of a single dose of PEG-rhG-CSF in comparison with 7 daily-dose of rhGCSF in patients after chemotherapy by a multicenter, randomized, open-label phase I study designed. Methods: Patients with breast cancer or non-small-cell lung cancer 48 hours after chemotherapy received a single subcutaneous injection of PEG-rhGCSF (60, 100, or 120 mg/kg) per chemotherapy cycle or daily subcutaneous injections of rhG-CSF (5 mg/kg/day) for up to 7 days. Forty patients were enrolled in this study, 10 of whom were randomised to receive rhG-CSF (5 mg/kg/day) and 30 to receive PEG-rhG-CSF (n ¼ 10, n ¼ 9, and n ¼ 11 for the 60-, 100-, and 120 mg/kg dose groups respectively). The serum concentrations of PEG-rhG-CSF and rhG-CSF were determined using an enzyme-linked immunosorbent assay (ELISA kit, R&D Systems, USA; kit number, SCS50). The pharmacokinetic parameters were calculated by DAS 3.0 software. Results: For PEG-rhG-CSF, a greater than proportional increase in both the peak concentration (Cmax) and the area under the concentration-time curve (AUC) was observed with increasing dose,while the rate of serum clearance decreased,which is attributed to saturation of the neutrophilmediated clearance pathway. The time to reach peak concentration (Tmax) and elimination half life were longer with 120 mg/kg dose compared to all others. Tmax and elimination half life of PEG-rhG-CSF averaged 14–19 h and 41–57 h at all doses, approximately 3-fold and 6-fold longer respectively than with rhG-CSF. Comparison of PEG-rhG-CSF with rhG-CSF indicates that PEG-rhG-CSF is clearly eliminated more slowly, with a 3-fold decrease. Conclusions: The pegylated protein, compared with

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rhG-CSF, exhibits improved pharmacokinetic properties, decreased system clearance, enhanced drug exposure and sustainedefficacy in accelerating recovery from chemotherapy-induced neutropenia.

References [1] Simona B, Cristina R, Luca N, et al. A single dose of Pegfilgrastim versus daily Filgrastim to evaluate the mobilization and the engraftment of autologous peripheral hematopoietic progenitors in malignant lymphoma patients candidate for high-dose chemotherapy. Transfus Apher Sci. 2010;43(3):321–326.

P391. A MODELING FRAMEWORK FOR THE ASSESSMENT OF [(18)F]-FLUORODEOXYGLUCOSE POSITRON EMISSION TOMOGRAPHY IN PREDICTING OVERALL SURVIVAL IN NON-SMALL CELL LUNG CANCER


Ahmed A. Suleiman1, Sebastian Frechen1, Matthias Scheffler2, Thomas Zander2, Ju¨rgen Wolf2, Lucia Nogova2 and Uwe Fuhr1 1 Department of Pharmacology, Clinical Pharmacology Unit, University of Cologne, Cologne, Germany, 2Department I of Internal Medicine, Center for Integrated Oncology Ko¨ln Bonn, University of Cologne, Cologne, Germany Statement of Hypothesis: Evaluating the metabolic activity of tumors rather than their anatomical sizes was promoted for evaluating the response to newer cancer therapies which are cytostatic rather than being cytocidal. Positron emission tomography (PET) using [(18)F]-fluorodeoxyglucose (FDG) has been used for this purpose [1]. Our aim was to develop a model linking exposure to erlotinib (a tyrosine kinase inhibitor acting on epidermal growth factor receptor, EGFR) to the time course of the metabolic activity of the tumors assessed by FDG uptake in patients with non-small cell lung cancer (NSCLC), which in turn will be coupled to a survival model to evaluate the usefulness and statistical significance of using FDG for early prediction of survival in NSCLC. Data & Methods: Data including overall survival (OS) was available from 34 patients with newly diagnosed stage-IV NSCLC treated with erlotinib (150 mg/day) in a phase-II study [2]. As no erlotinib concentrations were available, a previously published erlotinib pharmacokinetic model [3] was used to generate the individual pharmacokinetic parameters and plasma concentrations to drive the pharmacodynamic model. FDG uptake was measured at baseline, one and six weeks after treatment initiation. The average metabolic activity of the most active lesions at each visit was calculated. Plasma concentration driven disease-drug models with different characteristics of tumor metabolic kinetics and treatment effects were tested. Different covariates (sex, age, EGFR and KRAS mutational statuses, histology, smoking) were evaluated at a ¼ 0.05 in the likelihood-ratio test. A time-to-death analysis was conducted by testing exponential, Weibull, Gompertz and log-logistic distributions for the best fit to OS data. Relative changes in FDG uptake together with the covariates and the baseline uptake were tested as OS predictors. Non-linear mixed effects modeling using NONMEM 7.2 was used for analysis. Results: Tumor metabolic kinetics was best described using Gompertzian kinetics. A concentration-driven drug-effect model was included to describe the inhibitory effect of erlotinib on the tumor’s metabolic activity. The treatment effect was shown to decrease with time due to resistance. Related to its mechanism, patients expressing an activating EGFR mutation (n ¼ 5) were more sensitive to erlotinib and slower in resistance development by 62%. An exponential distribution was found to be the best distribution describing OS. The relative change in FDG uptake after week-1 and the baseline FDG uptake itself were found to be the most significant predictors of OS (p ¼ 0.006 and p ¼ 0.014, respectively). The relative change in FDG uptake was only found to be significant after accounting for baseline uptake demonstrating the power of models in maximizing the statistical power in detecting an effect. The hazard of death increased exponentially by a constant of 0.234 for every unit increase in the baseline FDG uptake and decreased exponentially by a constant of 0.206 for every 10% reduction in FDG uptake after week-1. Conclusions: We developed a modeling framework linking erlotinib exposure to a surrogate biomarker, FDG uptake, which itself was linked successfully to the most relevant clinical outcome in oncology, OS. The model has proven the appropriateness of early FDG assessment in predicting response.

References 1. Wahl, R.L., et al., From RECIST to PERCIST: Evolving Considerations for PET response criteria in solid tumors.J Nucl Med, 2009. 50 Suppl 1: p. 122S–50S. 2. Zander, T., et al., Early prediction of nonprogression in advanced non-small-cell lung cancer treated with erlotinib by using [(18)F]fluorodeoxyglucose and [(18)F]fluorothymidine positron emission tomography.J Clin Oncol, 2011. 29(13): p. 1701–8. 3. Lu, J.F., et al., Clinical pharmacokinetics of erlotinib in patients with solid tumors and exposure-safety relationship in patients with non-small cell lung cancer. Clin Pharmacol Ther, 2006. 80(2): p. 136–45.

P392. MILK TRANSFER AND TOXICOKINETICS OF VALPROIC ACID IN LACTATING CYNOMOLGUS MONKEYS Jong-Hwa Lee1, Kyoung-Sik Moon1, Tae-Sung Koo2, Eun Ju Jeong1, Moon-Koo Chung1 and Sang-Joon Lee1 1 Toxicology Center, Korea Institute of Toxicology, Daejeon, South Korea, 2Graduate School of Drug Discovery and Development, Chungnam National University, Daejeon, South Korea


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Studies on milk transfer of drugs in non-human primates (NHPs) are among the crucial components in the assessment of periand postnatal toxicity because of the similarity between NHPs and humans. There may be chances of Valproic acid (VPA) transferring to the fetuses in pregnant women or to the infants by breast-feeding in lactating women after intake of VPAcontaminated foods. However, the probability for VPA crossing the placental barrier and mammary gland has not been studied so far. To evaluate the milk transfer of valproic acid (VPA) in NHPs, VPA was administered once daily to pregnant cynomolgus monkeys at doses of 0, 30, 90, and 270 mg/kg by oral gavage from Day 100 of gestation (GD 100) to Day 31 of lactation (LD 31). Concentrations of VPA and its metabolite, 4-ene-VPA, in the maternal plasma on GD 100, GD 140, and LD 30, and concentrations of VPA and 4-ene-VPA in the offspring plasma and milk on LDs 30 and 31, respectively, were quantified using liquid chromatography-tandem mass spectrometry (HP1100-API 3200 Qtrap). After administration of a single oral dose of VPA to pregnant monkeys on GD 100, the concentrations of VPA and 4-ene-VPA were generally quantifiable in the plasma of all treatment groups up to 24 h after administration, which showed that VPA was absorbed and that the monkeys were systemically exposed to VPA and 4-ene-VPA. After administration of multiple doses of VPA to the monkeys, VPA was detected in the pup’s plasma and milk taken on LD 30 and LD 31, respectively, and the concentration of VPA in the milk increased with an increase in the dose. The detection of VPA in milk suggest the possibility of transfer of VPA through breast-feeding from dams to pup dose level of 30 270 mg/kg/ day, and the VPA concentrations in pups was 3 8% of those of maternal plasma. Extremely low concentrations (50.5 mg/mL) of 4-ene VPA were detected in the milk and the pup plasma. In conclusion, pregnant monkeys were exposed to VPA and 4-ene-VPA after oral administration of VPA at doses of 30, 90, and 270 mg/kg/day. VPA transferred via milk, and the VPA exposure to the pup increased with an increase in the dose of VPA. The metabolite, 4-ene VPA, was present in extremely low concentrations (50.5 mg/mL) in the milk and the pup plasma. In this study, we established method to elucidate toxicokinetics using cynomolgus monkeys.

P393. A PHYSIOLOGICALLY-BASED POPULATION PHARMACOKINETIC MODEL FOR SIMVASTATIN AND ITS ACTIVE METABOLITE SIMVASTATIN ACID Nikolaos Tsamandouras1, Gemma Dickinson2, Stephen Hall2, Amin Rostami-Hodjegan1, Aleksandra Galetin1 and Leon Aarons1 1 Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, The University of Manchester, Manchester, United Kingdom, 2Eli Lilly and Company Simvastatin (SV), a commonly used HMG-CoA reductase inhibitor, is a prodrug with complex pharmacokinetics due to the interconversion between the parent drug and its main active metabolite, simvastatin acid (SVA). Both SV and SVA are subject to drug-drug interactions and are differentially affected by genetic variation in enzyme/transporter proteins relevant for their disposition [1]. Therefore, an operating model that mechanistically describes the pharmacokinetics of both these forms has numerous applications and its development is the scope of this work. Plasma concentrations of both SV and SVA from 34 healthy volunteers were analysed under a population approach using nonlinear mixed effects software (NONMEM 7.2). A model with physiologically realistic compartmental structure was developed allowing interconversion between the lactone and acid form of the drug in different tissues. Parameter estimation was performed combining the information from the clinical data with prior distributions informed from physiology literature, in vitro experiments and in silico methods [2]. An independent dataset [3] was used for the external validation of the SV-SVA mechanistic model. The developed model was employed to simulate SV and SVA concentration-time profiles in plasma, liver and muscle tissues to investigate the impact of SLCO1B1 polymorphism. The mechanistic model provided a good fit to the plasma concentrations of both SV and SVA and adequately described their variability in the population. In addition, the model predictions were also in close agreement with the observed SV-SVA data used for external validation. The proof of concept simulations representing the scenario of a SLCO1B1 polymorphism recovered a 168% increase in exposure of SVA in plasma [4], while also predicted a comparable fold increase in the muscle exposure, with a minimal impact on SVA liver AUC. The current work illustrates the feasibility of parameter estimation in complex physiologically based models, where estimation is performed without neglecting: i) the variability in key system and drug related parameters and ii) the uncertainty on the results of in vitro experiments that are used to inform model parameters. The developed mechanistic model can be used in the future to predict the impact of genetic polymorphisms or potential drug-drug interactions on both SV and SVA.

References 1. Wilke RA, et al. The clinical pharmacogenomics implementation consortium: CPIC guideline for SLCO1B1 and simvastatininduced myopathy. Clin Pharmacol Ther 2012; 92: 112–7. 2. Langdon G, et al. Linking preclinical and clinical whole-body physiologically based pharmacokinetic models with prior distributions in NONMEM. Eur J Clin Pharmacol 2007; 63: 485–98. 3. Polli JW, et al. Evaluation of drug interactions of GSK1292263 (a GPR119 agonist) with statins: From in vitro data to clinical study design. Xenobiotica 2013; 43: 498–508.

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4. Pasanen MK, et al. SLCO1B1 polymorphism markedly affects the pharmacokinetics of simvastatin acid. Pharmacogenet Genomics 2006; 16: 873–9.

P394. DRUG-DRUG INTERACTION STUDY TO ASSESS WHETHER A PHARMACOKINETIC BASIS EXISTS FOR PREVIOUSLY REPORTED COGNITIVE DEFICITS WITH A COMBINATION OF 5-FLUOROURACIL AND LOW DOSE METHOTREXATE


Vaishnavi Ganti, Ellen Walker and Swati Nagar Pharmaceutical Sciences, Temple University, Philadelphia, PA In the past we reported increased cognitive deficits in mice receiving a combination of 5-fluorouarcil (5-FU, 75 mg/kg) and low dose methotrexate (MTX, 3.2 mg/kg). These cognitive deficits were absent or less prominent when either drug was administered alone or when 5-FU was dosed with other higher doses of MTX (10 mg/kg, 32 mg/kg). The purpose of this study was to: i) determine whether the reported drug-drug interaction (DDI) was of a pharmacokinetic (PK) nature; ii) explore probable mechanisms for any observed PK interactions, and iii) postulate a relationship between observed PK interactions and reported cognitive deficits. For this, a PK study was conducted in mice receiving 5-FU and MTX individually and in combinations in plasma, brain and urine for a period of 5 min–24 h. Also, for animals receiving 5-FU, we determined the plasma and brain concentrations (5 min-24 h) of a-fluoro, b-alanine, a known neurotoxic metabolite of 5-FU. There was no significant increase in concentrations of either drug/metabolite in the brain. However, volume of distribution at steady state increased for 5-FU from 0.9 0.3 dosed alone to 12 3 L/kg dosed with 3.2 mg/kg MTX. Additionally, for 3.2 mg/kg MTX, systemic clearance increased from 0.05 0.02 dosed alone to 0.21 0.02 L/min/kg when in combination with 5-FU. For the groups receiving higher doses of MTX (10 mg/kg, 32 mg/kg), non-linear PK was observed. Hence, non-compartmental analysis for PK parameters determination could not be performed. For the group receiving 5-FU with 3.2 mg/kg MTX, we observed significant increase in fraction excreted unchanged in the urine (fe) for 3.2 mg/kg MTX from 0.2 0.006 (MTX dosed alone) to 0.4 0.2 (MTX dosed with 5-FU). Based on the above results, we made the following conclusions: i) changes in primary PK parameters establish PK basis for the reported DDI; and, ii) increase in fe is indicative of probable interactions at the site of active transporters. To follow-up the latter possibility, we are actively investigating interactions for uptake organic anion transporters in-vivo and in-vitro. Additionally we are investigating possible changes in intrinsic clearance for 5-FU and MTX using cryopreserved murine hepatocytes and liver cytosols. Also, based on our PK results we hypothesize changes in systemic drug exposure are suggestive of processes affecting cognition indirectly, such as systemic oxidative stress. In future studies, we aim to monitor reduced glutathione levels in plasma as an indicator of oxidative stress. Ultimately a mechanistic PK model addressing observed DDI will be developed. (Supported by NIHR01-CA129092 and NIHR03-CA159389)

P395. PHYSIOLOGICALLY-BASED PHARMACOKINETIC AND PHARMACODYNAMIC MODEL FOR THE INHIBITION OF ACETYLCHOLINESTERASE BY ACOTIAMIDE, A NOVEL GASTROPROKINETIC AGENT FOR THE TREATMENT OF FUNCTIONAL DYSPEPSIA, IN RAT STOMACH Kazuyoshi Yoshii, Minami Iikura, Masamichi Hirayama, Ryoko Toda, Yukinori Mera and Yoshihiro Kawabata Zeria Pharmaceutical Co., Ltd., Saitama, Japan The novel gastroprokinetic agent acotiamide is an inhibitor of acetylcholinesterase (AChE) activity in vitro and is often used for the treatment of functional dyspepsia. In rats and dogs, acotiamide enhances gastric emptying, increases gastrointestinal tract motility, and is expected to inhibit AChE in gastric muscle. However, whether or not these phenomena are direct acotiamide inhibition effects on AChE remains unclear. Physiologically-based pharmacokinetic and pharmacodynamic (PBPK/PD) analysis could be useful for clarifying this matter, as this analysis method quantitatively describes the relationship between exposure, response, and physiological factors by constructing a valid model. As such, we attempted to clarify the relationship between acotiamide and acetylcholine (ACh) concentrations in the stomach. A two-compartment model was fitted to the blood concentration profiles of acotiamide, and a PBPK model was used to describe the time course of acotiamide concentration in the stomach, including precursor pool and deep pool compartments. A PD model was used as the indirect response model. The simulated AChE activity using in vitro kinetics parameters for MATPþ hydrolysis in the PBPK model agreed well with the observed AChE activity ex vivo. The PBPK/PD model well described the stomach concentrations of acotiamide and ACh. Additionally, the estimated IC50 value from the model when assuming acotiamide concentration in the precursor pool was similar to the Ki value estimated in vitro. Taken together, these results indicate that acotiamide in the precursor pool does indeed help preserve ACh concentration via AChE inhibition in rat stomach.

P396. UNIQUE ROLE OF PXR IN THE HEPATOCYTE PROLIFERATION IN MICE Ryota Shizu, Satoshi Benoki, Miki Takahashi, Susumu Kodama, Yasushi Yamazoe and Kouichi Yoshinari Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan


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Xenobiotic-responsive nuclear receptors PXR, CAR and PPARa are transcription factors involved in the xenobioticinduced expression of genes associated with the drug metabolism and transport, and energy metabolism. While CAR or PPARa activation promotes hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In this study, we have investigated the role of PXR in the hepatocyte proliferation with in vivo and in vitro models. Mice were treated with PCN (a mouse PXR activator) with or without TCPOBOP (a mouse CAR activator) or Wy-14643 (a PPARa activator). TCPOBOP or Wy-14643 treatment (48 h) resulted in the increased mRNA levels of cell cycle-related genes and the number of Ki-67-positive cells in livers, suggesting the increased hepatocyte proliferation as expected. In contrast, no proliferation was observed in mice treated with PCN alone. PCN treatment, however, augmented the TCPOBOP- or Wy-14643-dependent hepatocyte proliferation in wild-type mice but not in PXR-deficient mice. AML-12 cells, immortalized mouse hepatocytes, were synchronized at G0 phase by culturing in serum-free medium for 48 h and then cultured in fresh medium containing 10% serum for cell cycle progression. Reentry into cell cycle was confirmed by the decreased and increased ratios of G0/G1-phase and S- or G2/M-phase cells, respectively, by FACS analysis. PCN treatment following infection with recombinant adenovirus expressing mouse PXR accelerated this serum-induced cell cycle progression. Moreover, PXR activation augmented the cell population after serum addition. FACS analysis of mouse primary hepatocytes demonstrated that pre-treatment of mice with PCN increased the RNA content of hepatocytes, suggesting that PCN treatment moves G0-phase hepatocytes into G1 phase. PCN treatment of mice reduced hepatic mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation, p27 and p130, respectively. In reporter assays, PXR suppressed the transactivation of mouse Cdkn1b and Rbl2 mediated by FOXO3, known as a transcriptional activator of these genes. Our present findings suggest that hepatocyte proliferation mediated by different stimuli is accelerated by PXR co-activation despite that PXR activation alone does not cause the cell proliferation of mouse hepatocytes. This enhancing effect might result from the down-regulation of the suppressors of G0/G1-phase transition, p27 and p130. Thus PXR may play a unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics.

P397. AHR INACTIVATION BY ALPHA-NAPHTHOFLAVONE AND RESVERATROL INHIBITS CELL PROLIFERATION BY INCREASING P53 AND APOPTOSIS LEVELS IN HELA CELLS Alegre Flores, Maria Cabanãs and Guillermo Elizondo Cell Biology, CINVESTEV-IPN, Mexico, Mexico The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates the expression of genes encoding biotransformation enzymes. However, in recent years it has been associated with the regulation of several cell processes others than biotransformation such as cell proliferation and apoptosis. Deregulation of these processes is known to contribute to tumor promotion and cancer development. In particularly, it has been showed that AhR is overexpressed in several cancer types such as human lung carcinoma, hepatocarcinoma, and pancreatic cancer. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an AhR ligand, increases hepatocytes proliferation while inhibit UV light and diethylnitrosamine (DEN) –induced apoptosis. Other studies have shown that hepatocytes and embryonic fibroblasts from AhR-null mouse presented an increase in apoptotic cells compared to wild type cells. More recently, we have showed that TCDD treatment blocks apoptosis by decreasing p53 levels in mouse hepatocytes. On the other hand, it has been observed that AhR is constitutively active in several glioma cell lines, while untreated HeLa cells presented high levels of AhR in the nucleus compared to Hepa I cells. The above data prompted us to study whether AhR antagonists suppress cell proliferation and modify apoptosis levels in HeLa cells. For this purpose, HeLa cells were treated with known AhR antagonists, alpha naphthoflavone and resveratrol, and cell proliferation, cell cycle profile, p53 and apoptosis levels were determined. The present data indicates that AhR antagonists inhibit cell proliferation while increase p53 and apoptosis levels in a dose dependent manner. On the other hand, resveratrol treatment results in a G1 arrest while alpha naphthoflavone treatment increase the percentage of cells in S phase compared with controls. In conclusion, the AhR inactivation promotes a decrease in cell proliferation accompanied by an increase in p53 and apoptosis levels. These data implies that AhR may be a novel drug-target for cancer.

P398. THE EFFECT OF ERK SIGNALING PATHWAY INHIBITION ON PREGNANE X RECEPTOR (PXR)MEDIATED CYP3A SUB-FAMILY GENES EXPRESSION Tomas Smutny and Petr Pavek Department of Pharmacology and Toxicology, Charles University in Prague, Hradec Kralove, Czech Republic Members of the cytochrome P450 (CYP) represent super-family of enzymes with capacity to carry out both anabolic and catabolic metabolism. Of the 57 CYP genes present in humans cytochromes from families CYP1, CYP2 and CYP3 are mainly involved in metabolism of xenobiotics. Of these, the CYP3A sub-family appears as the most important group due to high quantity in human liver and substrate promiscuity, meaning that up 60% of therapeutics in use today are metabolized by CYP3A sub-family members [1].


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The pregnane X receptor (PXR) belongs to the nuclear receptor super-family and it is well known xenobiotic sensor which regulates wide variety of genes involved in xenobiotic biotransformation [2]. The transcriptional activity of PXR is governed by direct binding of structurally diverse PXR ligands. In addition, numerous studies have indicated that cellular signaling pathways modulate the functions of PXR in transactivation of CYP genes [3]. ERK signaling pathway plays a key role in integrating extracellular stimulations into various cell functions including cell proliferation, survival and migration [4]. Several reports showed that expression of individual CYP isoforms was changed in response to alteration in ERK signaling cascade activity [5]. In our study, we have investigated whether selective small molecule MEK1/2 inhibitors (U0126, PD0325901 and PD184352) regulates expression of CYP3A sub-family members in hepatocellular carcinoma cells. We observed that these MEK inhibitors significantly induced mRNA of major isoforms of CYP3A sub-family. Next, we aimed to shed the light on the mechanism involved in upregulation of CYP3A genes by MEK inhibitors. First, we suggested that MEK inhibitors could be direct ligands of PXR what we demonstrated using PXR competitive binding assay. In addition, we used dominant negative MEK1 protein to suppress endogenous activity of ERK signaling. In these experiments, we found that CYP3A4 promoter was also activated in absence of small molecule MEK inhibitors which implies ligand-independent regulation of CYP3A4 gene through ERK signaling. There are several lines of evidence that EKR signaling pathway regulates small heterodimer partner (SHP), a corepressor of PXR-dependent CYP expression, by increasing SHP stability and inhibiting proteasomal degradation [6]. Our further studies however indicated that MEK inhibitors downregulated SHP mRNA expression and suppressed SHP luciferase reporter construct. These data indicate that upregulation of CYP3A sub-family genes by small molecule MEK1/2 inhibitors is mediated via multiple mechanisms including suppression of SHP at the transcriptional level. This work is supported by GACR P303/12/G163 and P303/12/0472 projects.

References 1. Plant N (2007) The human cytochrome P450 sub-family: Transcriptional regulation, inter-individual variation and interaction networks. Biochim Biophys Acta 1770(3):478–88. 2. Wang YM, Ong SS, Chai SC, Chen T (2012) Role of CAR and PXR in xenobiotic sensing and metabolism. Expert Opin Drug Metab Toxicol 8(7):803–17. 3. Pondugula SR, Dong H and Chen T (2009) Phosphorylation and Protein-protein interactions in PXR-mediated CYP3A Repression. Expert Opin Drug Metab Toxicol 5(8):861–873. 4. Min L, He B, Hui L (2011) Mitogen-activated protein kinases in hepatocellular carcinoma development. Semin Cancer Biol 21(1):10–20. 5. Braeuning A (2009) Regulation of cytochrome P450 expression by Ras- and b-Catenin-Dependent Signaling. Curr Drug Metab 10(2):138–158. 6. Miao J, Xiao Z, Kanamaluru D, Min G, Yau PM, Veenstra TD, Ellis E, Strom S, Suino-Powell K, Xu HE, Kemper JK (2009) Bile acid signaling pathways increase stability of Small Heterodimer Partner (SHP) by inhibiting ubiquitin-proteasomal degradation. Genes Dev 23(8):986–96.

P399. SELECTION AND CHARACTERIZATION OF HUMAN CELL LINE SUITABLE FOR ASSESSEMENT OF THYROID RECEPTOR ACTIVATION Peter Illes and Zdenek Dvorak Department of Cell Biology and Genetics, Faculty of Science Palacky University Olomouc, Olomouc, Czech Republic Thyroid hormones (THs), triiodothyronine and thyroxine (T3 and T4), regulate gene expression by binding to high-affinity thyroid hormone receptors (TRs) and play a crucial role in growth, development and energy homeostasis. TRs belong to the subfamily of nuclear receptors that recognize specific response elements (TREs) in promoter regions of TH-regulated genes and activate or repress transcription in response to hormone. It is known that large number of exogenous ligands, including natural and synthetic compounds, drugs and environmental pollutants, may disrupt thyroid hormone mode of action and thus affect normal function of endocrine system. Therefore, the development of stable reporter system for assessment of TR transcriptional activity allowing high-throughput screening of effects of various compounds on TR-dependent regulation of gene expression, would be of great benefit. In our recent work we focused on selection and characterization of human cell line, meeting the criteria for successful construction of such stably transfected reporter cell line. Using western blot immunodetection analyses we investigated the presence of TRs (TRa and TRb) in protein cell lysates of 15 human cell lines. Based on the results we preselected 5 cell lines (A2780 – human ovarian carcinoma, FTC-133 – human follicular thyroid carcinoma, G-361 – human malignant melanoma, HeK293T – human embryo kidney and HepG2 – human hepatocellular carcinoma) with the highest levels of endogenously expressed TRs. In respect to fact, that only the presence of TRs in cells does not provide the functionality of mechanism responsible for TR-dependent regulation of gene expression, we performed quantitative real time PCR studies in selected cell lines. We observed changes in levels of mRNA expressions of human Spot 14gene (TH-inducible gene playing role

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in lipogenesis) after treatment of cells by 0.2 nM, 2 nM and 20 nM T3. We found out, that among 5 selected lines the most prominent induction of mRNA expression was in the FTC-133 cell line and the level of expression was T3 dose dependent. These results indicate that human FTC-133 cell line is a suitable tool for construction of human stable transfected reporter cell line for assessment of TR transcriptional activity. Research was supported by project CZ.1.07/2.3.00/30.0004

P400. LXR MEDIATED UP-REGULATION OF ABCA1 AND ABCG1 THROUGH DOXORUBICIN IN CARDIOMYOCYTES


Judith V. Monzel1, Henriette Meyer zu Schwabedissen1, Thomas Budde1, Sandra Bien-Moeller1, Stefan Oswald2, Heyo K. Kroemer1 and Markus Grube1 1 Department of Pharmacology, University of Greifswald, Greifswald, Germany, 2Department of Clinical Pharmacology, University of Greifswald, Greifswald, Germany Cardiotoxicity is still an important dose-limiting factor in anthracycline based chemotherapy. In this context formation of reactive oxygen species (ROS) is assumed to be responsible for the development of cardiac side effects. Previously, using a murine model for acute doxorubicin-induced cardiotoxicity we found elevated cholesterol and oxysterol levels in the serum and cardiac tissue accompanied by enhanced expression of the cholesterol transporters Abca1 and Abcg1. In the present study we focus on the underlying mechanism of this observation using HL-1 murine cardiomyocytes. Treatment of HL-1 cells with doxorubicin and other anthracyclines resulted in an enhanced expression of Abca1 and Abcg1 on mRNA and protein level. Furthermore, Abca1 and Abcg1 function was examined measuring cholesterol efflux. Therefore, HL-1 cells were treated with doxorubicin over 48 h and additionally loaded with tritium-labeled cholesterol (1mCi/ml) after 24 h incubation. The cholesterol efflux was measured over 4 h using transport buffers containing 10 mg/ml ApoA1 (Abca1) or 50 mg/ml HDL (Abcg1) in the presence or absence of the ABC-transporter inhibitor Diisothiocyanostilbenedisulfonic acid (DIDS, 100 mM). The Abca1 (Abcg1) mediated transport was 4.0 0.5-fold (2.0 0.4-fold) higher in doxorubicin treated cells compared to control and was significantly inhibited in presence of DIDS. Similar results were obtained using the known LXR ligand TO-091317. Since expression of both cholesterol transporters Abca1 and Abcg1 is known to be regulated by liver-x-receptor (LXR) an involvement of this transcription factor in the doxorubicin-dependent upregulation of Abca1 and Abcg1 was analyzed. Here, we were able to demonstrate by confocal laser scanning microscopy that doxorubicin-treatment leads to a translocation of LXR. In addition, luciferase reporter gene assays using a LXR-binding site containing promotor construct revealed a concentration dependent induction of the reporter gene expression (Doxorubicin 300 nM: 1.8 0.2-fold; TO-091317 10 mM: 4.0 0.5-fold), which was significantly inhibited by cotransfection of the endogenous LXR inhibitor SHP-1 by about 60%. Taken together, anthracycline (doxorubicin) dependent induction of the cholesterol transporters ABCA1 and ABCG1 is mediated by LXR-activation probably due to doxorubicin induced formation of oxysterols.

P401. METABOLISM AND DISPOSITION OF MELENGESTROL ACETATE IN HUMANS EXTRAPOLATED FROM THE PHARMACOKINETICS IN CHIMERIC MICE WITH HUMANIZED LIVER AND PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELING Hiroshi Yamazaki1, Hiroshi Suemizu2, Ai Tsukada1, Shigeto Sota1, Norie Murayama1, Ryohji Takano1, Makiko Shimizu1 and Masato Nakamura2 1 Showa Pharmaceutical Univ, Machida, Japan, 2Central Institute for Experimental Animals, Kawasaki, Japan It is of global interest to develop simple, advanced, and accurate risk assessment systems to support the interpretation of data from human biomonitoring studies. Sex hormones administered to farm animals are of particular interest because of their regulatory role in developmental processes. To predict concentrations in humans of the synthetic growth promoter melengestrol acetate (17a-acetoxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione), a forward dosimetry approach was carried out using data from no-observed-adverse-effect-level doses orally administered to rats, mice or chimeric TK-NOG mice with humanized liver and from in vitro human and rodent experiments. Human liver microsomes preferentially mediated 2-hydroxylation of melengestrol acetate, but rodent livers produced additional unidentified hydroxymetabolites. Melengestrol acetate elimination in humans was estimated to be slow compared with elimination in rodents with a simplified physiologically based pharmacokinetic (PBPK) model (basically consisting of a chemical receptor compartment, a metabolizing compartment, and a central compartment); and actual slow drug disposition of melengestrol acetate were seen in chimeric TK-NOG mice with humanized liver. Although difference in early elimination kinetics might have some limitations in assessment of fully chronic exposure of melengestrol acetate, together with the present results that melengestrol acetate clearance in humans may be slower than that in rodents, a species difference factor should be considered in setting the uncertainty factor to take into account the possibly slower clearance in humans. In conclusion, adjusted animal biomonitoring equivalents for melengestrol acetate from animal studies with humanized liver could be scaled to human biomonitoring equivalents using known species allometric scaling factors and human metabolic data with the simplified PBPK model using another bottom up system.

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P402. MIR-137 REGULATES THE CONSTITUTIVE ANDROSTANE RECEPTOR (CAR) AND MODULATES DOXORUBICIN SENSITIVITY IN CANCER CELLS


Apana Agha L. Takwi1, Yue-Ming Wang1, Jing Wu1, Martin Michaelis2, Jindrich Cinatl Jr.3 and Taosheng Chen1 1 Chemical Biology and Therapeutics, St. Jude Children Research Hospital, Memphis, TN, 2School of Biosciences, University of kent, Canterbury, United Kingdom, 3Clinics of the Goethe-University, Institute for Medical Virology, Frankfurt am Main, Germany Multidrug resistance (MDR) remains a major obstacle to effective chemotherapy? the most common treatment for cancer. CAR functions in xenobiotic detoxification by regulating the expression of phase I drug metabolizing enzymes and ATP-binding cassette (ABC) transporters, whose overexpression in cancers and whose role in drug resistance make them potential therapeutic targets for reducing MDR. MicroRNAs are endogenous negative regulators of gene expression and have been implicated in drug resistance. Here we report the inversely related expression of miR-137 and CAR in neuroblastoma cells. Interestingly, we show for the first time a negative feedback loop whereby miR-137 down-regulates CAR expression and CAR down-regulates miR-137 expression. We found that miR-137 is down-regulated and CAR is up-regulated in doxorubicin-resistant neuroblastoma cells. miR-137 over-expression resulted in down-regulation of CAR protein and mRNA (via mRNA degradation), and its transcriptional targets MDR1 and CYP2B6, leading to re-sensitization of doxorubicin-resistant cells to doxorubicin (as shown by reduced proliferation, increased apoptosis, and increased G2-phase cell cycle arrest) and reduced the in vivo growth rate of neuroblastoma xenografts. We observed similar results in cellular models of hepatocellular and colon cancers, indicating that the doxorubicin-sensitizing effect of miR-137 is not tumor type-specific. Finally, the promoter of miR-137 is hypermethylated in doxorubicin-resistant cancer cells. Hypermethylation of miR-137 promoter and negative regulation of miR-137 by CAR contribute to reduced miR-137 expression and increased CAR and MDR1 expression in doxorubicin-resistant neuroblastoma cells. These findings demonstrate that miR-137 is a crucial regulator of cancer response to doxorubicin, and identify miR-137 as a promising target to reduce CAR-driven doxorubicin resistance.

P403. EFFECT OF P,P-DDE ON PROLIFERATION AND DIFFERENTIATION OF PROMYELOCITIC CELLS HL-60 Nallely A. Torres1, Leonor Acosta2, Ana Laura Luna2 and Emma S. Calderon2 1 Toxicologia, Centro de Investigacioń y Estudios Avanzados del IPN, Me´xico City, Mexico, 2Toxicologıá, Centro de Investigacioń y Estudios Avanzados del IPN, Me´xico City, Mexico Dichlorodiphenyltrichloroethane (DDT) is a potent insecticide used worldwide for agricultural and public health purposes since the 1940s. Because DDT toxic effects on wildlife and humans, and its environmental persistence, its use was restricted in several countries since 1970s. In Me´xico DDT was banned in 2000 but its breakdown product p,p0 -DDE and the compound p0 p-DDT are still detected in the environment and human samples. Due to the liposolubility, p,p0 -DDE has been found in adipose tissues like and in bone marrow; in fact, in this tissue the range of concentration of the p,p0 -DDE found is from 0.019 to 1.9 mg/ml, and its presence has been associated with myelodysplastic syndromes that implicate alterations in myeloid proliferation and differentiation. Since that in the differentiation process JAK2 has a relevant role, and our group recently demonstrated that p,p0 DDE induces the activation of JAK2 in murine macrophages, the aim: of this study was to investigate the effect of low concentrations of p,p0 -DDE on proliferation and differentiation of a promyelocytic cells line (HL-60) to monocytes/macrophages or to granulocyte Results: The exposure to 0.019, 0.19 and 1.9mg/ml of p,p0 -DDE during 5 days do not affect the viability and the proliferation of non-stimulated HL-60 cells. In cells stimulated to monocytic/macrophage and granulocytic differentiation with TPA and DMSO, respectively, p,p0 -DDE reduced significantly the viability and proliferation by more than 20% only in DMSO co-exposition. The effect of the p,p0 -DDE on cell non-stimulated or stimulated to differenciation was evaluated by morphologic characteristics, by adherent cells (monocyte/macrophages), and by presence of CD11b, CD14 and CD16 by flow cytometry. The p,p0 -DDE treatment at five days induced the presence of morphologic characteristics (metamyelocytes). The p,p0 -DDE decreased the percentage of differentiation to metamielocyte in cells treated with DMSO compared with the control (only DMSO). The p,p0 -DDE decreased the percentage of adherent cells in cultures treated with TPA, compared with the control (only treated with TPA). The presence of CD11b was detected only in cultures exposed to 1.9 mg/ml of p,p0 -DDE. The p,p0 -DDE in cells treated with TPA during 8 days decreased the presence of CD11b and CD14 compared to control (only TPA). In cells treated with DMSO it was not detected CD11b or CD16, whereas in cells co-treated with p,p0 -DDE (0.19 and 0.019mg/ml) CD11b was detected. Nowadays we are evaluating the presence of CD11b, CD14 and CD16 in cultures co-treated with p,p0 -DDE with TPA or DMSO, at shorter time as upon other stimulation schedules.(Supported by Conacyt - 152491)

P404. NOVEL APPROACH TO IDENTIFYING CYP2C9 RESIDUES RESPONSIBLE FOR STEREOSELECTIVE BINDING AND OXIDATION OF COMPOUNDS Grover P. Miller1, Timothy M. Horton2, Shelby C. Cobb2 and Martin D. Perry Jr2 1 Dept of Biochem & Molec Biol, Univ of Arkansas for Med Sci, Little Rock, AR, 2Chemistry, Ouachita Baptist University, Akadelphia, AR


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Cytochromes P450 (CYP for isoforms) play a central role in metabolism of chiral biological molecules; thus, development of computational methods to predict the role of P450 structure in recognizing and metabolizing chiral compounds is critical for advancing this field. In this study, we employed a novel approach to identify CYP2C9 residues responsible for stereoselective binding and oxidation of substrate enantiomers. Specifically, we docked twelve enantiomeric pairs of CYP2C9 substrates to the enzyme and subjected the complexes to Molecular Dynamic simulations to minimize structure. For each residue, enzyme contacts with bound molecules were identified within four angstroms and the energy of interactions calculated using LennardJones potential energy equations. The difference in energy values for each residue between enantiomeric substrate pairs served as a scoring system for its role in stereoselective interactions. These computational studies were carried out with CYP2C9 in the ground state (Fe3þ) and activated state in which perferryl iron (Fe ¼ O) is poised for catalysis as models of different CYP2C9 states during the catalytic cycle. Overall, the substrate dockings indicated that many of the interactions with CYP2C9 were specific to a particular pair of enantiomers; however, there were exceptions. In ground state complexes, Ala297 and Phe476 were often observed selectively interacting with the preferred substrate enantiomer, while Leu208 and Leu366 favored contact with the other enantiomer. The transition to an activated complex led to significant changes in the active site in which contacts were gained/lost or interestingly, switched from favoring one enantiomer to the other. For activated complexes, Leu208 now favored binding the preferred substrate enantiomer along with Phe100 and Ala297, but Arg108 and Trp212 preferentially bound the other enantiomer. Importantly, binding energy totals from substrate interactions for activated complexes indicated that more favorable complexes mostly correlated with stereoselective oxidation of CYP2C9 substrates. As further validation of this approach, CYP2C9 residues involved in critical interactions are being currently targeted for mutagenesis to assess the effects of those changes on stereoselective oxidation of substrates. Taken together, the identification of CYP2C9 residues involved in stereoselective metabolism yields insights for interpreting and predicting the importance of CYP2C9 in metabolism of chiral compounds, and in a broader sense, demonstrates the utility of a strategy for revealing the contributions of protein structure in that function.

P405. STEREOSELECTIVE N-OXIDATION OF BI-A BY CYP3A4 AND FMO3 Regina Nardi, Holly Maw, Donald Tweedie and Hongbin Yu Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT Prior to the First in Human (FIH) clinical trial, it is important to conduct CYP450 reaction phenotyping studies to determine whether polymorphic CYP450s (CYP2C9, 2C19 and 2D6) contribute significantly to the metabolism of a development compound. Preliminary data from Drug Discovery indicated that CYP2D6 contributed to the metabolism of BI-A (see Figure 1 for the partial structure). However, the extent of contribution was not determined due to the slow turnover of BI-A in human liver microsomes (HLM). In this study, a metabolite formation approach was used to determine the contributions of CYP450 isoforms (CYP2D6 in particular) to the metabolism of BI-A. Firstly, five metabolites were identified by high-resolution LC/MS (see Figure 1 for structures and abundance (% of total metabolites by MS peak areas) of 3 regioisomeric hydroxylation metabolites and 2 diastereomeric N-oxides). Secondly, the CYP450 isoforms responsible for the formation of these five metabolites were determined using recombinant human CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4 and 3A5) and CYP450 isoformselective chemical inhibitors with HLM. CYP3A4 was the main CYP450 isoform contributing to the formation of the

Figure 1 Enzymes responsible for the formation of the hydroxylation and N-oxide metabolites of BI-A.

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hydroxylation metabolites (with minor contribution from CYP2C8 or CYP2D6). CYP3A4 was the only CYP450 isoform contributing to the formation of N-oxide/A and N-oxide/B. In HLM ketoconazole inhibited the formation of the three hydroxylation metabolites and N-oxide/B to a significant extent. Interestingly ketoconazole did not inhibit the formation of N-oxide/A despite the fact that it was formed by rCYP3A4. Based on these findings, the potential role of FMOs in the formation of N-oxide/A was considered. Results from two additional experiments confirmed that N-oxide/A was mainly formed by FMO3: 1. The rate of formation of N-oxide/A was decreased by 60% when HLM was treated at 50 C for 2 minutes while the rate of formation of N-oxide/B was not affected; 2. N-oxide/A was formed when BI-A was incubated with recombinant human FMO3 and FMO5. N-oxide/B was not detected and the rate of formation of N-oxide/A with FMO3 was 10-fold more than that in FMO5. Since N-oxide/A comprised almost half of the metabolites of BI-A in HLM, FMO3 is considered a major enzyme contributing to the metabolism of BI-A. In summary, we have determined that CYP3A4 and FMO3 are the two major enzymes responsible for the metabolism of BI-A in vitro. Additionally these two enzymes demonstrated stereoselectivity in the formation of the two N-oxides, with FMO3 mainly responsible for the formation of N-oxide/A and CYP3A4 exclusively responsible for the formation of N-oxide/B.


P406. USE OF EFAVIRENZ ANALOGS TO PROBE CYP2B6 SUBSTRATE SPECIFICITY Philip M. Cox1 and Namandje´ N. Bumpus2 1 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, 2 Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD Cytochrome P450 2B6 (CYP2B6) is primarily responsible for the metabolism of the anti-HIV drug efavirenz ((S)-1-(2-Amino-5-chlorophenyl)-1-(trifluoromethyl)-3-cyclopropyl-2-propyn-1-ol). In order to probe the structural properties of efavirenz that render it a substrate for CYP2B6, four efavirenz analogs ((E)-Dihydroefavirenz, (aR)-Chloro-a-(cyclopropylethynyl)-2-[[(4-methoxyphenyl)methyl]amino]-a-(trifluoromethyl)benzenemethanol, as well as the C4 stereoisomers (aS)-2-Amino-5-chloro-a-(2-cyclopropylethynyl)-a-(trifluoromethyl)benzenemethanol and (aR)-2Amino-5-chloro-a-(2-cyclopropylethynyl)-a-(trifluoromethyl)benzenemethanol) were employed. Metabolism of these analogs by human liver microsomes (HLM) and cDNA-expressed cytochrome P450s (CYPs) was assessed using ultra-performance liquid chromatography–tandem mass spectrometry. HLM metabolism revealed two mono-oxygenated metabolites of (E)-Dihydroefavirenz, indicating oxygenation at position 7 or 8 and position 5 on the benzene ring. From the C4 stereoisomers, only one identical mono-oxygenated metabolite representing oxygenation at position 7 or 8 on the benzene ring was formed. CYP2B6 was able to catalyze oxygenation at position 7 or 8 on the benzene ring of (E)-Dihydroefavirenz but was unable to metabolize the C4 stereoisomers. Metabolism assays performed using the methoxyphenyl analog did not result in the formation of oxygenated products, although an N-dealkylatedspecies was detected from CYP1A2-catalyzed metabolism. To aid in assigning structures to fragmentation patterns, HLM assays were also performed in the presence of H218O. Peaks corresponding to fragments retaining a single oxygenation event were shifted by 2 mass units compared to the peaks observed using H216O. Together these data suggest the metabolism of efavirenz by CYP2B6 is dependent upon the integrity of the triple bond, the oxazine ring, and the stereochemistry of C4. (Funding: NSF Fellowship DGE-1232825 and NIH R01GM103853)

P407. COMBINED EFFECT OF SILYBUM MARIANUM AND GLYCYRRHIZA GLABRA ON CCL4 INDUCED LIVER INJURY IN RATIS NORVAGICUS Andleeb Shahzadi1, Mohsin Farooq2, Shazia Anwer Bukhari2, Sadia Javed2, Fozia Anjum2 and Haseeb Anwar3 1 Department of MedicalPharmacology, Istanbul University, Istanbul, Turkey, 2Department of Chemistry, Government College University, Faisalabad., Pakistan, 3Department of Physiology, Government College University, Faisalabad., Pakistan Liver disorders are more common through-out the world. Medicinal plants have been used for many years as a remedy for liver disorders. Silybum marianum and Glycyrrhiza glabra have been used in treatment of various disorders including liver disorders. Both plants have hepatoprotective, antioxidant and anti-inflammatory effects. Studies were conducted to investigate the combined hepatoprotective effect of Silybum marianum and Glycyrrhiza glabra on the carbon tetrachloride (CCl4) induced liver injury in albino rat. Albino rats were divided into four groups: control, induced, experimentally treated with different doses of Silybum marianum and Glycyrrhiza glabra. Three groups except control were administrated with 1 ml CCl4 in olive oil according to their body weight twice a week. Hepatic damage was revealed by activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). AST, ALT and ALP levels were increased while serum total protein (TP) and glutathione (GSH) levels were decreased in rats. In rats treated with S. marianum and G. glabra extracts, levels of AST, ALT and ALP were decreased while TP and GSH levels were increased. Statistical analysis of data demonstrated that combined effects of Silybum marianum and Glycyrrhiza glabra against carbon tetrachloride induced liver injury in rats were more significant (p 0.05) than that observed with individual plant’s extract or in combination with extract of other plants.

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P408. PREVENTION OF CISPLATIN-INDUCED NEPHROTOXICITY BY DICHLOROACETATE


Angelo Theodoratos1, Jane Dahlstrom2, Jonathan Smiles2, Anneke Blackburn1, Luyang Tian1, Ramindhu Galgamuwa1 and Philip Board1 1 Molecular Bioscience, John Curtin School of Medical Research, Canberra, Australia, 2Anatomical Pathology, ACT Pathology and ANU Medical School, The Canberra Hospital, Canberra, Australia There is a continuing need to improve the efficacy of anticancer agents and to reduce the toxicity associated with them. Recently, reversal of the glycolytic phenotype of cancer cells with novel agents such as dichloroacetate (DCA) has been recognised as an important new target for cancer therapy. DCA is currently being assessed as a novel anti-cancer agent in clinical trials and it is anticipated that DCA will be used in combination with other well-established chemotherapeutic drugs (1). It is therefore critically important to determine the effects of DCA on therapeutic efficacy and off-target effects of existing frontline anti-cancer drugs. We have recently discovered that DCA induces glutamate cysteine ligase, the rate-limiting step in glutathione (GSH) synthesis, a major cellular antioxidant (2). We hypothesised that the ability of DCA to stimulate GSH-synthesising capacity could reduce the dose limiting nephrotoxicity associated with cisplatin, which is thought to be mediated in part by oxidative stress. We therefore sought to determine whether DCA could attenuate cisplatin-induced nephrotoxicity and whether DCA influences the anti-cancer properties of cisplatin. Balb/c mice were treated with DCA prior to and after administration of a nephrotoxic dose of cisplatin. DCA prevented increases in serum creatinine, BUN, and renal proximal tubule apoptosis evident in cisplatin-only treated mice. When injected subcutaneously with the syngeneic 4T1 tumour cell line, tumour size in mice in the co-treated group decreased at the same rate as the cisplatin-only treated mice. We have concluded that DCA significantly reduces the development of cisplatin-induced nephrotoxicity and does not attenuate its anti-cancer properties.

References 1. Michelakis, E. D., Sutendra, G., Dromparis, P., Webster, L., Haromy, A., Niven, E., Maguire, C., Gammer, T. L., Mackey, J. R., Fulton, D., Abdulkarim, B., McMurtry, M. S., and Petruk, K. C. (2010) Metabolic modulation of glioblastoma with dichloroacetate. Sci Transl Med 2, 31ra34 2. Theodoratos, A., Blackburn, A. C., Cappello, J., Tummala, P., Dahlstrom, J. E., and Board, P. G. (2011) Dichloroacetic acid up-regulates hepatic glutathione synthesis via the induction of glutamate-cysteine ligase.Biochem Pharmacol 83, 427–433

P409. PHARMACOKINETICS OF SORAFENIB CORRELATES WITH ADVERSE EFFECTS UPON DOSAGE REDUCTION OR WITHDRAWAL IN PATIENTS WITH HEPATOCELLULAR CARCINOMA Miki Shimada1, Hoshimi Okawa2, Takahiro Maejima1, Yasuteru Kondo3, Tooru Shimosegawa3, Kanehiko Hisamichi1, Masaki Matsuura1 and Nariyasu Mano1 1 Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan, 2Division of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan, 3Division of Gastroenterology, Tohoku University Hospital, Sendai, Japan Background: Sorafenib, an oral multi-kinase inhibitor, was approved for treatment of hepatocellular carcinoma (HCC) judged to be unresectable or unresponsive to transarterial chemoembolization and radiofrequency ablation. Continuation of administration is important because sorafenib is the final therapy prior to palliative care for HCC patients. However, adverse effects forced 20% of patients to discontinue sorafenib within 1 month after first administration. Bioavailability of sorafenib is influenced by the activity of the excretory transporters ABCB1 and ABCG2, and cytochrome P450 3A4 in the small-intestinal mucosa or liver. Then, we investigated pharmacokinetics of sorafenib in order to identify predictive factors that correlate with adverse effects upon dosage reduction or withdrawal in patients with HCC. Method: HCC patients taking 200–800 mg of sorafenib once or twice day at Tohoku University Hospital were enrolled in this study. Peripheral blood samples were collected 0, 2, 6, and 12 h post-first administration, continuously at trough in morning every other day in hospital stay and at visit every 2 to 4 weeks in outpatient clinic. Serum concentration of sorafenib and sorafenib N-oxide was determined with a liquid chromatography system (Agilent 1100, Agilent Technologies, Inc., CA, USA). In this case, we connected trough values with a line and calculated under the concentration–time curve (AUC) by the trapezoidal method. The association of adverse effect-related sorafenib administration with AUC of sorafenib during days 1–7 (AUCsorafenib), sorafenib N-oxide during days 1–7 (AUCN-oxide), or AUCN-oxide/ AUCsorafenib (AUC ratio) was evaluated by Student’s t-test as comparison between dosage reduced or withdrawn group and dosage maintained group. Results and discussion: We observed that one adverse effect, hand-foot reaction (G2 according to the National Cancer Institute Common Terminology Criteria for adverse events version 4.0.), was accompanied by an increase in the serum sorafenib concentration in one patient. This result suggests that the serum concentration of sorafenib is related to this adverse effect of the drug. We, therefore, investigated pharmacokinetics of sorafenib in order to identify predictive factors that correlate with adverse effects upon dosage reduction or withdrawal of sorafenib in 14 patients with HCC. AUC ratio was significantly higher in group with dosage reduction or withdrawal than in that with dosage maintenance (0.21 0.021 vs 0.13 0.024). From the results of our statistical analysis (p ¼ 0.040), AUC ratio was a predictive factor for an adverse effect of

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sorafenib. Furthermore, in patients treated with the recommended daily dose (400 mg b.i.d.), the values of AUCsorafenib and AUCN–oxide correlated well with the corresponding concentrations at 6 h after first administration (r ¼ 0.82 [p50.01, n ¼ 10] and r ¼ 0.81[p50.01, n ¼ 9], respectively), as well as with laboratory data from a hepatic function test performed on the day prior to the beginning of treatment. These results suggest that we might be able to predict the adverse effects of sorafenib before and shortly after first treatment.

P410. THE DEVELOPMENT OF A UPLC-MS METHOD FOR THE DETECTION OF 17SS-ESTRADIOL AND ITS METABOLITES IN CELL SAMPLES TO EXAMINE METABOLISM-TRANSPORTER INTERACTION


Joanna H. Sier, Alfred E. Thumser and Nick J. Plant Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom The role of active drug transport in determining chemical fate is becoming increasingly clear, with efflux transporters such as MDR1, MRP2 and BCRP recognised as key determinants of drug action. These efflux transporters may impact upon both compound bioavailability, through first-pass effects, and drug resistance, through their up-regulation following chronic drug exposure. While the role of these transporters is well established, there is less information on other transporters, such as MRP3 and MRP4, which may act to move chemicals back into the systemic circulation rather than target them for elimination. The endogenous steroid 17b-estradiol (E2) and its major metabolites represent good probe substrates to investigate this relationship, allowing us to test the hypothesis that the relative activity of basolateral transporters (MRP3 and MRP4) compared to apical transporters (MDR1, MRP2, BCRP) in the liver is a key determinant of drug fate and potential site for drug-drug interactions. To achieve this aim we have developed an ultra-pressure liquid chromatography - mass spectrometry (UPLC-MS) method to detect estradiol and it metabolites in both cell lysate and medium samples of primary human hepatocytes. We utilise a 15 minute solvent gradient between water (solvent A) and methanol (solvent B), run on an Acquity C8 column 1.7 mm particle size, 2.1 100 mm: The gradient run consists of 1 minute at 98% A, 2–8 minutes 55% A, 9–11 minutes 10% A, 11–15 minutes 98% A and is run at a flow rate of 0.5 ml/min. The Xevo-TQS system was used in negative electrospray ionisation (ESCI-) mode with a capillary voltage of 1.65 kV and cone voltage of 30 V. The method was set up to detect Estradiol (E2) along with two of its metabolites; estrone (E1) and estrone-3-sulphate. We have also optimised detection for an internal standard of ethynylestradiol (EE2), chosen as a common synthetic analogue of E2 often used in oral contraceptives. Multiple reaction monitoring (MRM) pairs for each of these compounds were selected; 271.24145, 269.24145.8, 348.94106.9 and 295.34158.9, respectively. These could all be separated off the column with retention times of; E2 10.02mins, E1 9.98mins EE2 10.05mins and E3S 6.74mins in methanol standards and cell samples. Lower flow rates on the UPLC caused poorer peak resolution and reduced the limit of detection within samples. We looked at the impact of DMSO in sample preparation, with 0.1% DMSO (v/v) in methanol causing an overall improvement in detection for the four different structures. With this method we work within suitable limits of detection to allow us to test the central hypothesis.

P411. DOES A CHANGE IN THE EXPRESSION OF AQUAPORIN-3 IN THE INTESTINAL TRACT AFFECT DRUG ABSORPTION? Nobutomo Ikarashi, Chika Nagoya, Satoshi Kitaoka, Sayuri Kajiwara, Masayo Saito, Risako Kon, Yoshiki Kusunoki, Wataru Ochiai and Kiyoshi Sugiyama Dept of Clin Pharmacokinet, Hoshi Univ, Shinagawa-ku, Japan There are many transporters in the intestinal tract. Changes in the expression levels and activity of transporters affect the absorption of substrate drugs from the intestinal tract. The water channel aquaporin-3 (AQP3) is expressed at a high level in the intestinal tract and plays an important role in water transport. The rate of water transport in the intestinal tract may affect the unstirred water layer and may somehow affect drug absorption; however, the details are not at all clear. This study examined how drug absorption changed when the activity or expression level of AQP3 in the intestinal tract was altered. Warfarin alone, or warfarin and either mercury chloride (HgCl2; inhibitor of AQP3 activity) or prostaglandin E2 (PGE2; suppressor of AQP3 expression) were added to the apical side of differentiated cells from the human intestinal cell line Caco-2 in trans-well plates. The apparent permeability coefficient (Papp) was calculated by measuring the warfarin concentration on the basal side. The Papp of warfarin when HgCl2 was added to inhibit AQP3 activity by approximately 60% was almost the same as when warfarin alone was added. However, the Papp of warfarin when PGE2 was added to reduce the AQP3 protein expression to approximately 70% was about twice as high as that when warfarin alone was added. These results demonstrated that the absorption of drugs that were passively transported from the intestinal tract did not change when the expression level of AQP3 remained unchanged, and only the transport rate of water was reduced. However, it was found that drug absorption from the intestinal tract increased when the transport rate of water was reduced by reducing AQP3 expression. This study suggests that drug absorption from the intestinal tract may change when the expression of AQP3 is only altered, regardless of the rate of water transport.

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P412. SPECIES COMPARISON OF BCRP (ABCG2) MEDIATED EFFLUX ON PHARMACOKINETICS OF SUBSTRATE DRUGS USING BCRP KNOCK-OUT RATS AND MICE


Christoffer Bundgaard, Lassina Badolo and Mats Garmer Discovery DMPK, H. Lundbeck A/S, Copenhagen-Valby, Denmark Breast cancer resistant protein (BCRP) (ABCG2) acts to decrease the oral bioavailability and systemic exposure of its substrates as it is considered to be localized on the canalicular membrane of the hepatocytes, kidney and intestine where it mediates the elimination of compounds from these tissues. Genetically modified transporter knock-out mice offer an in vivo tool to assess the role of BCRP on drug absorption and disposition and has been widely applied in pre-clinical ADME research. Recently, Bcrp knock-out rats became commercially available offering an additional model to the common mice knock-out model. With use of these knock-out models the current study compared the functional role of Bcrp mediated efflux on in vivo pharmacokinetics between rats and mice for a set of known substrates. Four Bcrp substrate drugs (Sulfasalazine,Topotecan, Imatinib and Methotrexate) were administered orally or intravenously to Bcrp knock-out rats and mice and their gene-competent wild-type counterparts and the pharmacokinetic plasma profile was compared between genotypes and species. In addition, in vitro membrane permeability and intrinsic clearance data in rat and mice liver microsomes and hepatocytes were obtained to assess the role of metabolism and intestinal permeability. Following oral administration in rats all compounds tested were shown to have significantly higher AUC in Bcrp knock-out rats compared to wild-types with Sulfasalazine exhibiting the most pronounced difference. Following intravenous administration, only a minor increase in AUC was observed for Sulfasalazine and Topotecan in knock-out rats with the other compounds having similar AUC between knock-out and wild-type rats. This suggests that Bcrp primarily influences intestinal uptake and/or hepatic first-pass metabolism of these compounds. After oral dosing in mice, a significant increase in AUC in Bcrp knockout mice was observed for Sulfasalazine, Topotecan and Methotrexate with Sulfasalazine also in this species being the most prominent substrate. However for all compounds, the difference in plasma exposure between knock-out and wild-type mice was smaller than observed in rats. For Imatinib, no difference was observed between AUCs from Bcrp knock-out and wildtype mice after oral dosing. This could likely be attributed to the high intrinsic clearance observed for Imatinib in mice liver microsomes and hepatocytes. In conclusion, Bcrp knock-out rats was proven to be a useful model to study the functional effects of Bcrp on pharmacokinetics and was shown to be a more sensitive species towards BCRP mediated effects compared to mice for the substrates tested here. However, to understand the functional impact of these transporter effects on drug absorption and disposition, and translating this between species, information on the rate determining process of a substrate in the overall absorption and clearance pathways is required, as exemplified by Imatinib.

P413. ENHANCEMENT OF NASAL ABSORPTION OF A PROMISING NOVEL TACRINE DIMER HLS-3 BY COADMINISTRATION WITH P-GLYCOPROTEIN INHIBITOR VERAPAMIL Shuai Qian1, LS He2, Marvin Mak3, CY Ho2, Yifan Han3 and Zhong Zuo1 1 School of Pharmacy, The Chinese University of Hong Kong, Hong Kong, Hong Kong, 2Center of Novel Functional Molecules, The Chinese University of Hong Kong, Shatin, Hong Kong, 3Department of Applied Biology & Chemical Technology Institute of Modern Chinese Medicine, The Hong Kong Polytechnic University, Hung Hom, Hong Kong Purpose: As a novel tacrine dimer with high potency of anti-acetylcholinesterase activity (IC50: 1.2 nM) for the treatment of Alzheimer’s disease, HLS-3 has a poor permeability and low oral bioavailability. Present study aims to investigate potential efflux transporters involved in absorption process of HLS-3 and the effect of related efflux tranporter inhibitor on intranasal delivery of HLS-3. Methods: Bidirectional transport studies of HLS-3 in presence and absence of various inhibitors/substrates including verapamil, mitoxantrone and MK571 on Caco-2 cell monolayer model were employed to explore possible efflux transporters involved in its absorptive process. For pharmacokinetic evaluations, rats were divided into two groups (n ¼ 5/group). Rats in Group 1 were firstly pretreated with 10 ml of 12 mg/ml verapamil (effective P-gp inhibitor identified in Caco-2 cell studies) solution by instilling it into each nostril. After 20 min, 30 ml of solution containing HLS-3 (4.2 mg/ml, equivalent to 0.5 mg/kg) and verapamil (12 mg/ml) was then intranasally administered to rats. Rats in Group 2 were intranasally administered with only HLS-3 at 0.5 mg/kg and severed as reference. At different post-dose times, blood was collected through a catheter inserted in the right jugular vein. HLS-3 plasma concentration was analyzed by a validated LC/MS/MS method, and its pharmacokinetic parameters were calculated with WinNonlin software and compared between the two treatment groups. Results: HLS-3 with a low absorptive permeability of 0.11 106 cm/s had a medium efflux ratio of 8.3. Both verapamil and MK571 could significantly inhibit the secretive transport of HLS-3, suggesting important roles of P-gp and MRPs in the efflux transport of HLS-3. In comparison to intranasal administration of HLS-3 solution, coadministration with the P-gp inhibitor verapamil significantly improved the Cmax and AUC0–1 of HLS-3 for 2.2-fold and 1.7-fold, respectively. Meanwhile, Tmax of HLS-3 was significantly decreased from 30 min to 10 min in presence of verapamil. No significant difference was detected on the elimination half-life of HLS-3 between two tested groups. These results suggested that verapamil could significantly improve the nasal absorption of HLS-3 without alteration of its elimination. Conclusion: HLS-3 was a low permeable drug with P-gp

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and MRPs efflux transporters involved in its absorption process. Coadministration with P-gp inhibitor verapamil could significantly improve the nasal absorption of HLS-3. [CUHK 480809 from the Research Grants Council of the Hong Kong SAR].

P414. CONTRIBUTION OF OATPS TO DRUG ABSORPTION IN HUMAN INTESTINE IS MUCH LESS THAN PREDICTED


Hidenori Takada, Michael J. Knauer, Rommel G. Tirona and Richard B. Kim Division of Clinical Pharmacology, The University of Western Ontario, London, ON, Canada Organic anion transporting polypeptides (OATPs) mediate the cellular uptake of various endogenous substances and prescribed drugs. OATPs are known for broad substrate specificity including anionic (e.g. HMG-CoA reductase inhibitors and angiotensin II receptor antagonists), neutral (e.g. HIV protease inhibitors and anticancer agents), cationic (e.g. b-adrenergic blocking agents and b-lactam antibiotics) and zwitterionic compounds (e.g. fexofenadine). Our group and others had reported that fruit juice such as grapefruit juice, orange juice, and apple juice, decrease the systemic exposure to drugs such as fexofenadine, celiprolol, and talinolol through inhibition of intestinal OATP(s). However, the role and relevance of intestinal OATPs in their contributions to drug absorption remains to be clarified. We assessed mRNA and protein expression of OATPs from a large number of human intestinal biopsy samples. Only OATP2B1 was significantly expressed in human duodenum, terminal ileum, and colon when mRNA levels were measured using quantitative real-time PCR. OATP1A2, 1B1 and 1B3 mRNA levels were negligible or undetectable in most of the intestinal samples. In addition, we were able to verify expression of OATP2B1 protein among the OATPs tested, from intestinal samples using an LC-MS/MS-based protein absolute quantification method. It should be noted that intestinal OATP2B1 protein concentration was significantly lower than those of OATP1B1, 1B3, and 2B1 expressed in human hepatocytes. We then evaluated OATP2B1-mediated uptake of rosvastatin, fexofenadine, and talinolol at concentrations seen in the intestinal lumen using a novel adenovirus-based expression system. We observed modest or absence of transport for these drugs at pH5.5 and pH7.4. Our findings suggest OATP2B1 is unlikely the main mechanism that mediates the uptake of these drugs from the intestine. In conclusion, in the human intestine, only OATP2B1, but not OATP1A2, 1B1 or 1B3 is significantly expressed. Moreover, OATP2B1 appears to have limited affinity for rosvastatin, fexofenadine, or talinolol uptake. Therefore it remains possible there may be additional transporter(s) that mediate the intestinal absorption of such compounds.

P415. STUDY ON TRANSCELLULAR TRANSPORT MECHANISMS OF ACETYLBOSWELLIC ACID IN VITRO Xiaoyan Ci, Yuanyuan Xia, Yong Zeng, Xiulin Yi, Changxiao Liu and Duanyun Si State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, China Acetylboswellic acid (ABA) is a pentacyclic triterpenoid compounds, extracted from Boswellia serrate, used for the treatment of inflammatory diseases and cancer1. Extensive researches show that the oral bioavailability of most natural pentacyclic triterpenoids is low (53%). But the absolute bioavailability of ABA was 30% when gavaged to rats, and it kept high exposure in brain and liver tissues. Therefore, the transcellular transportation mechanisms of ABA are needed to be investigated. An LC-MS/MS assay was established to determine the concentration of ABA in Hank’s buffer. Cell models of CACO-2, MDCK, MDCK-MDR1 and cell lines expressing human drug transporter were applied to study the transportation mechanism of ABA. In CACO-2 and MDCK-wild cell models, Papp(AP ! BL) of 50 mM AKA was 0.82 106 cm/s and 0.26 106 cm/s, respectively The ratio of Papp(BL ! AP)/Papp (AP ! BL) in MDCK-MDR1 cell monolayer of 50 mM acetylboswellic acid is smaller than 2. The IC50value of ABA was determined to be 24.5 mM and 1.5 mM for hOATP1B1 and hOATP2B1, respectively, and the intake of 50 mM acetyl-boswellic acid mediated by OATP1B1-HEK293 and OATP2B1HEK293 compared with negative control in MOCK-HEK293 cells was 4 time higher. These data suggested that transcell permeation ability of ABA was in medium level. ABA was not a substrate or a inhibitor of P-gp, and it was the substrate and the inhibitor of OATP1B1 and OATP2B1. These results show that ABA was via passive transcellular transport and OATP1B1 and OATP2B1 transporter -mediated uptake through intestinal epithelia, Blood-brain barrier and hepatocytes.

Reference [1] Mona Abdel-Tawab, Oliver Werz, Manfred Schubert-Zsilavecz. Boswellia serrate: an ocerall assessment of in vitro, preclinical, pharmacokinetic and clinical data. Clin Pharmacokinet. 2011, 50(6):349–369.

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P416. CANCER-TYPE OATP1B3: FURTHER EVIDENCE ON ITS EXISTENCE AND EXPRESSION PROFILES IN HUMAN LUNG AND COLON CANCER TISSUES


Yuchen Sun1, Tomomi Furihata1, Seiya Ishii1, Shinichiro Motohashi2, Ichiro Yoshino3, Osamu Shimozato4, Takehiko Kamijo4, Miki Nagai1, Atsuko Kamiichi1 and Kan Chiba1 1 Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University., Chiba-Shi, Japan, 2Dapartment of Medical Immunology, Guraduate School of Medicine, Chiba, Japan, 3Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan, 4Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba, Japan Organic anion transporting polypeptide 1B3 (OATP1B3) is a transporter that is exclusively expressed in the human liver at the basolateral membrane of hepatocytes under normal conditions. However, it has been reported that OATP1B3 is also expressed in various solid cancer tissues, such as lung, colon, breast, and pancreas cancer. Recently, we first identified a new OATP1B3 mRNA isoform (hereafter referred to as cancer-type OATP1B3, Ct-OATP1B3), which showed a predominant expression profile over the liver-type OATP1B3 (Lt-OATP1B3) mRNA in several human colon and lung cancer tissues. However, because only a limited number of samples were examined in our previous study, additional evidence for existence of Ct-OATP1B3 as well as its expression profile in cancer tissues should be provided to establish Ct-OATP1B3 as a bona fide cancer-specific mRNA isoform. Therefore, in this study, we aimed to provide concrete evidence for predominant expression of Ct-OATP1B3 mRNA in a larger number of lung and colon cancer tissues along with their expression profiles. Thirty pairs of human lung cancer and adjacent normal tissues were collected from lung cancer patients who had undergone surgery at the Chiba University Hospital upon the approval from the institutional ethics committee. Ct-OATP1B3 and Lt-OATP1B3 mRNA levels were separately analyzed by real-time PCR. As a result, Ct-OATP1B3 mRNA expression was detected in all lung cancer tissues, and among them, ten cancer tissues were classified as the high Ct-OATP1B3 group because the median value of the Ct-OATP1B3 mRNA expression level of the group showed approximately 50-fold higher than that of the remaining cancer tissues. In the high Ct-OATP1B3 group, the median value of Ct-OATP1B3 mRNA expression levels was 32-fold higher than that of the matched normal tissues. Furthermore, it was found that Ct-OATP1B3 mRNA expression was strikingly predominant (approximately 15-fold difference) over Lt-OATP1B3 mRNA expression in the high Ct-OATP1B3 group. We also examined Ct-OATP1B3 mRNA expression profile in colon cancer tissues. The preliminary results of real-time PCR using seven commercially available colon cancer total RNA specimens showed that Ct-OATP1B3 mRNA was abundantly expressed, and that the expression levels were significantly higher than those of Lt-OATP1B3 mRNA in four colon cancer tissues. To summarize, we firmly demonstrate that Ct-OATP1B3, but not Lt-OATP1B3, is the primary OATP1B3 mRNA isoform expressed in lung and colon cancer tissues. Furthermore, our results identify that several samples show markedly higher Ct-OATP1B3 mRNA expression than others in human lung cancer (10/30) and in colon cancer tissues (4/7). Therefore, special attention must be paid to Ct-OATP1B3 mRNA expression in future studies on cancer-associated OATP1B3. We are currently examining Ct-OATP1B3 mRNA expression in a larger number of colon samples from the Chiba Cancer Center Research Institute, and the results will be compared with those obtained from the lung cancer.

P417. EXPRESSION OF ORGANIC ANION-TRANSPORTING POLYPEPTIDES (OATP) 2A1, 4A1, 5A1 AND 6A1 AS POSSIBLE BIOMARKERS FOR HUMAN OVARIAN CANCER Juliane Riha1, Stefan Brenner1, Felicitas Mungenast2, Sirag Sultan2, Johann Gruensteidel2, Dan Cacsire Castillo-Tong3, Robert Zeillinger3, Paul Speiser3, Reinhard Horvat4, Walter Jaeger1 and Theresia Thalhammer2 1 Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria, 2Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, 3Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria, 4Department of Clinical Pathology, Medical University of Vienna, Vienna, Austria Ovarian cancer is the deadliest of all gynecological tumors because it is usually diagnosed at an advanced stage. Additionally, after an initially successful chemotherapy, ovarian cancer cells develop drug resistance quite early. Therefore, new markers are necessary for diagnosis and improved treatment of ovarian carcinoma. A novel approach might be organic anion-transporting polypeptides (OATPs) as markers for targeted anticancer therapy. We therefore investigated the expression of OATP2A1, OATP4A1, OATP5A1 and OATP6A1 in human ovarian cancer specimens by using immunohistochemistry. Double immunofluorescence staining was done to identify the localization of individual OATPs in tumor, stroma and immune cells. We showed that OATP2A1, OATP4A1 und OATP5A1 were strongly represented in the majority of cancer samples, while OATP 6A1 was only marginally detectable. The prostaglandin transporter OATP2A1 was detectable in tumor cells, but its highest expression was in blood vessels in the tumor areas. OATP2A1 was not detectable in blood vessels of normal ovarian tissue. In all samples, OATP4A1 and OATP5A1 levels were significantly higher (p ¼ 0.002 and p ¼ 0.03) in the tumor areas compared to the tumor surrounding stroma. Overall, the expression of both OATPs was high in tumor cells, even in those which lost the epithelial marker for CK19 as part of the epithelial-mesenchymal transition. Both OATPs were also present in cells in the stroma and in immune cells (CD45-positive leukocytes, macrophages, B-cells). Surprisingly, OATP4A1 showed higher immunoreactive levels

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in the stroma of endometrial tumors than in serous tumors. Its expression was also significantly higher (p ¼ 0.008) in patients with a longer recurrence free interval than in those with an early recurrence of the tumor. However, OATP5A1 levels were higher in patients with a considerable amount (4500 ml) of ascites fluid in the peritoneal cavity. In conclusion, our data demonstrated a distinct expression pattern of OATP2A1, OATP4A1 and OATP5A1 in ovarian cancer samples. Whether these transporters might be considered as novel tumor biomarkers has to be studied in a larger collective of patients.

P418. SPECIFIC OATP EXPRESSION PATTERN IN PRIMARY SMALL CELL LUNG CANCER (SCLC) CELL LINES: IMPACT ON DIAGNOSIS AND THERAPY


Stefan Brenner1, Juliane Riha1, Madeleine Schoelm2, Veronika Buxhofer-Ausch3, Christoph Ausch3, Erika Bajna2, Angelika Reiner-Concin4, Gerhard Hamilton5, Lukas Klameth2, Walter Jaeger1 and Theresia Thalhammer2 1 Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria, 2Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, 3Cluster for Translational Oncology, Ludwig Boltzmann Society, Vienna, Austria, 4Department of Pathology, Donauspital, Vienna, Austria, 5Cluster for Translational Oncology, Medical University of Vienna, Vienna, Austria Small-cell lung cancer (SCLC) is a highly aggressive tumor with neuroendocrine features that develops resistance to chemotherapy quite early resulting in low 5 year survival rates. Hence the role of OATPs as transporters for chemotherapeutical drugs and endogenous transmitters may be considered to be crucial for tumor therapy. Therefore, using quantitative real time PCR (qPCR), we investigated mRNA-expression of OATP1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4C1, 4A1, 5A1 and 6A1 in 13 primary SCLC and carcinoid cell lines isolated from patients with different response to chemotherapy. Non tumorous lung tissue was used as control. OATP3A1 was highly present in the control, whereas its expression was reduced (5–30% of control values) in all cell lines except in etoposide-resistant DMS-456 cells. OATP4A1 and OATP4C1 were again highly expressed in the control, but only found at very low levels in all SCLC cell lines tested. OATP5A1 was increased (up to 30-fold) in 9/13 cancer cell lines. Interestingly, expression of OATP5A1 was strongly reduced in the resistant DMS-456 cell line. Similarly, the expression levels in chemoresistant GLC-16 and GLC-19 cells were significantly lower than in the GLC-14 cell line obtained from the same patient before chemotherapy. mRNA expression of other OATPs (OATP1A2, 1B1, 1B3, 1C1, 2A1, 2B1, and 6A1) was below or near the detection limit in the cell lines. Additionally, immunoreactive OATP3A1 and 5A1 were also investigated in paraffin-embedded tumor specimens and brain metastasis from SCLC patients by immunohistochemistry and doubleimmunofluorescence staining also showing a distinct expression pattern of both transporters. In conclusion these data demonstrate altered expression of OATP3A1, 4A1, 4C1, and 5A1 in SCLC cell lines at the mRNA level and of immunoreactive OATP4A1 and OATP5A1 protein in lung cancer tissue samples and brain metastasis. As expression of OATP5A1 was significantly reduced in SCLC chemotherapy-resistant cell lines, this transporter may act as a novel biomarker for neuroendocrine tumors.

P419. DIFFERENTIAL EXPRESSION OF OATPS, MRP1 AND BCRP TRANSPORTERS AS WELL AS STS AND HSD-1 ENZYMES IN HORMONE DEPENDENT AND HORMONE INDEPENDENT HUMAN BREAST CANCER TISSUES Nilasha Banerjee1, Naomi Miller2, Christine Allen1 and Reina Bendayan1 1 Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada, 2Pathology, University Health Network, Toronto, ON, Canada The objective of this research is to investigate the intra-tumoral fate of estrone-3-sulphate (E3S), a predominant source of postmenopausal tumour estrogen [1–3], in clinical breast tumour tissues. Prior in-vitro characterization demonstrated the role of Organic Anion Transporting Polypeptides (OATPs), a family of membrane associated transporters in E3S cellular uptake [4]. Increased functional expression (measured as a ratio of Vmax/Km) of OATPs in hormone dependent (HRþ) (MCF-7) as compared to hormone independent (HR) (MDA-MB-231, MDA-MB-468) breast cancer cells, suggested their potential as novel molecular targets for HRþ breast cancers. Furthermore, in vivo E3S biodistribution studies conducted in murine models of HRþ (MCF-7) and HR (MDA-MB-231) breast cancers demonstrated significant differences between the tumoral uptake in MCF-7 (13.9 3.1%ID/g) and MDA-MB-231 (10.4 1.1%ID/g) xenografts which in turn could be explained partially by the significantly higher OATP1A2 expression observed in MCF7 as compared to MDA-MB-231 tumour sections [5]. Herein, we compared the expression of: i) OATP transporters (i.e. OATP1A2, OATP2B1, OATP3A1, OATP4A1: isoforms that showed significantly higher gene and protein expression in HRþ breast cancer cells) known to facilitate E3S uptake [6], ii) Breast Cancer Resistance Proteins (BCRP) and Multidrug Resistance Proteins (MRP1), involved in E3S efflux [7,8], and iii) Sulphatase (STS) and 17b-Hydroxy steroid dehydrogenase-1(HSD-1) enzymes, known to be involved in E3S intra-tumoral conversion to estradiol [9,10]; among clinical samples of HRþ (n ¼ 20) and HR- breast tumours (n ¼ 20). We hypothesize that by characterizing the differential expression/function of these transporters and enzymes their clinical potential as novel molecular targets for HRþ tumours can be elucidated. Tumour sections (4 mm) were incubated with respective primary antibodies followed

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by AlexaFluor488 (fluorophore) or diaminobenzidine (DAB) (chromophore) labelled secondary antibodies. Fluorescent and brightfield images were acquired at 4x and 20x magnification, respectively. Marker densities were measured as the total area of positive signal divided by the surface area of the tumour section analyzed and was reported as % area (ImageJ software). Algorithms were developed for applying automated pattern recognition to quantify histologic tumor, stroma and non-tumor tissue areas in tumours (Inform software). The ratio of optical intensity per histologic area reported as %area/tumour, %area/ stroma and %area/non-tumour was also determined. Significantly higher OATP and STS total expression (p50.001) was observed in HRþ tumours. In contrast, significantly (p50.001) lower BCRP expression was observed in HRþ tumours. MRP1 total expression was found significantly higher (p50.001) in LNþ (lymph node) as compared to LN- sections in both HRþ/ HR tumours. All markers had significantly (p50.05) higher % area/tumour than % area/stroma or % area/non-tumour when compared among each category. The significantly higher total tissue and specific tumour cell expression of OATPs in HRþ tumours suggest their role in intra-tumoral estradiol production and their potential as novel molecular targets.


References 1. Geisler J (2003) Breast cancer tissue estrogens and their manipulation with aromatase inhibitors and inactivators. J Steroid Biochem Mol Biol 86: 245–253. S0960076003003649 [pii]. 2. Pasqualini JR, Chetrite G, Blacker C, Feinstein MC, Delalonde L, Talbi M, Maloche C (1996) Concentrations of estrone, estradiol, and estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients. J Clin Endocrinol Metab 81: 1460–1464. 3. Van Landeghem AA, Poortman J, Nabuurs M, Thijssen JH (1985) Endogenous concentration and subcellular distribution of estrogens in normal and malignant human breast tissue. Cancer Res 45: 2900–2906. 4. Banerjee N, Allen C, Bendayan R (2012) Differential role of organic anion-transporting polypeptides in estrone-3-sulphate uptake by breast epithelial cells and breast cancer cells. J Pharmacol Exp Ther 342: 510–519. jpet.112.192344 [pii];10.1124/ jpet.112.192344 [doi]. 5. Banerjee N, Fonge H, Mikhail A, Reilly RM, Bendayan R, Allen C (2013) Estrone-3-sulphate, a potential novel ligand for targeting breast cancers. PLoS One 8: e64069. 10.1371/journal.pone.0064069 [doi];PONE-D-12-36323 [pii]. 6. Hagenbuch B, Meier PJ (2003) The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 1609: 1–18. S0005273602006338 [pii]. 7. Imai Y, Asada S, Tsukahara S, Ishikawa E, Tsuruo T, Sugimoto Y (2003) Breast cancer resistance protein exports sulfated estrogens but not free estrogens. Mol Pharmacol 64: 610–618. 10.1124/mol.64.3.610 [doi];64/3/610 [pii]. 8. Conrad S, Kauffmann HM, Ito K, Leslie EM, Deeley RG, Schrenk D, Cole SP (2002) A naturally occurring mutation in MRP1 results in a selective decrease in organic anion transport and in increased doxorubicin resistance. Pharmacogenetics 12: 321–330. 9. Stanway SJ, Purohit A, Woo LW, Sufi S, Vigushin D, Ward R, Wilson RH, Stanczyk FZ, Dobbs N, Kulinskaya E, Elliott M, Potter BV, Reed MJ, Coombes RC (2006) Phase I study of STX 64 (667 Coumate) in breast cancer patients: the first study of a steroid sulfatase inhibitor. Clin Cancer Res 12: 1585–1592. 12/5/1585 [pii];10.1158/1078-0432.CCR-05-1996 [doi]. 10. Ng JH, Nesaretnam K, Reimann K, Lai LC (2000) Effect of retinoic acid and palm oil carotenoids on oestrone sulphatase and oestradiol-17beta hydroxysteroid dehydrogenase activities in MCF-7 and MDA-MB-231 breast cancer cell lines. Int J Cancer 88: 135-138. 10.1002/1097-0215(20001001)88:15135::AID-IJC2143.0.CO;2-S [pii].

P420. MRP4 (ABCC4) PROTECTS CELLS FROM ARSENIC THROUGH THE EXPORT OF DIMETHYLARSINIC ACID (DMAV) AND THE DIGLUTATHIONE CONJUGATE OF MONOMETHYLARSONOUS ACID (MMAIII) Michael W. Carew1, Mayukh Banerjee1, Xiufen Lu2, Hua Naranmandura3, X. Chris Le2 and Elaine M. Leslie1 1 Physiology, University of Alberta, Edmonton, AB, Canada, 2Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada, 3Institute of Pharmacology and Toxicology and Biochemical Pharmaceutics, Zhejiang University, Hangzhou, China Millions of people world-wide are exposed to the environmental carcinogen arsenic (as inorganic arsenite [AsIII] and arsenate [AsV]) in drinking water. Chronic exposure to arsenic is associated with increased incidences of skin, lung and bladder tumours. In humans, arsenic is extensively methylated to monomethylarsonous acid (MMAIII), monomethylarsonic acid (MMAV), dimethylarsinous acid (DMAIII) and dimethylarsinic acid (DMAV), mainly in the liver. Arsenic elimination from the body occurs primarily through urinary excretion. Chemical forms found in urine are inorganic (10–30%), monomethylated (10–20%) and dimethylated (60–80%). The multidrug resistance proteins (MRPs) are ATP-binding cassette transporter proteins critical for the cellular efflux of drugs, carcinogens and environmental toxicants. MRP1 (ABCC1) has been established to play an important role in the cellular efflux of inorganic arsenic as the triglutathione conjugate As(GS)3 and MMAIII as the diglutathione conjugate MMA(GS)2. In vivo studies of Mrp2(Abcc2)-deficient (TR) Wistar rats show Mrp2 is responsible for the biliary excretion of arsenic as As(GS)3 and MMA(GS)2. In vitro studies have shown that As(GS)3 and the seleno-bis(S-glutathionyl) arsinium ion


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[(GS)2AsSe] are substrates for human MRP2. Despite the important role MRP1 and MRP2 play in protecting cells from arsenicals, neither protein is localized to the basolateral surface of hepatocytes. Thus, the transport protein(s) responsible for arsenic efflux from the liver (into blood), ultimately for urinary elimination are unknown. MRP4 (ABCC4) is localized to the basolateral surface of hepatocytes and the apical surface of renal proximal tubule cells making it an ideal candidate for arsenic elimination. Determining the ability of MRP4 to transport arsenic species was the focus of the current study. Tetrazolium based cytotoxicity assays using HEK293 cells expressing empty vector (HEK-V) or MRP4 (HEK-MRP4) showed that MRP4 increased the EC50 values for AsV, MMAIII, MMAV, DMAIII, and DMAV by 2.8-, 1.7-, 1.6, 2.0-, and 1.6-fold, respectively. In contrast, MRP4 did not protect cells from AsIII. Protection against AsV and MMAIII was GSH-dependent while protection from MMAV, DMAIII, and DMAV was not. Consistent with the cytotoxicity data, HEK-MRP4 cells accumulated significantly less total arsenic after treatment with AsV, MMAV, MMAIII, DMAIII, and DMAV (but not AsIII) than HEK-V cells. Arsenic speciation analysis of HEK293 cells treated with AsV revealed that490% of arsenic was methylated, suggesting that MRP4 confers cellular protection through the export of methylated arsenic species. ATP-dependent transport of AsIII, AsV, MMAV, MMAIII, DMAIII, and DMAV (GSH) was measured using MRP4-enriched membrane vesicles and total arsenic quantified by inductively coupled plasma mass spectrometry. AsIII, AsV, MMAV and DMAIII were not MRP4 substrates. Transport of MMAIII (1 mM) was GSH dependent but not supported by the non-reducing GSH analog, ophthalmic acid, suggesting that MMA(GS)2 was the transported form. DMAV (1 mM) was transported by MRP4 and the addition of GSH (3 mM) had no effect. MMA(GS)2 and DMAVinhibited MRP4-dependent transport of other substrates. These results suggest that MRP4 could be critical for hepatic basolateral transport and ultimately urinary elimination of arsenic.

P421. RAT IN VIVO FETAL-TO-MATERNAL TRANSFER CLEARANCES OF VARIOUS XENOBIOTICS BY UMBILICAL PERFUSION Tomohiro Nishimura, Masatoshi Tomi and Emi Nakashima Faculty of Pharmacy, Keio University, Tokyo, Japan It is important to address the tissue permeability of drugs, particularly in tissues that have a barrier function, in terms of both lipophilicity and the contribution of transporters. Here, we employed umbilical perfusion in rats at gestational day 19 to evaluate in vivo fetal-to-maternal transfer clearances of various xenobiotics. We measured fetal-to-maternal clearance (CLfm) of 23 compounds, which have a broad range of lipophilicity. Drugs for which CLfm was more than 300 mL/min/placenta belonged exclusively to Biopharmaceutical Drug Disposition Classification System (BDDCS) class 1 (highly permeable) and those for which CLfm was less than 50 mL/min/placenta belonged exclusively to BDDCS class 3 (poorly permeable). For BDDCS class 1 and 2 drugs, CLfm values were consistent with lipophilicity and significant regression curve was obtained. CLfm of verapamil and quinidine was much higher than the regression curve, indicating that the involvement of efflux transporters such as P-glycoprotein(P-gp) in their fetal-to-placental permeabilities in addition to the lipophilicity. Glibenclamide is a substrate of OATP2B1 and BCRP that are expressed in the apical and basolateral membrane of syncytiotrophoblasts in human, respectively. Glibenclamide also showed high CLfm, implying the involvement of the interplay of Oatp2b1 and Bcrp1 in fetal-to-maternal transfer. Pravastatin showed a low CLfm below the regression line, comparable with that of inulin, a paracellular flux marker. Compared with pravastatin, rosuvastatin was efficiently eliminated from fetal circulation. These observations could be explained by higher affinity of rosuvastatin for OATP2B1 and BCRP than that of pravastatin. To study the possible involvement of efflux transporters, effect of inhibitors on the representative substrates of P-gp and BCRP was measured. CLfm of digoxin was saturable and was inhibited by verapamil, suggesting that P-gp-mediated efflux has a substantially effect on measured clearance. CLfm of mitoxantrone slightly increased at high concentration of mitoxantrone, but placental-to-maternal clearance of mitoxantrone was saturable, implying that Bcrp1 contributes to mitoxantrone efflux across the placenta. In conclusion, we showed fetal-to-maternal transfer clearances of various xenobiotics in rats. Classification by BDDCS can be useful to understand the placental permeability of drugs.

P422. SANDWICH CULTURED HEPATOCYTES: UTILITY IN DELINEATING THE HEPATIC DISPOSITION OF NINE DRUGS SELECTED FROM DIFFERENT BDDCS CLASSES Carina Cantrill and J. Brian Houston School of Pharmacy and Pharmaceutical Sciences, Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, United Kingdom Sandwich-cultured hepatocytes (SCH) constitute an in vitro liver model that allows the assessment of multiple kinetic processes simultaneously, namely uptake and biliary efflux transport functions. However, the reduction of many liver-specific functions in rat hepatocytes in culture, in particular cytochrome P450, restricts the use of this system for long term studies The purpose of this study was to assess the feasibility of using primary rat SCH to estimate both uptake and biliary clearances and to examine whether the reported loss of sinusoidal transporter expression results in a substantial change in the unbound hepatocyte-to-media partition coefficient (Kpu) and the contribution of passive permeability, when compared to other hepatocyte systems.


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Compounds showing active uptake of 80% of total CLuptake were selected for chemical diversity using the BDDCS classification. Since class 1 and 2 compounds exhibit high passive diffusion, active uptake is unlikely to be the rate determining process in clearance as these compounds easily penetrate the sinusoidal membrane. In contrast, for BDDCS class 3 or 4 compounds; hepatic clearance predominantly relies on the function of active uptake and efflux transport proteins. Uptake clearances were determined in RSCH for 9 compounds: selected class 3 compounds comprised rosuvastatin, valsartan, fexofenadine and pravastatin, class 2 compounds included repaglinide, telmisartan, atorvastatin and saquinavir and class 1 compound, quinidine (a passive compound) was examined and compared to published, in-house values using conventional monolayer and suspension hepatocyte systems [1, 2]. CLint,uptake ranged from 1.05–40.56 ml/min/106 cells in SCH and were significantly reduced compared to the other systems (up to 15- and 115-fold less than those observed in plated [1] and suspended hepatocytes [2], respectively). These results indicate a time-dependent loss in uptake transport activity, evident even for the class 2 compounds. Since differences in activity and/or expression levels of uptake transporters will influence intracellular drug concentration, and consequently hepatic clearance, CLint,biliary was estimated using intracellular drug concentrations. Kpu values determined in SCH ranged between 0.7–59 which were lower when compared to other hepatocyte systems (estimates ranged between 7.7–246 in plated [1] and 8.7–281 in suspended hepatocytes [2], respectively). Estimates of CLint,biliary were lower using intracellular concentration for all class 3 compounds, ranging between 0.09–1.53, compared to 0.32–10.8 mL/min/kg based on media concentrations. These predictions of CLint,biliary, scaled by hepatocellularity, were lower but consistent in rank order with observed in vivo data with the exception of pravastatin. In conclusion, these studies demonstrate reduced drug uptake in RSCH which limits drug access to canalicular efflux transport proteins and highlights the importance of elucidating the interplay between these transport proteins for accurate prediction of hepatic clearance for compounds of chemical diversity.

References 1. Menochet, K., et al., Simultaneous assessment of uptake and metabolism in rat hepatocytes: a comprehensive mechanistic model. J Pharmacol Exp Ther, 2012. 341(1): p. 2–15. 2. Yabe, Y., A. Galetin, and J.B. Houston, Kinetic characterization of rat hepatic uptake of 16 actively transported drugs. Drug Metab Dispos, 2011. 39(10): p. 1808–14.

P423. FUNCTIONAL ASSESSMENT OF OATP1B1 AND BCRP POLYMORPHISMS IN AN OATP1B1/ BCRP CO-EXPRESSING MODEL Mark Warren, Mirza Jahic, Xuexiang Zhang, Ilene Kaufman, Jane Huang and Yong Huang Optivia Biotechnology Inc., Menlo Park, CA Summary: An in vitro model concomitantly expressing two major hepatic transporters OATP1B1 and BCRP, or their genetic variants, in polarized MDCK cells has been developed and characterized. Vectorial transport of common substrates of OATP1B1 and BCRP was demonstrated. Effects of BCRP_Q141K variant on vectorial transport and intracellular accumulation of transporter substrates, including endogenous compounds and drugs, were demonstrated. Abstract: OATP1B1 and BCRP, two major hepatic transporters that are expressed on basolateral and apical membranes of hepatocytes respectively, mediate vectorial transport/hepatic clearance of various organic anion compounds. Genetic variants of the two transporters have been linked to altered pharmacokinetic profiles of various drugs, including statins. Although polarized cell lines co-expressing OATP1B1 and BCRP have been developed for evaluating vectorial transport of their common substrates, there is lack of ability in evaluating genetic variants of these transporters in a co-expressing context as each variant requires the creation of a cell line and there is considerable difference in transporter expression levels among different clones. We have demonstrated in our previous studies that polarized cell monolayers, such as MDCK cells, can be in situ transfected, effectively and consistently, using a novel Opti-Expression technology. Several multiple-transporter expressing models have been developed using this method. Moreover, not only consistent expression levels of each transporters have been achieved in different experiments, the relative expression levels of individual transporters can be quantitatively fine tuned to certain extent. Using this method, we have developed and characterized an OATP1B1/BCRP co-expressing MDCK-II model. Linear and dose-dependent vectorial transport of the common substrates of the two transporters, such as Estradial-17-b-Glucuronide (E17bG) and Estrone-3-Sulfate (E3S), has been demonstrates. Contributions of OATP1B1 and BCRP to the overall transport of the substrates were assessed in cells expressing one transporter alone. Furthermore, a high prevalence BCRP variant, C421A/ Q141K, was expressed along with OATP1B1. Compare to OATP1B1/BCRP-wt, OATP1B1/BCRP-Q141K expressing cells exhibited substantially reduced B4A flux of E17bG, but had increased E17bG intracellular accumulation. The data demonstrated that the BCRP variant had reduced activity compared to BCRP wild type. Similar trends were also observed in atorvastatin B4A transport and intracellular accumulation, which substantiated clinical observations on reduced atorvastatin clearance in subjects that have BCRP_Q141K SNP. Our studies demonstrated that consistent and robust assays can be achieved by transiently expressing OATP1B1/BCRP or their genetic variants in polarized MDCK-II cells. The models can be useful to evaluate the synergistic effect of OATP1B1 and

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BCRP on vectorial transport of their common substrates and to study the functional effects of OATP1B1 and BCRP polymorphisms in a double transporter context.

P424. SPECIES DIFFERENCES OF CANALICULAR AND SINUSOIDAL EFFLUX OF MYCOPHENOLIC ACID O-GLUCURONIDE IN SANDWICH-CULTURED HEPATOCYTES


Kazuhiro Tetsuka1, Nicolas Gerst1, Kenji Tabata2, Toshio Teramura2, Jeffrey N. Masters1 and Kouichi Tamura1 1 Astellas Research Institute of America LLC, Skokie, IL, 2Astellas Pharma Inc., Tsukuba-shi, Japan Purpose: Mycophenolic acid (MPA) is an immunosuppressive agent to prevent acute rejection in organ transplantation. MPA shows enterohepatic circulation in both rat and human resulting from biliary excretion of O-glucuronide of MPA (MPAG) followed by deconjugation to MPA in the gastrointestinal tract and absorption into the blood. However, MPAG is reported to exhibit higher urinary recovery in human compared to rat. This study aimed to elucidate the vectorial transport of MPAG in rat and human sandwich-cultured hepatocytes (SCH). Methods: The unique characteristics of tight junctions were utilized in this study, where calcium-containing buffer maintains tight junctions while calcium-free buffer disrupts the tight junctions. After incubation of substrates onto SCH, sinusoidal efflux is modeled by the extracellular concentration of the compounds of interest in the presence of calcium, while net efflux (sinusoidal plus canalicular) is modeled by their concentration in the absence of calcium. In detail, SCH from rats and humans were incubated with MPA at 37 C for 20 min followed by two washes. Fresh buffer in the presence or absence of calcium was added to the SCH for predetermined times up to 20 min, then the culture supernatant was harvested for LC-MS/MS measurement of the extracellular concentrations of MPAG as well as the concentrations of the acyl glucuronide of MPA (AcMPAG). In addition, intracellular MPAG and AcMPAG concentrations were determined at time zero culture. Results and discussion: Intracellular MPAG levels were over 100-fold higher than intracellular AcMPAG levels in rat and human SCH. Increase of extracellular MPAG concentration in the presence of calcium was observed in both rat and human SCH, suggesting sinusoidal efflux. The extracellular net MPAG concentration in the absence of calcium was significantly higher than that in the presence of calcium in rat and human SCH, suggesting that canalicular efflux is also an event in both species. However, the percentage of sinusoidal efflux was significantly higher in human SCH (78-81%) than in rat SCH (45–55%; p50.05, n ¼ 3). These results suggest that MPAG clearance in human hepatocytes is dominated by sinusoidal efflux over canalicular efflux. Conclusion: Different directionality of MPAG efflux was observed between human and rat SCH. Sinusoidal-oriented MPAG efflux in human is a unique characteristic of hepatic MPA disposition and can be one of the reasons for different urinary recovery of MPAG between human and rat.

P425. FUNCTION OF OAT4/SLC22A11 AS A 16A -HYDROXY DEHYDROEPIANDROSTERONE SULFATE TRANSPORTER AND ITS ASSOCIATION WITH PLACENTAL ESTRIOL SYNTHESIS Tomohiro Tawara, Hiromi Eguchi, Mayuko Ozaki, Sachika Nishimura, Tomohiro Nishimura, Masatoshi Tomi and Emi Nakashima Faculty of Pharmacy, Keio University, Minato-ku, Tokyo, Japan Estriol is a pregnancy hormone that is only found in large amounts during pregnancy. The estriol synthesis is occurred in placental syncytiotrophoblasts constituting the placental barrier from 16a-hydroxy dehydroepiandrosterone sulfate (16a-OH DHEAS) which is mostly produced by CYP3A7 (16a-hydroxylase) in fetal liver. In order to attain estriol synthesis, syncytiotrophoblasts are required to be equipped with 16a-OH DHEAS uptake transporter(s) at the basolateral membrane facing fetal circulation. It is known that organic anion transporter (OAT) 4 (SLC22A11), organic anion transporting polypeptide (OATP) 2B1 (SLCO2B1), and organic solute carrier protein (OSCP) 1 are localized at the basolateral membrane of the placental syncytiotrophoblasts. However, the involvement of these transporters in 16a-OH DHEAS uptake is not clear. The purpose of this study is to clarify transport mechanism(s) of 16a-OH DHEAS at the placental syncytiotrophoblasts. Forskolin-induced differentiated JEG-3 human choriocarcinoma cells were used as an in vitro model of the placental syncytiotrophoblasts. OAT4 and OSCP1 mRNAs were expressed in the differentiated JEG-3 cells, while OATP2B1 mRNA was not. The uptake of [3H]16aOH DHEAS by the differentiated JEG-3 cells was significantly inhibited by 1 mM DHEAS, estrone sulfate (E1S), bromosulfophthalein (BSP), and probenecid, but not by OSCP1 substrates, tetraethylammonium (TEA) and p-aminohippuric acid (PAH), suggesting the involvement of OAT4 in the uptake of 16a-OH DHEAS. The uptake of [3H]16a-OH DHEAS by human OAT4-expressing COS-7 cells was significantly higher than that by mock cells. The OAT4-mediated uptake of [3H]16aOH DHEAS exhibited concentration-dependence with a Km of 7.4 mM, and was significantly inhibited by DHEAS, E1S, BSP, and probenecid but not by TEA. This inhibitory characteristics of [3H]16a-OH DHEAS uptake by OAT4-expressing COS-7 cells was consistent with that by the differentiated JEG-3 cells. On the other hand, the uptake of [3H]16a-OH DHEAS by human OATP2B1-expressing COS-7 cells was not observed. The secretion of estriol from the differentiated JEG-3 cells was detected by incubating with 16a-OH DHEAS for 12 hours and was inhibited by co-incubating with 50 mM BSP, indicating that the inhibition of [3H]16a-OH DHEAS uptake decreased estriol synthesis. In conclusion, OAT4 appears to play a predominant role in the

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uptake of 16a-OH DHEAS into the placental syncytiotrophoblasts from fetus and contribute to the estriol synthesis during pregnancy.

P426. MITOXANTRONE EXPOSURE INDUCED EXPRESSION OF ABCG2 THROUGH ESTROGEN RECEPTOR A AT THE PLACENTAL BARRIER


Masatoshi Tomi, Kenji Oda, Tomohiro Nishimura and Emi Nakashima Faculty of Pharmacy, Keio University, Tokyo, Japan ABCG2 (breast cancer resistance protein: BCRP) is localized in the apical membrane of placental syncytiotrophoblasts, and plays a role in forming the placental barrier. Substrate-induced upregulation of ABCG2 has been well studied in cancer cells, but it is also important to understand whether ABCG2 is upregulated by its substrates in tissues in which it is constitutively expressed. This is of interest, because it could influence the fetal disposition of ABCG2 substrate drugs. The purpose of the present study is to clarify the regulatory mechanism of Abcg2 expression by its substrate, mitoxantrone at the placental barrier. Rat placental TR-TBT 18d-1 cells were used as an in vitro model of the placental barrier, which constitutively expresses Abcg2. Abcg2 mRNA expression in TR-TBT 18d-1 cells treated with mitoxantrone at more than 1 mM for 24 hrs was increased, compared with that in non-treated cells, while 10 mM pheophorbide-a had no effect. Methylated CpG level in the promoter region of the Abcg2 gene was less than 10% and was not altered by mitoxantrone. The presence of unmethylated CpG in the Abcg2 promoter region of TR-TBT 18d-1 cells is in good agreement with the constitutive expression of Abcg2. Of nuclear receptors such as estrogen receptor (ER) a and b, progesterone receptor (PR) B, aryl hydrocarbon receptor (AhR), and hypoxia inducible factor 1 (HIF-1), mitoxantrone markedly increased the expression of ERa and PRB. Fulvestrant, an ER antagonist, attenuated the mitoxantrone-induced increase of Abcg2 mRNA expression, while mifepristone, a PR antagonist, had little effect. The protein expression of Abcg2 and ERa was also increased by 10 mM mitoxantrone treatment, but this increased expression of Abcg2 protein was markedly attenuated in the presence of fulvestrant. 17b-Estradiol, an ER ligand, positively regulated the mitoxantrone-induced increase of Abcg2 expression. These results suggest that mitoxantrone-mediated Abcg2 transcription is mediated predominantly by increased expression of ERa. DNA demethylation by 5-aza-2-deoxycytidine treatment increased ERa expression, but mitoxantrone failed to facilitate the demethylation of ERa promoter in TR-TBT 18d-1 cells. In conclusion, ERa induced by mitoxantrone exposure appears to play a key role in increasing Abcg2 expression in rat placental TR-TBT 18d-1 cells. The findings in the present study provide valuable information for developing optimum chemotherapeutic strategies, especially for pregnant women.

P427. TRANSPLACENTAL PERMEABILITY OF NUCLEOSIDES AND NUCLEOSIDE-ANALOGUE DRUGS EVALUTED BY RAT FETUS UPTAKE INDEX METHOD Yuichiro Sano, Tomohiro Nishimura, Masatoshi Tomi and Emi Nakashima Faculty of Pharmacy, Keio University, Tokyo, Japan A quantitative understanding of carrier-mediated transplacental transport is important for predicting efficacy and toxicity of drugs in feto-placental unit. Although equilibrative nucleoside transporter (ENT)-mediated transport processes have been known to be present at the placental barrier, it is not fully evaluated the transplacental permeability of ENT substrates such as nucleosides and nucleoside-analogue drugs. The purpose of this study was to evaluate rat transplacental permeability of nucleosides and nucleoside-analogue drugs. We measured the permeability by fetus uptake index (FUI) method, which has an advantage that the transfer is less affected by plasma components. Radiolabeled test compound and antipyrine as a reference compound were rapidly co-injected into the abdominal aorta in pregnant Sprague-Dawley rats at the gestational day of 19. At 10 seconds after administration, fetuses were collected for measuring the radioactivity. FUI value (%) was determined by dividing the ratio of the test compound and antipyrine in fetuses by that in the injectate. FUI values of [3H]thymidine(67%) and [3H]uridine (67%) were much higher than those of the paracellular transfer markers, [14C]D-mannitol (15%) and [14C]inulincarboxyl (3.3%), supporting the a previous report that ENT is involved in thetransplacental transfer of these nucleosides. FUI value of [3H]adenosine (28%) was significantly lower than that of [3H]uridine. This is probably because adenosine is metabolized to less permeable form(s) in the placental barrier more quickly than uridine. FUI values of [3H]zidovudine (AZT) (58%), [3H]didanosine (ddI) (37%) and [3H] zalcitabine (ddC) (34%) were also much higher than those of the paracellular transfer markers. The observed maternofetal permeability of [3H]AZT in rat is higher than that of [3H]ddI and [3H]ddC, which is consistent with the reported human maternofetal permeability of these drugs in the perfused term human placenta. We have previously observed that the uptake mechanism of AZT by TR-TBT 18d-1 cells, an in vitro model of the rat placental barrier, was distinct from the ENT-mediated process, while ddI and ddC were taken up into the cells mainly by ENT2. Therefore, the difference in transplacental permeability between AZT and ddI/ddC may be explained by the difference in transport processes. In conclusion, the in vivo transplacental permeability of nucleosides and nucleoside-analogue drugs has been successfully evaluated by using FUI method, and the permeability data suggested that the transporters including ENT play a predominant role in the transplacental transfer of these compounds.

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P428. SYSTEM-DEPENDENT INHIBITION OF BCRP TRANSPORT BY HIGH- AND LOW-PERMEABLE COMPOUNDS IN BI-DIRECTIONAL MDCKII CELLS AND MEMBRANE VESICLES


Joel Widmer, Garrett Tibbets, Caleb Isringhausen, Alison Coons, Kevin C. Lyon, Andrea Wolff, Amanda Hays, Greg Loewen and David B. Buckley XenoTech, LLC, Lenexa, KS The EMEA and FDA guidelines recommend investigation of the inhibitory potential of drugs on transporters involved in clinically relevant DDIs, including the efflux transporter breast-cancer resistant protein (BCRP). Compounds with high or low permeability could potentially exhibit system-dependent inhibition when different BCRP expression systems are employed for in vitro experiments. In this study, we evaluated the system-dependent effects of both a high- and low-permeable compound on BCRP-mediated efflux, namely Ko143 and sulfasalazine. Ko143 is a potent and specific inhibitor of BCRP with moderate ˚ 2). Sulfasalazine, an anti-inflammatory agent, permeability (predicted logD7.442 and total polar surface area (TPSA) of 98 A was reported to cause potent inhibition of BCRP transport (IC50 1 mM) in membrane vesicles. However, sulfasalazine, with a ˚ 2, is poorly absorbed with limited permeability across physiological membranes. We predicted logD7.4 0 and TPSA of 4140 A hypothesize that, due to limited membrane permeability, sulfasalazine would cause significantly less inhibition of BCRP transport in cell-based assays. Inhibition of BCRP transport was evaluated in two test systems 1) bidirectional transport in BCRP-expressing MDCKII cells and 2) BCRP-expressing membrane vesicles. The bi-directional permeability of prazosin in BCRP-expressing MDCKII cells in 24-well trans-well plates was determined in the presence of each inhibitor added to both the apical and basolateral chambers. Prazosin was analyzed by LC-MS/MS. Similarly, membrane vesicles were incubated with radio-labeled estrone-3-sulfate in the presence or absence of each inhibitor for one minute and samples were analyzed by liquid scintillation counting. The high permeable compound, Ko143, inhibited BCRP-mediated efflux in BCRP transfected MDCKII cells and membrane vesicles, with IC50 values of 12 nM and 7 nM, respectively. However, the low permeable compound, sulfasalazine, did not inhibit BCRP-mediated prazosin efflux in BCRP-transfected MDCKII cells (up to 1 mM), whereas, it caused potent inhibition of BCRP in membrane vesicles (IC50 1 mM). Thus, sulfasalazine was identified as a systemdependent inhibitor of BCRP. In conclusion, compounds with high or low permeability may exhibit system-dependent transporter inhibition with common in vitro test systems. Therefore, it is imperative to consider the expression system and the physiochemical properties of the compound when evaluating the inhibition of efflux transporters.

P429. STUDY OF TRANSPORT OF ANTIRETROVIRAL DRUGS ZIDOVUDINE AND LAMIVUDINE ACROSS THE RAT TERM PLACENTA Lukas Cerveny, Zuzana Neumanova and Frantisek Staud Department of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic Mother-to-child-transmission (MTCT) of HIV represents the main source of HIV infection occurring in 20–45% of children born to infected mothers. Currently available antiretroviral regimens contribute to reduction of HIV transmission rate to 1–2%. Combination therapy based on administration of two nucleoside reverse transcriptase inhibitors (NRTIs), zidovudine (AZT) and lamivudine (3TC) is, nowadays, the most commonly prescribed therapy in HIV-positive pregnant women. As both drugs are listed in pregnancy category C by FDA, knowledge on drug transplacental kinetics and interactions with ATP-binding cassette (ABC) transporters is inevitable to guarantee adequate and safe medication for the fetus throughout gestation. ABC drug efflux transporters such as P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are known to affect transplacental kinetics since they pump their substrates back to mother’s circulation, thus limiting permeation of a drug across the placenta. The aim of this study was to describe the transplacental transport of AZT and 3TC employing the method of dually perfused rat term placenta. Using open-circuit perfusion system, transport of AZT or 3TC was investigated in fetal-to-maternal (fm) and maternalto-fetal (mf) directions. At a low, non-saturating concentration (9 nM) of AZT and 3TC no asymmetry in mf and fm transplacental clearances was observed, indicating ABC transporters do not affect penetration of AZT and 3TC across the placenta. On the other hand, employing closed-circuit perfusion system we observed that AZT (9 nM) is transported against the concentration gradient from the fetus to mother’s blood, proposing active transport of AZT across the placenta. Saturability of the transport mechanisms was subsequently confirmed by application of AZT at a concentration of 500 mM that resulted in linearity in transplacental permeation. Moreover, application of a dual inhibitor of both ABCB1 and ABCG2 (GF120908) led to significant suppression of AZT active transport from fetal circulation to mother. In the case of 3TC, capacity-limited active transport mechanism was not observed. In summary, our findings suggest that AZT but not 3TC transplacental pharmacokinetics is affected by activity of Abcb1 and/or Abcg2. This should be taken into account when thinking of co-medication of pregnant women on AZT based therapy. Concomitantly administrated drugs that are substrate/inhibitor of ABCB1 and/or ABCG2 such as abacavir, NRTI used for prevention of MTCT in combination with AZT and 3TC, can lead to unpredictable drug concentrations in the fetus. The study was supported by Czech Science Foundation (GACR 170/53/25303).

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P430. ABSENCE OF P-GLYCOPROTEIN TRANSPORT IN THE PHARMACOKINETICS AND TOXICITY OF THE HERBICIDE PARAQUAT


Sarah E. Lacher1, Julia Gremaud2, Kasse Skagen1, Emily Eickholt1, Rachel Dalton1, Kent Sugden2, Catherine M.T. Sherwin3, Fernando Cardozo Pelaez1 and Erica L. Woodahl1 1 Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT, 2Chemistry, University of Montana, Missoula, MT, 3Department of Pediatrics, University of Utah, Salt Lake, UT P-glycoprotein (P-gp) is an efflux transporter encoded by the ABCB1 (or MDR1) gene. ABCB1 genetic variation has been associated with Parkinson’s disease, which may be due to altered neurotoxicant disposition. Our goal was to investigate the role of P-gp transport in the disposition of the herbicide paraquat, a Parkinson’s-associated neurotoxicant. We utilized in vitro transporter methods of ATPase, cytotoxicity, transepithelial permeability, and rhodamine-123 inhibition. We also measured paraquat pharmacokinetics and brain distribution in FVB wild-type and P-gp-deficient (mdr1a(/)/mdr1b(/)) mice following 10, 25, 50, and 100 mg/kg oral doses. In vitro data showed that: 1) paraquat failed to stimulate ATPase activity; 2) paraquatinduced cytotoxicity was unchanged in P-gp-expressing cells in the absence or presence of P-gp inhibitors verapamil and GF120918 (37.0 1.14, 46.2 3.57, and 34.0 0.87 mM, respectively); 3) transepithelial transport of paraquat in P-gp-expressing cells was not altered by GF120918 (efflux ratios of 1.55 0.39 and 1.58 0.36, respectively); and 4) paraquat did not inhibit rhodamine-123 transport. We observed no difference in pharmacokinetic parameters between FVB wild-type and mdr1a(/)/mdr1b(/) mice: clearances of 0.78 (95% CI: 0.58–0.98) and 0.47 L/hr (0.42–0.52), respectively; and volume of distributions of 3.36 (2.39–4.33) and 1.77 L (1.50–2.04), respectively. Additionally, paraquat brain accumulation was the same across all doses. Together these studies indicate that paraquat is neither a substrate nor an inhibitor of P-gp. Therefore, the association between ABCB1genetic variation and risk of Parkinson’s disease is not due to alterations in efflux of paraquat.

P431. COMPARISON OF KI AND IC50 VALUES FOR PROTOTYPICAL AND CLINICALLY-RELEVANT PROBE SUBSTRATES OF THE HEPATIC TRANSPORTERS OATP1B1 AND OATP1B3 Garrett Tibbets, Caleb Isringhausen, Joel Widmer, Alison Coons, Kevin C. Lyon, Andrea Wolff, Amanda Hays, Greg Loewen and David B. Buckley XenoTech, LLC, Lenexa, KS The recent EMEA Guideline on the Investigation of Drug Interactions (2012) recommends the calculation of Ki values when evaluating the inhibition of a transporter by investigational drugs. Although Ki values have been reported for inhibitors of various uptake transporters, the difficulty of experimental design coupled with vigorous mathematical extrapolation can make the determination of intrinsic Ki values difficult and oftentimes reverts to the determination of extrinsic IC50 values. Additionally, it is important to consider clinically relevant substrates rather than prototypical probe substrates when evaluating potential DDIs of investigational drugs at the transporter level. In this study, Ki and IC50values were determined for one prototypical (estradiol-17b-glucoronide (E2G)) and one clinically-relevant (pravastatin) substrate of the hepatic uptake transporters OATP1B1 and OATP1B3 with an experimental design incorporating multiple substrate and inhibitor concentrations. In brief, HEK293 cells over-expressing OATP1B1 or OATP1B3 were cultured in 24-well plates. Cells were pre-incubated with four concentrations of the inhibitor rifampin (bracketing the IC50 value) for 15 min followed by incubation with multiple concentrations of substrate (six concentrations up to 3-fold Km) and inhibitor. Uptake was terminated by washing the cells three times with cold PBS. For analysis by scintillation counting, cells were lysed with 0.1 M NaOH. For analysis by LC-MS/MS, cells were extracted with 50:50 v/v methanol:water. Ki values were determined with GraFit software. Inhibition of OATP1B1mediated uptake by rifampin with E2G as a probe substrate yielded comparable Ki and IC50 values (1.1 and 0.8 mM, respectively). Similarly, inhibition of OATP1B3-mediated uptake of E2G by rifampin yielded nearly identical IC50 and Ki values (0.19 mM). Likewise, inhibition of OATP1B1- and OATP1B3-mediated uptake of the clinically-relevant probe substrate pravastatin by rifampin also yielded comparable IC50 and Ki values. Substrate concentrations used in IC50 experiments were much lower than Km and, therefore, the IC50 values were expected to be similar to the Ki value. Overall, Ki values were similar to IC50 values (within two-fold) for inhibition of OATP1B1 and OATP1B3. However, substrate-dependent inhibition was observed in OATP1B1 cells but not in OATP1B3 cells; rifampin inhibited OATP1B1-mediated pravastatin uptake with approximately five-fold more potency than observed with E2G. Although Ki determination experiments are time-consuming and expensive compared to IC50 determinations, intrinsic Ki values may be more accurate for DDI predictions and, in some cases, should be determined with a clinically relevant substrate.

P432. IN VITRO TRANSPORTER ASSESSMENT OF GDC-0068, AN ATP-COMPETITIVE AKT INHIBITOR IN PHASE 1B/II CLINICAL TRIALS Emile G. Plise, Yuzhong Deng, Laurent Salphati and Bianca M. Liederer Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA


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Akt functions as a pivotal node in the PI3K–Akt–mTOR pathway and constitutive activation or overexpression of Akt isoforms have been identified in a wide variety of cancers1,2. GDC-0068 is a potent small molecule pan-Akt inhibitor that is currently in the clinic for the treatment of a broad range of tumors as a single agent and in combination. The objective of these in vitro studies was to assess GDC-0068 as a potential substrate or inhibitor of select transporters at clinically observed total plasma concentrations. The experimental systems employed included flat-bottom 24-well plates seeded with CHO cells transiently transfected with OATP2B1 and transwell plates (24-well, 1 mm PET membrane) with confluent monolayers of stably transfected MDCKII-MDR1 or MDCKII monolayers transiently transfected with the desired uptake transporter. The potential of GDC-0068 to inhibit the transport of radiolabeled prototypical probe substrates was tested in triplicate over a concentration range of 0.0123– 9.0 mM for OATP1B1 ([3H]-estradiol-17b-D-glucuronide), OATP1B3 ([3H]-CCK-8), OATP2B1([3H]-estrone-3-sulfate), OAT1 ([3H]-p-aminohippurate), OAT3 ([3H]-estrone-3-sulfate), OCT1 ([14C]-metformin), and OCT2 ([14C]-metformin). MDR1 inhibition was tested at 0.0123–729 mM GDC-0068 using [3H]-digoxin as the probe substrate. GDC-0068 was tested in triplicate as a substrate of MDR1, OATP1B1, OATP1B3, OATP2B1, OAT1 and OAT3 at the observed Phase I (600 mg/QD) clinical Cmax (1.8 mM3) and one-fifth the Cmax (0.36 mM). GDC-0068 was quantitated by LC/MS/MS for all substrate assays and the probe substrate levels for the inhibition assays were determined by radiometric detection. GDC-0068 appears to be a substrate of MDR1 with B-A/A-B ratios 42.1 for both concentrations tested. Upon the addition of the MDR1 inhibitor verapamil (100 mM) the B-A/A-B ratios were markedly reduced to 52, further demonstrating the involvement of MDR1. GDC-0068 does not appear to be transported by OATP1B1, OATP1B3, OATP2B1, OAT1 and OAT3 as the difference in intracellular accumulation between transfected cells, transfected cells treated with reference inhibitor, and mock transfected cells was not statistically different (p value40.05). At the concentrations tested, GDC-0068 did not inhibit probe substrate flux in MDR1, OATP1B1, OATP1B3, OATP2B1, OAT1, OAT3, OCT1 and OCT2 transfected cell by more than 50%, thus IC50 values could not be calculated. The results of the in vitro transporter studies suggest GDC-0068 is not a substrate or inhibitor of various clinically relevant uptake transporters. However, it is a likely substrate, but not an inhibitor of MDR1 at concentrations observed in the clinic.

References 1. Cheng, J. Q., et al., Oncogene 24 (2005) 7482–7492. 2. Altomare, D. A., et al., Oncogene 24 (2005) 7455–7464. 3. Takahashi, R., et al., ISSX Annual Meeting (2012) Poster 73.

P433. LEVERAGING HIGH-THROUGHPUT BSEP INHIBITION ASSAYS TO DRIVE DECISION-MAKING AND ENHANCE UNDERSTANDING OF THE CHOLESTATIC POTENTIAL OF NEW CHEMICAL ENTITIES Kathleen W. Mosure1, Lisa L. Elkin2, Jeremy Hurley2, Mian Gao3, Dianlin Xie3 and Matthew G. Soars1 1 Metabolism and Pharmacokinetics, Bristol-Myers Squibb, Wallingford, CT, 2Lead Profiling, Bristol-Myers Squibb, Wallingford, CT, 3Protein Science and Structure, Bristol-Myers Squibb, Princeton, NJ Cholestasis is one of the primary causes of drug induced liver injury and has been shown to be a major factor in the withdrawal from the market of a number of drugs1. In vivo studies in pre-clinical species are often used to assess the potential toxicology of new chemical entities (NCEs), however animal models have been shown to be poor predictors of cholestasis in humans. Recent studies have highlighted a major role of the bile salt export pump (BSEP) in the transport of bile from the liver and therefore the inhibition of this transporter by NCEs has been implicated as a potential indicator of cholestasis. To this end, several groups have developed high-throughput screens assessing the ability of NCEs to inhibit human BSEP2,3. However, it has become clear that the sole use of these assays in their current format will not be sufficient to derisk the potential for cholestasis. The aims of this work were two-fold: (1) To determine if the difference in cholestatic potential observed between the pre-clinical species and humans is due to differences in the inhibition of BSEP and (2) to determine if the inhibiton of taurocholate uptake into vesicles expressing BSEP is representative of the inhibition observed with bile acids that are more physiologically relevant to humans. A fully automated 384-well inhibition assay was developed to assess the effects of NCEs on the uptake of [3H]-taurocholate into vesicles expressing either rat or human BSEP. Our results demonstrate this assay is robust (Z’ of40.5 and a signal to bacground of 410 fold) and reproducible in a high-throughput, automated setting. The potential of over 50 compounds to inhibit taurocholate uptake into vesicles expressing either rat or human BSEP was investigated. Several compounds inhibited both rat and human BSEP to similar extents (cyclosporine A rat IC50 0.34 uM, human IC50 0.5 uM; tacrolimus rat IC50 4.6 uM, human IC50 2.4 uM). However, the majority of compounds inhibited human BSEP more potently than rat BSEP (ketoconazole rat IC50 18 uM, human IC50 3 uM). These data suggest that for many compounds the reduced BSEP potency (rat compared to human) may contribute to the reduced prevalence of cholestasis observed in rat toxicology studies. A similar analysis using dog BSEP vesicles is ongoing. In addition, high-throughput inhibition assays have been developed to investigate the uptake of unlabelled bile acids (glycocholate and glycochenodeoxycholate) into vesicles expressing human BSEP. An analysis of the inhibition profile of over 50 compounds in these assays in comparison to data obtained using taurocholate will be presented. In summary, the investigation into inter-species and inter-bile acid differences in BSEP inhibition using a significant number of compounds will

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allow an informed decision on the appropriate conditions to use to translate in vitro BSEP data. These data, in conjunction with physiologically relevant parameters such as unbound liver levels, will aid our predictions of cholestatic risk in early drug discovery.

References 1. Abboud and Kaplowitz (2007) Drug Induced liver injury. Drug Saf 30:277–294 2. Morgan RE, Trauner M, Van Staden CJ, Lee PH, Ramachandran B, Eschenberg M, Afshari CA, Qualls CW Jr, LightfootDunn R and Hamadeh HK (2010) Interference with bile salt export pump function is a susceptability factor for human liver injury in drug development. Toxicol Sci 118: 485–500. 3. Dawson S, Stahl S, Paul N, Barber J and Kenna JG (2012) In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab Dispos 40: 130–138.


P434. CHARACTERIZATION OF A NOVEL TRANSPORTER MODEL FOR STUDYING DRUG INTERACTION WITH ORGANIC ANION-TRANSPORTING POLYPEPTIDE (OATP) Na Li, Jie Wang, Kirsten Cooper, Joanne Bourgea, Charles L. Crespi and Christopher J. Patten Corning Life Sciences, Bedford, MA Organic anion-transporting polypeptide (OATPs) expressed in liver play a vital role in drug disposition, metabolism and excretion. OATP1B1 and OATP1B3 have been implicated in a significant number of clinical drug-drug interactions (DDI); both are recommended to study by FDA’s 2012 Draft Guidance on Drug Interactions and EMA 2012 final drug interaction guidelines. OATP1B1 and 1B3, localized on the sinusoidal membrane of hepatocytes, are major players of hepatic uptake. Understanding of hepatic uptake clearance and drug-drug interaction (DDI) involving heptic uptake transporter is a key component of investigating drug pharmacokinetics profiles. Appropriate in vitro characterization of drug interaction with hepatic uptake transporter facilitates better predication of in vivo pharmacokinetics and DDI. In this study, a novel cell based model was developed and validated for in vitro evaluation of transporter (OATP)-based interactions. Time dependent and concentration dependent uptake of selective prototypical substrates was characterized for OATP1B1 and OATP1B3. Inhibitory studies were performed using prototypical inhibitors recommended by drug transporter white paper published by International Transporter Consortium (ITC). The kinetic profiles of selected probe substrates and IC50s of selected inhibitors are comparable to the published data. Together, the data demonstrates the model is a compliant and useful in vitro tool to study solute carrier transporter involved interactions.

P435. VALIDATION OF A NOVEL TRANSPORTER MODEL TO STUDY ORGANIC ANION/CATION TRANSPORTERS Na Li, Jie Wang, Kirsten Cooper, Joanne Bourgea, Charles L. Crespi and Christopher J. Patten Corning Life Sciences, Bedford, MA Per regulatory authority drug interaction guidelines, two organic anion transporters, OAT1 and OAT3, and two organic cation transporters, OCT1 and OCT2, are recommended for investigation as key transporters with clinical implications. OAT1, OAT3 and OCT2 are the major basolateral uptake transporters in kidney proximal tubule epithelia, serving for renal excretion. OCT1 is mainly expressed on the sinusoidal membrane of hepatocytes where it is responsible for cation drug uptake. Inhibition of these transporters can cause reduced renal/hepatic clearance of potential ‘‘victim’’ drugs. Understanding of these OATs and OCTs involved drug-drug interactions (DDI) is a key component of investigating drug efficacy and safety profiles. A new cell-based model has been developed to evaluate OAT/OCT-based interactions. This study focuses on the application and validation of this model in evaluating OAT/OCT-based interactions. Time dependent and concentration dependent uptake of selective prototypical substrates was characterized for both OAT1/3 and OCT1/2. Inhibitory parameters of prototypical inhibitors (recommended by the ITC drug transporter white papers) were determined using this model as well. The Km of selected substrates and IC50s of selected inhibitors are comparable to literature reports. The study demonstrates the model is a compliant and useful in vitro tool to assess OAT/OCT involved drug interaction.

P436. IS INHIBITION OF P-GLYCOPROTEIN (P-GP) SUBSTRATE DEPENDENT? Steven W. Louie1, Peng Hsiao2 and Jashvant D. Unadkat2 1 Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, CA, 2Pharmaceutics, University of Washington, Seattle, WA Introduction: P-glycoprotein (P-gp) has multiple binding sites (Aller et al., 2009). Therefore, using a single substrate for in vitro drug interaction screening may not accurately predict the magnitude of in vitro and in vivo P-gp based drug interactions mediated


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by a new molecular entity (NME). This is of concern because the Food and Drug Administration (FDA) guidelines suggest that pharmaceutical companies utilize a single P-gp probe substrate to assess the IC50 (drug concentration at 50% P-gp inhibition) of a NME. Therefore, we determined the IC50 of several prototypical P-glycoprotein inhibitors, using LLCPK1 cells overexpressing human P-glycoprotein (LLCPK1-MDR1) and a panel of well-established P-glycoprotein substrates, namely [3H]-digoxin, [3H]verapamil, [3H]-prazosin, [3H]-quinidine or [3H]-quinine. Methods: Parental LLC-PK1 and human MDR1-transfected LLC-PK1 cells were cultured and seeded as in Booth-Genthe et al. (2006). Cells were washed with HanksBalance Salt Solution containing 10 mM Hepes prior to dosing with radiolabeled probes in the presence and absence of increasing concentrations of the inhibitor. Transwells were incubated for 2 hours with orbital shaking at 37 C, 5% CO2 and 95% humidity. Radiolabeled samples were withdrawn from apical and basolateral chambers and transferred to liquid scintillation vials containing scintillation fluid and counted. The apparent permeability coefficient (Papp) of all tested agents was calculated using the following equation: Papp ¼ VR*CR(t)/A*t*CD (cm/s), where VR is the receiver compartment volume (mL), CR is receiver concentration at the end of incubation (mM), A is the surface area of the insert filter, t is incubation time (s), and CD is the initial concentration of the test compound (mM). The IC50 values were calculated by nonlinear regression analysis of the data using the Hill Equation SigmaPlot V.11.0: f ¼ V / (1 þ (x/IC50)^n) where f is the % inhibition of control, and x is the inhibitor concentration, and n is the Hill coefficient. IC50 values of each inhibitor, across the substrates used, were compared using the unpaired Student’s t-test. Results: Loperamide inhibited P-gp transport of quinidine, quinine, or verapamil (IC50 8.2 2.7, 10.1 3.1, and 10.6 2.9 mM, respectively) more potentely (p50.05) than that of prazosin (IC50 22.1 3.7 mM). Likewise, quinine inhibited P-gp transport of quinidine (IC50 42.3 14 mM) more potently (p50.05) than that of prazosin (IC50 112.9 18 mM). In addition, verapamil inhibited P-gp transport of quinidine or quinine (IC50 6.6 2.5, and 5.5 1.2 mM, respectively) more potently (p50.05) than that of digoxin or prazosin (IC50 21.0 15 and 14.5 1.9 mM, respectively). Conclusions: Although the potency of P-gp inhibition by the inhibitors tested was substrate dependent, the effect was modest. Where a signficant difference was observed, P-gp was most potently inhibited when quinine was used as a substrate. Therefore, we propose that quinine be used as a substrate to estimate the maximal potential of a NME to inhibit P-gp. Supported by ITHS/TL1/Multidisciplinary Predoctoral Clinical Research Training Program, PHS2271, NIH NS068404.

P437. INTERACTIONS OF ENOBOSARM (OSTARINE, GTX-024) WITH A PANEL OF HUMAN DRUG TRANSPORTERS Gang Luo1, Manuela De Avila1, Michele Hamrick1, Kristen Cardinal1, Kris Horabik1, Bonnie Jung1, Amy Nehmer1, Donald McKenzie1, Amanda Jones2 and James T. Dalton2 1 Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI, 2GTx, Inc., Memphis, TN Enobosarm is an oral selective androgen receptor modulator that GTx, Inc. is developing for the prevention and treatment of muscle wasting in patients with non-small cell lung cancer. Enobosarm is currently at Phase III clinical trial and has been evaluated in ten clinical trials involving over 1300 subjects including five efficacy studies. In this study, enobosarm was assessed as a substrate and inhibitor of a panel of human transporters including OAT1, OAT3, OATP1B1, OATP1B3, OCT1, OCT2, and breast cancer resistant protein (BCRP). 14C-Enobosarm (1 and 10 mM) was incubated with Chinese hamster ovary (CHO) cells stably expressing uptake transporters and CHO vector control cells at 37 C for 5, 15, and 30 minutes. Uptake of 14C-enobosarm by the cells was then determined. The results indicate that enobosarm is not a substrate of these uptake transporters. The inhibitory effects of enobosarm on uptake transporters were determined by measuring the uptake of known substrates in the presence of vehicle and enobosarm. Enobosarm demonstrated little to no inhiibition of OAT1, OAT3, OATP1B1, OATP1B3, OCT1, and OCT2 with IC50 values 42 mM. In Caco-2 cells, the apparent permeability of 14C-enobosarm (1 and 10 mM) was determined in both the apical to basolateral and the basolateral to apical directions in the presence of vehicle and Ko143. In the presence of vehicle, 14C-enobosarm was found to have permeability ranging from 5.70 to 7.65 106 cm/s in either direction with efflux ratio ranging from 0.854 to 1.12. The efflux ratio of 14C-enobosarm was not altered by the BCRP-specific inhibitor Ko143 (1 mM) indicating that enobosarm is not a substrate of transporters expressed in Caco-2 cells. The inhibitory effect of enobosarm on BCRP was determined by measuring the efflux ratio of 3H-estrone-3-sulfate, a known BCRP substrate, in the presence of vehicle and enobosarm. In the preliminary assessment, efflux ratios of 3H-estrone-3-sulfate were 20.9, 6.74, and 1.21 in the presence of enobosarm at 0, 2, and 20 mM, respectively, suggesting that enobosarm is an inhibitor of BCRP. The IC50 value was determined to be 1.31 mM from the definitive assessment. In summary, enobosarm is not a substrate of human uptake transporters and human efflux transporters expressed in Caco-2 cells. Enobosarm demonstrated little to no inhibition of OAT1, OAT3, OCT1, OCT2, OATP1B1, and OATP1B3 and was a weak inhibitor of BCRP.

P438. PREDICTION OF RENAL TRANSPORTER-MEDIATED CLINICAL DRUG-DRUG INTERACTIONS Bo Feng, Yasong Lu, Manthena Varma and Charles J. Rotter Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Inc., Groton, CT Kidney plays a critical role in the elimination of xenobiotics. Drug-drug interactions (DDIs) via inhibition of renal organic anion (OAT) and organic cation (OCT) transporters have been observed in the clinic. This study examined the quantitative

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predictability of renal transporter-mediated clinical DDIs based on basic and mechanistic models. In vitro transport substrate and inhibition assays using transporter-transfected HEK293 cells were carried out to identify renal transporter substrates and measure the inhibition potency of transporter inhibitors. In vitro transporter data and clinical pharmacokinetics parameters were used to quantitatively predict DDIs of victim drugs when co-administrated with OAT or OCT inhibitors, probenecid and cimetidine, respectively. The predicted changes in renal clearance (CLr) and area under the plasma concentration-time curve (AUC) were comparable to that observed in clinical studies. With probenecid, basic modeling predicted 61% cases within 25% and 94% cases within 50% of the observed CLr changes in clinic. With cimetidine, basic modeling predicted 61% cases within 25% and 92% cases within 50% of the observed CLr changes in clinic. Additionally, mechanistic model predicted 54% cases within 25% and 92% cases within 50% of the observed AUC changes with probenecid. Notably, the magnitude of AUC changes attributable to the renal DDIs is generally less than two-fold, unlike the DDIs associated with inhibition of CYPs and/or hepatic uptake transporters.


P439. LEVERAGING A PANEL OF HIGH-THROUGHPUT (HT) TRANSPORTER INHIBITION ASSAYS TO DRIVE DECISION-MAKING AND ENHANCE ADMET KNOWLEDGE Lisa L. Elkin1, Mary Ellen Cvijic2, Huaping Tang2, Kathy Mosure3, Ding Ren Shen2, Yong-Hae Han3, Andrew Wagner4, Wilson Shou4, Jonathan O’Connell5, David Rodrigues3 and Matthew Soars3 1 Lead Profiling, Bristol-Myers Squibb, Wallingford, CT, 2Lead Evaluation, Bristol-Myers Squibb, Wallingford, CT, 3 Metabolism and Pharmacokinetics, Bristol-Myers Squibb, Wallingford, CT, 4Synthesis and Analysis Technology Team, Bristol-Myers Squibb, Wallingford, CT, 5Lead Discovery & Profiling, Bristol-Myers Squibb, Wallingford, CT Over the last decade, it has become clear that transporters are expressed in numerous tissues and govern the ADMET (absorption-distribution-metabolism-excretion-toxicity) profile of many drugs. Transporter interactions can lead to altered disposition of both co-administered drugs and endogenous compounds, which has prompted regulatory agencies to request a detailed characterization of drug interactions with these proteins. While one strategy is to profile clinical candidates in preparation for regulatory submission, our strategy at Bristol-Myers Squibb has been to develop state-of-the-art, high-throughput (HT) assays to screen for potential transporter liabilities earlier in drug discovery in order to inform lead chemotype selection and guide clinical candidate nomination. To this end, a panel of relatively HT renal and hepatobiliary transporter inhibition assays was developed using clinically relevant probe substrates to assess compound inhibition of transport by OATP1B1, OATP1B3, NTCP, BSEP, MRP2, OAT1, OAT3, OCT2, and MATE. These assays complement existing cell-based epithelial monolayer assays, which have been designed to assess interaction with efflux transporters such as BCRP and Pgp (1, 2). The transporter assays described were developed in 384-well format using cryopreserved cell lines and/or vesicular expression systems and employ either Solid-Phase Extraction-Mass Spectrometry (SPE-MS/MS), LC-MS/MS, or microplate-based scintillation counting to assess probe substrate transport. Validation with reference compounds demonstrates that these assays are robust, reproducible, and generate data that are consistent with the literature. Here we present information related to assay methodologies, substrate selection, reference compound validation, assay statistics and long-term performance measures, as well as examples of how data can be used to enhance our understanding of transporter biology. This work demonstrates the value of developing a panel of HT transporter inhibition assays to enable more informed lead optimization and candidate selection. In addition, this strategy offers the opportunity to profile a wider range of compounds with distinct in vivo profiles, which can support in vitro-in vivo extrapolations and data translation.

References 1. Wagner AD, Kolb JM, Ozbal CC, Herbst JJ, Olah TV, Weller HN, Zvyaga TA, Shou WZ. Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P-glycoprotein (P-gp) inhibition screen. Rapid Commun Mass Spectrom. 2011 May 15;25(9):1231–40. 2. Cai X, Walker A, Cheng C, Paiva A, Li Y, Kolb J, Herbst J, Shou W, Weller H. Approach to improve compound recovery in a high-throughput Caco-2 permeability assay supported by liquid chromatography-tandem mass spectrometry. J Pharm Sci. 2012 Aug;101(8):2755–62.

P440. QUANTITATIVE EVALUATION OF UNBOUND INTRACELLULAR CONCENTRATIONS OF ANIONIC DRUGS IN RAT HEPATOCYTES Tomohisa Nakada1, Kei Ogawa1, Noriko Hisanaga1, Kazuya Maeda2 and Yuichi Sugiyama3 1 DMPK Research Laboratories, Mitsubishi Tanabe Pharma Corporation, Chiba, Japan, 2Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan, 3Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Research Cluster for Innovation, RIKEN, Yokohama, Japan Unbound intracellular concentration in the liver is an important determinant for predicting the risk of drug-drug interaction as well as efficacy and toxicity. Generally, it has been considered that blood unbound concentrations in the capillary beds are

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comparable to those in the liver assuming neither active transport nor extensive metabolism works. However, active transporters are often majorly involved in the membrane transport of charged and polar drugs. In this case, the intracellular and extracellular unbound concentrations of these drugs are not equivalent. Previously, it was reported that cell-to-medium unbound concentration ratio (Kp,uu) was estimated from the kinetic parameters obtained from the initial uptake clearance of each drug at several concentrations1). In the present study, we would like to propose a relatively simple method estimating the Kp,uu values in rat liver. We examined the uptake of five anionic drugs (atorvastatin, pitavastatin, pravastatin, rifampicin and rosuvastatin) in isolated rat hepatocytes at steady state. We estimated Kp,uu values and unbound fraction in the hepatocytes (fT) of these drugs with three different methods to stop active transport; (1) on ice, (2) inhibition by rifamycin SV (Oatp inhibitor), and (3) saturation with excess concentration of substrates. The difference in estimated Kp,uu and fT values of all these drugs was within 3-folds among these methods, suggesting our methods based on steady-state uptake allow us to easily determine the unbound intracellular concentration of anionic drugs in rat hepatocytes. Compared to the published data1, our estimated Kp,uu values tended to be lower. The possible mechanisms for the difference in Kp,uu values obtained from our methods and reported one will be discussed.

References Drug Metabolism Reviews Downloaded from informahealthcare.com by Dicle Univ. on 11/06/14 For personal use only.

1. Yabe Y et al., Drug Metab. Dispos., 39:1808–14 (2011).

P441. QUANTITATIVE FLUORESCENCE IMAGING OF TRANSPORTER-MEDIATED BILIARY EXCRETION OF TAURO-NOR-THCA-24-DBD IN SANDWICH-CULTURED RAT HEPATOCYTES Tom De Bruyn1, Wouter Sempels2, Hideaki Mizuno2, Johan Hofkens2, J. Snoeys3, Patrick Augustijns1 and Pieter Annaert1 1 Drug Delivery & Disposition, KU Leuven, Leuven, Belgium, 2Department of Chemistry, KU Leuven, Leuven, Belgium, 3 Johnson & Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V., Beerse, Belgium This study aimed to visualize transporter-mediated biliary excretion of the fluorescent bile acid derivative, Tauro-nor-THCA-24DBD in rats. The B-CLEARÕ technique was used to determine the in vitro biliary excretion index (BEI) and biliary clearance of nor-tauro-THCA-24-DBD in sandwich-cultured rat hepatocytes (SCRH) at day 4. The effect of cyclosporine on biliary excretion was measured to explore the involvement of the canalicular transporter bile salt export pump (Bsep). Biliary excretion was further visualized and quantified by live cell confocal imaging with a laser-scanning microscope. Biliary clearance and BEI of Tauro-nor-THCA-24-DBD (10 mM) in SCRH amounted to 1.56 mL/min/mg protein and 26.2 (5)% under control conditions. In presence of 10 mM cyclosporine, the BEI significantly decreased to 5 (3)%. Confocal imaging of the biliary excretion of Tauro-nor-THCA-24-DBD revealed a time-dependent increase in normalized fluorescence intensity in the bile pockets in rat hepatocytes seeded on glass bottom dishes for microscopy use. This increase was not present when Tauro-nor-THCA-24-DBD was co-incubated with cyclosporine. Our results indicate that the biliary excretion of nor-tauro-THCA-24-DBD can be visualized by confocal imaging. Furthermore, Bsep seems to be involved in the biliary excretion of Tauro-nor-THCA-24-DBD. We conclude that Tauro-norTHCA-24-DBD can be used to study Bsep-mediated interactions by confocal imaging.

P442. TOWARDS A MORE PREDICTIVE MODEL: EFFECT OF TELMISARTAN ON TAUROCHOLATE DISPOSITION IN B-CLEARÕ CRYOPRESERVED SANDWICH-CULTURED HEPATOCYTES COMPARED TO BSEP-EXPRESSING MEMBRANE VESICLES Tracy L. Marion, William R. Smith, Cassandra L. Hubert, Robert L. St. Claire III and Kenneth R. Brouwer Qualyst Transporter Solutions, Durham, NC Inhibition of BSEP-mediated bile acid transport has been implicated as a potential mechanism for drug-induced cholestasis. Initial correlations between BSEP inhibition and toxicity have been encouraging; however, there is significant room for improvement. Telmisartan (TEL), an angiotensin II receptor antagonist, reportedly demonstrates a low IC50 for BSEP inhibition in membrane vesicles (IC50 ¼ 16.4 mM), indicating that it is a potent BSEP inhibitor. However, there are no reports of clinical cholestasis/liver injury associated with TEL. Hypothesis: We hypothesized that the B-CLEARÕ model , an integrated, whole-cell system that maintains both uptake and efflux transporter functionality and metabolic capacity, can more accurately predict the potential for TEL to cause cholestasis as compared to BSEP membrane vesicles, which have limited transport function and no metabolic capacity. Methods: Transporter CertifiedÔ cryopreserved human hepatocytes cultured in sandwich configuration were treated with increasing concentrations of TEL (0.01–20 Cmax,total,ss) in the presence or absence of 4% bovine serum albumin (BSA) to approximate physiologic conditions (in vivo, TEL is 99.5% bound to plasma proteins) and the conditions of BSEP membrane vesicles experiments (no protein included). The uptake, intracellular accumulation, and biliary excretion of the BSEP probe substrate taurocholate (TCA) were determined using B-CLEARÕ technology. In addition, TEL and TEL-GLUC were measured by LC-MS/MS in the assay buffer and cell lysates. Results: TEL uptake was markedly decreased in incubations containing BSA compared to protein-free conditions; however, TEL accumulated within hepatocytes up to 85-fold and 225-fold

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higher than the corresponding exposure concentration with and without BSA, respectively, indicating that the free concentration was not the only driving force for hepatic uptake. At clinically relevant extracellular concentrations (up to 1 mM) there were no changes in TCA disposition despite the intracellular accumulation of TEL, indicating no inhibition of BSEP function. This is consistent with clinical observations. Changes in TCA disposition were seen only at the 10 and 20 mM extracellular exposures, which yielded high intracellular TEL concentrations (312–790 mM) that would not be observed clinically. Glucuronidation of TEL was low (520% of total) but consistent with clinical plasma concentration data, and TEL-GLUC did not appear to have an effect on BSEP function. Conclusions: Consistent with clinical data, under physiologically-relevant conditions, TEL did not inhibit BSEP-mediated taurocholate transport. The most pronounced effects on TCA disposition were seen only at the highest intracellular concentrations that were achieved in the absence of BSA. This suggests that B-CLEARÕ sandwich-cultured hepatocytes offer a better model than membrane vesicles for predicting the potential for a compound to interact with BSEP.


P443. TRANSPORTER INHIBITION BY HERBAL SUPPLEMENTS – POTENTIAL DRUG-DIETARY SUPPLEMENT INTERACTIONS Mark Warren1, Mirza Jahic1, Jane Huang1, Qiang Shi2, William Salminen3 and Yong Huang1 1 Optivia Biotechnology Inc., Menlo Park, CA, 2Division of Systems Biology, U.S. Food and Drug Administration (FDA), National Center for Toxicological Research (NCTR), Jefferson, AR, 3U.S. Food and Drug Administration (FDA), National Center for Toxicological Research (NCTR), Jefferson, AR Transporters are known to affect the absorption (A), distribution (D), metabolism (M), and elimination (E) of drugs. The presence of a second drug that inhibits those transporters can have a profound effect on the safety of the first drug, and has led to regulatory agencies putting increasing attention on transporter-mediated drug-drug interactions. Largely unregulated is the field of herbal supplements/neutraceuticals. Many of these compounds are touted as having drug-like benefits, while the potential for drug-like adverse events is left unsaid. Green Tea extract-based dietary supplements have been widely consumed for acclaimed beneficial health effects, such as weight reduction. However, tea polyphenols may also inhibit the activities of both drugmetabolizing enzymes and drug transporters, potentially resulting in adverse drug effects. In addition, tea catechins may upregulate or downregulate the expression of the drug-metabolizing enzymes and transporters, altering the ADME properties of co-medications. In order to investigate the action of supplements on drug transporters, we have studied the effects of a panel of widely used supplements on transporters shown to be clinically important for drug absorption and disposition. While the supplements studied had little to no effect on the organic cation transporter OCT2 and only modest effects on the efflux transporter p-glycoprotein, many of them had profound inhibitory effects on the organic anion transporting polypeptides OATP1B1 and OATP1B3, including green tea extract, black cohosh, ginkgo biloba, and St. John’s wort. Green tea extract was also shown to strongly inhibit the organic anion transporters OAT1 and OAT3. Since inhibition of these transporters by drugs can result in adverse drug effects for co-medications, inhibition of these transporters by supplements can potentially lead to adverse drug effects for co-medications as well.

P444. CHLOROTHIAZIDE AND TERIFLUNOMIDE AS POTENTIAL BCRP PROBES Erzse´bet Bee´ry, Bea´ta To´th, Zsolt Fekete, Zsuzsanna Rajnai, Remi Magnan, Ma´rton Jani, Emese Kis and Peter Krajcsi R&D, Solvo Biotechnology, Budao¨rs, Hungary Background: Breast cancer resistant protein (BCRP/ABCG2) plays a major role in absorption, disposition and excretion of clinically relevant drugs. BCRP is expressed at the apical membranes of epithelial/endothelial cells with barrier function. BCRP plays a role in clinically relevant drug-drug interaction and also has clinically relevant genetic polymorphisms. Therefore, regulatory agencies (FDA/EMA) recommend testing for BCRP-mediated drug interaction to assess the toxicity potential of coadministered drugs. However, there is still no consensus on an accepted BCRP probe to use in clinical studies and across the whole spectrum of the various in-vitro assays. Aim: To evaluate the potential of known BCRP substrates as a BCRP selective probe. Methods: based on literature data, five potential BCRP probe candidates, teriflunomide, chlorothiazide, topotecan, sulfasalazine, and rosuvastatin were selected and screened in an inhibitory vesicular transport assay with membranes overexpressing BCRP, P-gp and MRP2 and MRP4. These compounds were then tested in a direct vectorial transport assay on Caco-2 cells as well as on BCRP overexpressing MDCKII cells. Results: In our vesicular transport inhibition assay, sulfasalazine, chlorothiazide and teriflunomide showed a higher selectivity towards BCRP, unlike topotecan and rosuvastatin that also inhibited MRP4 mediated transport with a similar potency. Chlorothiazide and teriflunomide were both transported across Caco-2 and BCRP overexpressing MDCKII monolayers and were selective for BCRP. Sulfasalazine failed to be transported in BCRP-MDCKII cells which may be explained by its low passive permeability and the lack of uptake transporter on the baso-lateral membrane of these cells. In agreement with vesicular transport data neither topotecan nor rosuvastatin were selective for BCRP while being transported through both Caco-2 and BCRP-MDCKII monolayers. Conclusion: Based on regulatory agencies guidances, literature, clinical data, and our experimental data, we suggest that either chlorothiazide or teriflunomide can be used as a BCRP probe.

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P445. VECTORIAL TRANSPORT OF CHLOROTHIAZIDE BY MONOLAYERS MDCKII CELLS DOUBLY TRANSFECTED WITH HUMAN OAT1 AND BCRP OR WITH OAT3 AND BCRP


Vikto´ria Juha´sz, Erzse´bet Bee´ry, E´va Molna´r, Remi Magnan, Ma´rton Jani and Peter Krajcsi R&D, Solvo Biotechnology, Budao¨rs, Hungary Introduction: Chlorothiazide is used as diuretic to manage excess fluids associated with congestive heart failures, hepatic cirrhosis, corticosteroid/estrogen therapy, chronic renal failure and edema due to various forms of renal dysfunction such as nephrotic syndrome, acute glomerulonephritis. Chlorothiazide is also indicated in the management of hypertension either as the sole therapeutic agent or to enhance the effectiveness of other antihypertensive drugs. However, chlorothiazide intake is associated with adverse effects such as renal failure, renal dysfunction and interstitial nephritis. The mechanism of action of chlorothiazide-mediated diuretic effect and associated side effects remain poorly understood. Chlorothiazide is not metabolized but is eliminated rapidly by the kidney (half-life 45–120 min). We previously showed that chlorothiazide is a substrate of organic anion transporter 1(OAT1), organic anion transporter 3 (OAT3) and breast resistance cancer protein (BCRP). OAT1/3 and BCRP synergistically play an important role in the transport of organic anions by the kidney. We hypothesized that BCRP works in concert with OAT1 and/or OAT3 to transport chlorothiazide across the proximal tubule cells thus contributing to its rapid renal clearance. Aim: Generate MDCKII cells doubly transfected cell with human OAT1 and BCRP or OAT3 and BCRP in order to assess chlorothiazide vectorial transport. Results: MDCKII cells expressing both OAT1 and BCRP or OAT3 and BCRP were generated. Chlorothiazide vectorial transport was well detected on both OAT1-BCRP and OAT3-BCRP monolayers from the basolateral (B) to apical (A) direction, efflux ratios were superior to 10 and 100 fold, respectively. Both KO143, a BCRP specific inhibitor, and probenecid, an OATs specific inhibitor, were able to fully inhibit chlorothiazide vectorial transport. Furthermore, uric acid also partially inhibited chlorothiazide vectorial transport suggesting that chlorothiazide might alter uric acid secretion in physiological conditions. Conclusion: BCRP works in concert with OAT1 and OAT3 to transport chlorothiazide on our double transfectant models thus suggesting that chlorothiazide rapid renal secretion is likely to be achieved through the concerted action of basolateral OAT1/3 and apical BCRP transporters. These OAT1-BCRP and OAT3-BCRP double transfectant cell lines constitute 2 new cellular renal models for studying secretion of anionic drugs.

P446. CYCLIN-DEPENDENT KINASE INHIBITORS, FLAVOPIRIDOL, PD 0332991 AND ROSCOVITINE, INHIBIT ABCB1 TRANSPORTER IN VITRO AND SYNERGISTICALLY POTENTIATE THE CYTOTOXIC EFFECT OF DAUNORUBICIN IN MDCKII-ABCB1 CELL LINE Daniela Cihalova, Martina Ceckova and Frantisek Staud Department of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic ABCB1 (P-glycoprotein) efflux transporter contributes to multiple drug resistance, a major obstacle in cancer treatment. The cyclin-dependent kinase inhibitors (CDKi), flavopiridol, PD 0332991 and roscovitine, are novel anticancer agents in phase I/II clinical trials for the treatment of various cancers; their interactions with membrane efflux transporters have not been fully investigated to date. The aim of the present study was to characterize the inhibitory effect of the selected CDKi on ABCB1 efflux activity and to test the hypothesis that simultaneous administration of ABCB1-inhibiting CDKi with another cytotoxic agent, that is ABCB1 substrate, might result into synergistic antiproliferative effect. To investigate the inhibitory activity of CDKi on ABCB1, the intracellular daunorubicin accumulation in MDCKII-ABCB1 and control MDCKII parental cell lines was examined by flow cytometry. LY 335979, a model ABCB1 inhibitor, was used as a positive control. Intracellular accumulation of daunorubicin was increased 3.73-, 5.04- or 5.35-fold in the presence of flavopiridol, PD 0332991 or roscovitine at 50 mM concentration, respectively, in MDCKII-ABCB1 cells. All CDKi inhibited ABCB1-mediated efflux in a concentration dependent manner, but no significant effect on the parental MDCKII cell line was observed. We further employed the combination index method of Chou-Talalay to assess whether CDKi can synergistically potentiate the cytotoxic effect of daunorubicin in MDCKIIABCB1 cells and to quantify the dose-reduction index, representing the fold-decrease of individual agents used simultaneously contrary to being used separately to achieve a particular effect. When tested in the MDCKII-ABCB1 cell line, we observed significantly more pronounced synergistic effect of CDKi-daunorubicin combination in comparison with parental MDCKII cells. It is obvious that the more profound synergistic effect of this combination is directly related to the expression of ABCB1. The values of calculated dose-reduction index indicate that the presence of flavopiridol, roscovitine or PD 0332991 allows for 3.7-, 3.8- or 9.3-fold reduction of the dose of daunorubicin in MDCKII-ABCB1 cells to reach 75% effect, respectively. In conclusion, we observed that the inhibition of ABCB1 transporter by the CDKi could affect the pharmacokinetic behavior of ABCB1 substrates if administered simultaneously. We also suggest that the synergism observed in MDCKII-ABCB1 cells is, at least partly, caused by (i) higher intracellular retention of daunorubicin caused by CDKi-mediated ABCB1 inhibition and (ii) native cytotoxic activity of the CDKi, indicating that co-administration of the tested CDKi and ABCB1 substrates in the treatment of ABCB1-expressing tumors may allow for significant dose reduction of anticancer drugs. This work was supported by Zentiva Group, a. s. and by grants GAUK 700912/C/2012 and SVV/2013/267-003.

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P447. REDUCED PHYSIOLOGICALLY-BASED PHARMACOKINETIC MODEL FOR REPAGLINIDE: IMPACT OF OATP1B1 GENOTYPE AND SOURCE OF IN VITRO DATA ON THE PREDICTION OF DRUG-DRUG INTERACTION RISK


Michael Gertz, Carolina Sa¨ll, J. Brian Houston and Aleksandra Galetin School of Pharmacy and Pharmaceutical Sciences, Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, United Kingdom Physiologically-based pharmacokinetic (PBPK) modelling is increasingly applied for the prediction of enzyme-transportermediated pharmacokinetics and associated complex drug-drug interactions (DDIs). Analogous to trends seen for predominantly metabolised drugs, direct use of in vitro uptake transporter kinetic data results in under-prediction of hepatic clearance and a need for empirical uptake scaling factors to bridge the gap in transporter IVIVE. Repaglinide represents an example of a drug with complex hepatic disposition involving active uptake, relatively high passive diffusion and multiple metabolic pathways. In the current study, a reduced PBPK model was developed to investigate the effect of OATP1B1 genotype as a covariate on repaglinide pharmacokinetics and susceptibility to DDIs. A total of 29 mean plasma concentration-time profiles for the most prevalent OATP1B1 polymorphism, SLCO1B1 c.521T4C were collated, including TT (wild type, n ¼ 9), TC (n ¼ 3), CC (n ¼ 7) and unknown genotype (n ¼ 10). In total, 339 concentration-time points at different repaglinide dose levels (0.25 to 2 mg) were used in the analysis. The hepatic intrinsic uptake clearance (CLactive), absorption rate constant, central and peripheral volumes of distribution and absorption lag-time were estimated using a population approach in NONMEM v.7.2 (ADVAN 13 TRAN 1). A significant effect of OATP1B1 genotype as a covariate in the model was seen on CLactive (48% reduction for CC group); no differences were apparent for TC and unknown genotype groups relative to TT. Use of reported in vitro data for repaglinide metabolic clearance (CLmet) and passive diffusion (CLDiff) in the model resulted in estimated CLactive of 217 and 113 mL/(min.106 cells) for TT/TC and the CC group, respectively; up to 4.4-fold higher relative to in vitro data from plated hepatocytes (CLactive ¼ 49 mL/(min.106 cells)). The mechanistic model allowed assessment of repaglinide liver concentration and estimated liver accumulation (total Kp value of 3.9). In addition, the impact of applying a range of fixed CLmet and CLdiff(corresponding to 0.1 to 10-fold of the original parameter value, 25 parameter datasets tested) on the CLactive optimisation was assessed. This sensitivity analysis highlighted considerable uncertainty in the estimation of an empirical uptake scaling factor for repaglinide, as this parameter was highly sensitive to variation in CLmet and CLdiff. However, propagation of this uncertainty had only marginal impact on the predicted magnitude of repaglinide DDI with inhibitors of hepatic uptake; in contrast, sensitivity of the predicted fold change in repaglinide AUC associated with the metabolic component was high. The current study illustrates the application of reduced PBPK models as amenable tools for parameter optimisation; the limitations of the parameter optimisation based solely on plasma data as a surrogate for tissue profiles are highlighted.

P448. EFFECT OF PREGNANE X RECEPTOR AGONIST ON THE EXPRESSION OF PLASMA MEMBRANE MONOAMINE TRANSPORTER IN MICE Seokuee Kim1, Haejin Oh2, Sung Kweon Cho2, Joo-Youn Cho1, Howard Lee1 and Jae-Yong Chung3 1 Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, Seoul, South Korea, 2Department of Clinical Pharmacology, Yonsei University Severance Hospital, Seoul, South Korea, 3Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Bundang Hospital, Gyeonggido, South Korea Objective: We evaluated the effect of the pregnane X receptor (PXR) agonist, pregnenolone 16 alpha-carbonitrile (PCN) on the expression levels of plasma membrane monoamine transporter (PMAT) in the intestine and liver in mouse. Methods: Male C57/BL6 mice were divided into two 2 groups: mice in the PCN group (n ¼ 3) were administered PCN once a day for 4 days, while those in the control group (n ¼ 3) received the same volume of vehicle once a day for 4 days. After The mice were killed 24 h after administration of the last dose of PCN or vehicle, and the expression levels of PMAT in the liver and intestine tissues were isolated and measured the expression level of PMAT using immunohistochemical and western blotting analyses. Results: Injection of PCN significantly increased the number of PMAT-positive cells. The protein expression levels of PMAT in the liver and small intestine increased after PCN treatment compared to those of vehicle group. Conclusion: These results show that PXR agonist may play a clinically significant role by the induction of PMAT which increases the intestinal absorption and hepatic uptake of PMAT substrates such as metformin.

P449. ASSESSMENT OF AFFINITY OF ANTIPSYCHOTIC DRUGS FOR P-GLYCOPROTEIN AND THEIR POTENTIAL FOR DRUG-DRUG INTERACTION Satomi Matsui, Tomoko Inoue, Eiichiro Ogimura, Kiyoshi Natsui, Takao Watanabe and Masashi Yabuki Preclinical Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan


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A number of antipsychotic agents are currently used for the treatment of schizophrenia. Since many patients suffer from side effects such as hyperprolactinemia and extrapyramidal dysfunction, it is necessary to pay attention to drug-drug interactions (DDI) with concomitant drugs, which may increase the plasma levels of these antipsychotic drugs. P-glycoprotein (P-gp, MDR1) is the best known efflux transporter constitutively expressed in various tissues. P-gp expressed in cerebral microvascular endothelial cells and in the small intestine functions as a barrier against brain distribution and intestinal absorption of numerous substrate drugs. If a drug is a P-gp substrate, its brain penetration and GI absorption could be limited and there is a possibility of DDI via P-gp. Therefore, it is important to estimate the affinity of antipsychotic drugs for P-gp. In this study, we examined transcellular transport of nine typical antipsychotic drugs (Haloperidol:HPD, blonanserin:BNS, risperidone:RIS, paliperidone:PAL, clozapine:CLZ, olanzapine:OLZ, Quetiapine:QTP, Aripiprazole:APZ and Perospirone:PER) across LLCPK1 and human MDR1-expressing LLC-PK1 cells at their clinical efficacious concentrations. We also evaluated the inhibitory effect of these drugs on digoxin transport (typical P-gp substrate) across cells. The results demonstrated that only two drugs, RIS and PAL, were actively transported by P-gp as substrates (net flux ratio: 3.1 and 7.7, respectively), suggesting that the concentration of these drugs in the brain are restricted and there is a risk of DDI with P-gp inhibitors. All of these drugs have inhibition potency against P-gp at the following IC50s (APZ, 0.19 mM; QTP, 1.06 mM; CLZ, 1.62 mM; PER, 1.86 mM; HPD, 3.14 mM; RIS, 4.46 mM; BNS, 8.72 mM; PAL, 12.9 mM; and OLZ, 21.3 mM). In circulating blood, only three drugs (APZ, QTP and CLZ) had IC50s that were less than or comparable to their clinical concentration, indicating that these three drugs have a potential for DDI with P-gp substrates. Since concentrations of these nine drugs in the intestine are much higher compared with their plasma concentrations, the probability of DDI via P-gp in the intestine was also estimated using the drug-interaction number (DIN)-based approach proposed by Tachibana et al1. The DIN is the ratio of inhibitor dose to inhibition constant, and is used to index intestinal drug-drug interaction. Based on this approach, the DIN values of APZ, QTP and CLZ were larger than 27.9L and these three drugs were classified as having a high risk of DDI with P-gp substrates in the intestine, as well as in the circulating blood. The DIN values of the other drugs were smaller than 10.8L and these were classified as having a low risk of DDI with P-gp substrates in the intestine.

References 1. T. Tachibana, M. Kato, T. Watanabe, T. Mitsui, and Y. Sugiyama. Method for predicting the risk of drug-drug interactions involving inhibition of intestinal CYP3A4 and P-glycoprotein. Xenobiotica. 2009; 39(6):430–443.

P450. INVESTIGATION OF THE IMPORATANCE OF MULTIDRUG AND TOXIC COMPOUND EXTRUSION (MATE1) IN THE URINARY EXCRETION OF CATIONIC DRUGS IN MOUSE Hiroyuki Kusuhara1, Sumito Ito2, Tomoko Kitoh2, Katsuhisa Inoue3, Hiroaki Yuasa3, Yoshinori Moriyama4 and Yuichi Sugiyama5 1 Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan, 2the University of Tokyo, Tokyo, Japan, 3 Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan, 4Graduate School of Medicine, Density and Pharmaceutical Science, Okayama University, Okayama, Japan, 5Sugiyama Laboratory, RIKEN Innovation Center, RIKEN, Research Cluster for Innovation, RIKEN, Yokohama, Japan Purpose: Transporters play indispensable role in the tubular secretion of drugs in the kidney. The tubular secretion of organic cations involves transporters both in the uptake and subsequent efflux processes. In the tubular secretion of cationic drugs, proton/organic cation exchangers, MATE1 and MATE2-K, are considered to mediate the luminal efflux of various cationic drugs into the urine. We reported that pyrimethamine is a potent inhibitor of MATEs, and that it is useful to investigate the in vivo function of MATEs because its effect on OCT2 is negligible at the dose required to inhibit MATEs (Ito S et al., 2010). The purpose of this study is to elucidate the importance of MATEs in the urinary excretion of drugs in vivo using pyrimethamine. Methods: Pyrimethamine (20 ƒEˆmol/kg) was given to ddY mice by a bolus injection 30 min before starting infusion of test drugs (amiloride, fexofenadine, pilsicainide, pindolol, procainamide, ranitidine, triamuterene, varenicline, ASP, MIBG, sulpiride and famotidine). Drug concentrations in the plasma, urine and kidney were determined by LC-MS/MS. In vitro transport studies were conducted using HEK293 cells expressing MATE1, MATE2-K and Mate1 to confirm that the test drugs are their substrates. Results and Discussion: Amiloride, ASP, MIBG, procainamide, ranitidine, sulpiride, triamterene and rhodamine 123 showed higher significantly higher intracellular accumulation in Mate1 or MATEs expressing cells in the presence of Hþ gradient compared with mock vector-transfected cells, whereas specific transport of pilsicainide, pindolol and varenicline by MATEs was not observed. The renal clearance of all the test drugs is greater than the glomerular filtration rate in control condition. Pyrimethamine-treatment significantly increased the kidney-to-plasma ratios of amiloride, ASP, rhodamine123 ranitidine, and sulpiride, and decreased their renal clearance. Neither plasma concentration nor urinary excretion rate of MIBG was affected by pyrimethamine, however, its kidney-to-plasma ratio was significantly increased. Pyrimethamine-treatment affected neither renal clearance nor kidney-to-plasma ratio of other drugs. Mate1 accounts for the luminal efflux of amiloride, ASP, rhodamine123 ranitidine, and sulpiride in mouse. Conclusions: These data indicate significant contribution of Mate1 to the urinary excretion of drugs, however, other transporters may also mediate the luminal efflux of cationic drugs in the kidney.

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References Ito S. et al. J Pharmacol Exp Ther 333:341–50, 2010

P451. IDENTIFICATION OF THE PRIMARY HUMAN EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 1 MRNA ISOFORMS RESULTING FROM ITS ALTERNATIVE PROMOTER USAGE IN THE LIVER


Shogo Matsumoto, Tomomi Furihata, Misa Mizuguchi, Yuki Suzuki, Kaoru Kobayashi and Kan Chiba Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan Human equilibrative nucleoside transporter 1 (hENT1) transports various nucleoside analogues into cells, and thus plays an important role in determining their pharmacological potency. Although the single hENT1 promoter region (P1) and mRNA (a1) have been widely characterized, we have recently identified additional promoter regions (P2 and P3, which primarily generate hENT1c1/2/3 mRNAs and d1/2/3/4 mRNAs, respectively) in the human liver [1]. However, it remains unclear which mRNA isoforms are primarily expressed in the liver. Therefore, we aimed at identifying the primary hENT1 mRNA isoforms expressed in human hepatocyte, which is one of the target cells for nucleoside analogues, while simultaneously obtaining functional evidence of its alternative promoter usage of the hENT1 gene. Using quantitative real-time polymerase chain reaction (qPCR), we first showed that hENT1c1/2/3 and d1/2/3/4 mRNAs expression was strikingly predominant over hENT1a1 mRNA in human hepatocytes and three hepatoma cell lines. In addition, the results of 50 -rapid amplification of cDNA ends (50 -RACE) and reverse-transcriptase PCR (RT-PCR) further identified hENT1c1, d3 and (to a lesser extent) c2 mRNAs as primary hENT1 mRNA isoforms in the cells. To understand mechanisms underlying differential expression levels among hENT1 mRNA isoforms, transactivation property of alternative promoters (P1, 2 and 3) were examined by luciferase reporter gene assay. Consistent with the mRNA isoforms expression profile, the results showed that the P2 and P3 promoters were strongly activated in three different hepatoma cell lines while the P1 promoter was inert in the cells. When the extent of histone 3 lysine 9 acetylation (H3K9ac), which is one of hallmarks of active promoter regions, was examined by chromatin immunoprecipitation (ChIP) assays, H3K9ac was more enriched in the P2 and P3 promoters than in the P1 promoter or the 30 -intergenic region in the hepatoma cell lines. To summarize, our results demonstrate that, resulting from the manipulated usage of alternative promoters, hENT1c1 and d3 (and c2) mRNAs, but not a1 mRNA, are primarily expressed in human hepatocytes, which suggests that these mRNAs determine the hENT1 protein level in these cells. Furthermore, because such alternative promoter usage is likely to occur in other cell types, our findings are expected to provide significant insights into the molecular machinery of hENT1 expression in various cells that are the targets of nucleoside analogues as well.

Reference [1] Fukuchi Y, Furihata T, Hashizume M, Iikura M and Chiba K (2010) Characterization of ribavirin uptake systems in human hepatocytes. J Hepatol 52:486–492

P452. ROLE OF HYPOXIA-INDUCIBLE FACTOR 1A IN THE REGULATION OF CANCER-SPECIFIC VARIANT OF ORGANIC ANION TRANSPORTING POLYPEPTIDE 1B3 (CSOATP1B3) IN COLON AND PANCREATIC CANCER Nilay Thakkar1, Songhee Han2, Kyungbo Kim1, Donghak Kim2 and Wooin Lee1 1 Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, 2Biological Sciences, Konkuk University, Seoul, South Korea Purpose: Organic anion transporting polypeptide 1B3 (OATP1B3) plays an important role in the hepatic uptake of a variety of endogenous and xenobiotic substances. Over the past decade, several investigations reported that OATP1B3 is also expressed across multiple types of cancers. Only recently, our laboratory and others demonstrated the identity of cancer-specific OATP1B3 variants (csOATP1B3) arising from the use of an alternative transcription initiation site, different from the wildtype (WT) OATP1B3 expressed in the normal liver. Given that csOATP1B3 is detected only in cancer cells, but not in any other nonmalignant tissues, we hypothesized that csOATP1B3 expression may be influenced by molecular mediators of oncogenic signaling pathways. In our current study, we investigated the role of hypoxia and hypoxia inducible factor-1a (HIF-1a) in regulating the expression of csOATP1B3. Methods: RT-PCR, immunoblotting and immunohistochemical analyses were performed to determine the levels of csOATP1B3 and HIF-1a in established cancer cell lines and archived clinical tissue specimens of colon and pancreatic cancer. Cells were exposed to hypoxic conditions, by using either 1% O2 or cobalt chloride, a chemical inducer of HIF-1a. Transport activity of csOATP1B3 induced by hypoxia was assessed by measuring cellular accumulation of [3H]-cholecystokinin-8 (CCK-8, a prototype substrate for OATP1B3). To further identify the promoter sequence responsible for hypoxia-induced upregulation of csOATP1B3, we performed reporter assays using various constructs of csOATP1B3 promoter and electrophoretic mobility shift assays (EMSA) using the probes containing the putative hypoxia

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response element (HRE) site in the csOATP1B3 promoter. The role of HIF-1a on csOATP1B3 upregulation was further verified using siRNA-based knockdown of HIF-1a. Results: Our results indicated that csOATP1B3, but not WT OATP1B3, is upregulated in response to hypoxia, in a cell line-dependent manner. csOATP1B3 upregulated by hypoxia did not lead to an enhanced cellular uptake of CCK-8. Reporter assays with deletion and mutated constructs of the csOATP1B3 promoter revealed a functional HRE located in the proximal upstream region. The results from EMSA using a biotin-labeled probe containing the HRE sequence confirmed the physical interactions between HIF-1a and the HRE probe. Furthermore, siRNA-based knockdown of HIF-1a effectively blocked hypoxia-induced csOATP1B3 upregulation and decreased the basal expression levels of csOATP1B3 in colon and pancreatic cancer cells. Conclusions: We report for the first time that the transcription of csOATP1B3 is actively engaged during hypoxia, through a commonly utilized pathway involving HIF-1a. These findings on the regulatory mechanisms of csOATP1B3 may provide important clues about functional and clinical significance of csOATP1B3 in colon and pancreatic cancer.


P453. ABCG2 MRNA ISOFORM EXPRESSION AND DNA METHYLATION PROFILE IN THE MOUSE MAMMARY GLAND Alex Wu1, Mingdong Yang1, Darci Butcher2, Rosanna Weksberg2, Patricia A. Harper3 and Shinya Ito1 1 Physiology and Experimental Biology Program, Sickkids Research Institute, Toronto, ON, Canada, 2Genetics and Genome Biology Program, Sickkids Research Institute, Toronto, ON, Canada, 3Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada The multidrug transporter Breast Cancer Resistance Protein (BCRP/ABCG2) is highly expressed in the mouse and human mammary gland during lactation, and some drug resistant cancer cells. We have previously shown that the lactogenic hormone prolactin, via JAK2/STAT5, PI3K, and MAPK pathways, induces transcription of ABCG2 in T-47D human breast cancer cells. However, it is unclear if lactation-associated transcriptional upregulation of ABCG2 occurs through a similar mechanism in vivo. In order to gain insight into ABCG2 induction in the mammary gland in vivo, we investigated the expression profile of mouse Abcg2 mRNA isoforms E1A, E1B, and E1C, which are transcribed from alternative promoters, in the mammary gland of nonlactating (virgin) and lactating C57BL/6 and FVB mice. In both strains, the expression of all Abcg2 mRNA isoforms in nonlactating mammary tissue was extremely low. In contrast, the E1B and E1C Abcg2 mRNA isoforms were highly expressed in the mammary gland during lactation, with E1B being the predominant isoform. Interestingly, using the UCSC Genome Browser (mm10 assembly), we identified a hypothetical CpG island (231/þ178) at the promoter region of the E1B isoform. To determine the functional significance of this CpG island, we first characterized the basal promoter region of the E1B isoform in HC11 mouse mammary epithelial cells that express the E1B mRNA isoform under normal growth conditions. Promoter deletion analysis using luciferase reporter constructs driven by different lengths of the mouse E1B promoter region demonstrated that the basal promoter is within 71/þ199 bp of the transcription start site. This DNA region was subsequently cloned into a luciferase reporter construct that is free of CpG sites and then treated to specifically methylate CpG sites within the inserted 71/þ199 E1B promoter. As expected, methylation of the E1B promoter significantly reduced reporter activity. Similar results were observed using a construct that is driven by a longer motif of the E1B promoter region which contains the entire CpG island. This observation was compatible with our hypothesis that changes in the methylation status of the CpG island from being hyper- to hypo-methylated during the transition from virgin to lactation could potentially explain the lactation-associated induction of E1B promoter activity. However, when we examined the average DNA methylation status of five CpG sites in the E1B promoter by pyrosequencing of bisulfite converted genomic DNA, we discovered that the promoter is hypo-methylated even in the nonlactating mammary gland and liver, and remained hypo-methylated in the mammary gland of lactating mice. Taken together, our results suggest that the mouse Abcg2 gene is likely poised for expression in the virgin mammary gland but requires pregnancy/ lactation-associated hormones such as prolactin to induce its expression. This project is supported by a grant and graduate scholarship from CIHR.

P454. HEPATOBILIARY TRANSPORT OF ARSENIC IN SANDWICH CULTURED HUMAN HEPATOCYTES Barbara A. Roggenbeck1, Michael W. Carew1, Gregory J. Charrois2 and Elaine M. Leslie1 1 Physiology, University of Alberta, Edmonton, AB, Canada, 2Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada Arsenic (As) is a carcinogen associated with skin, lung, and bladder tumors. There are over 100 million people worldwide that rely on drinking water contaminated with As. Furthermore, As containing compounds are used clinically to treat hematological and solid tumors. The majority of As metabolism occurs in the liver and it is excreted predominantly in urine (60–80%). Despite this, little is known about the hepatic transport pathways involved in the disposition of As (particularly basolateral). Multidrug resistance proteins (MRPs/ABCCs) are ATP-binding cassette (ABC) transporters that are known to transport glutathione (GSH) conjugates. MRP1 (ABCC1) is known to transport As-GSH conjugates but is not expressed in healthy human liver. The current

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study used sandwich cultured human hepatocytes (SCHH) as a physiological model of As transport to elucidate cellular handling of this carcinogen. To confirm canalicular network formation and transport function of each SCHH preparation the biliary excretion index (BEI) for taurocholate was determined and found to be 47 10% at 10 min. Out of 10 SCHH preparations, we observed predominantly basolateral efflux of total As with the exception of one preparation (liver # 19), where both basolateral and biliary efflux occurred. Arsenic transport was temperature sensitive and decreased by 87 6% at 4 C, suggestive of an active transport process. Incubation of SCHH with buthionine sulfoximine (BSO, 0.5 mM), an inhibitor of the rate limiting step of GSH synthesis, g- glutamylcysteine synthetase, resulted in a 60% reduction of intracellular GSH and a corresponding 46 9% reduction in basolateral transport at 10 min. After GSH depletion in SCHH from liver #19, biliary excretion was not observed. In two human preparations with predominant basolateral efflux, the presence of MK-571 (0.1 mM), an LTC4 receptor antagonist, known to inhibit MRPs, decreased As efflux by 39% at 10 min. Overall, these results suggest an MRP plays a role in the basolateral efflux of As and that SCHH can show interindividual differences in As handling.


P455. P-GLYCOPROTEIN IS RESPONSIBLE FOR THE POOR INTESTINAL ABSORPTION AND LOW TOXICITY OF ACONITINE: THE IN VITRO, IN SITU, IN VIVO AND IN SILICO STUDIES Cuiping Yang, Tianhong Zhang, Zheng Li, Fei Liu, Jinxiu Ruan and Zhengqing Zhang Key Laboratory of Drug Metabolism and Pharmacokinetics, Beijing Institute of Pharmacology and Toxicology, Beijing, China Aconitine (AC) is an active and highly toxic alkaloid from plants with origin of genus Aconitum. Some of Aconite show excellent analgesic, cardiotonic and anti-rheumatism bioactivity and has been widely used as medicinal herbals for thousands of years. Meanwhile, the aconitine-poisoning events ocur frequently in East Asia and some also in Europe and America. To assess the mechanism of its low and variable oral bioavailability, the role of P-glycoprotein (P-gp) was systemically evaluated in this study. The bidirectional transport of AC across Caco-2 and MDCKII-MDR1 cells was investigated. The efflux of AC across Caco-2 and MDCKII-MDR1 cells was greater than its influx and P-gp inhibitors, verapamil and cyclosporin A, significantly decreased the efflux of AC. The in situ single pass intestinal perfusion study in rats indicated verapamil co-perfused with AC exhibited significant increase in intestinal permeability from (0.22 0.12) 105 cm/s to (2.85 0.85) 105 cm/s. The pharmacokinetic profile of AC was determined in rats following oral administration with or without pre-treatment of verapamil. After oral dosing, the maximum plasma concentration (Cmax) of AC increased sharply from 39.43 3.18 to 1490.67 248.3 ng/ml (p50.001). Accordingly, a 6.7-fold increase was observed in the area under the plasma–concentration curve (AUC0–t) of AC when coadministrated with verapamil (p50.001). The in silico docking study demonstrated AC exhibited similar recognition mechanism to verapamil when interacting with P-gp. This work demonstrated P-gp is involved to limit the intestinal absorption of AC and to attenuate its toxicity to human. Our data suggest that caution should be exercised to the P-gp-mediated drug-drug interaction in clinical application of aconite and their formulations containing AC.

P456. INHIBITORY EFFECTS OF HERBAL MONOMERS ON P-GLYCOPROTEIN IN VITRO AND IN VIVO Xue Li, Jinping Hu and Yan Li Dept of Drug Metabolism, Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China Herb-drug interactions have been increasingly reported because of the rise in concomitant use of conventional prescription drugs with herbal medicines. Inhibition and induction of drug transporters by coadministered herbal medicines may induce DDI through altering pharmacokinetics and pharmacodynamics of drugs. The present study was designed to investigate the inhibitory effects of 50 active monomers in 25 common used herbal medicines on P-glycoprotein (P-gp) in vitro and in vivo. The decreased Papp(BL-AP) and increased Papp(AP-BL) of digoxin were observed in the presence of emodin, 18b-glycyrrhetic acid (18b-GA), dehydroandrographolide, and 20 (S) -ginsenoside F1 [20(S)-GF1] in MDR1-MDCKII cells, which indicating the inhibitory effects of these monomers on P-gp in vitro. The structure analogues of above four monomers-chrysophanol, 18aGA,andrographolide, Rh1 and rest tested herbal compounds did not show significant inhibition on P-gp function in this cell model. Among four active compounds, emodin exhibited the strongest potency for the inhibition of P-gp(IC50 ¼ 9.42 mM), followed by 18b-GA (IC50 ¼ 21.78 mM), 20(S)-GF1 (IC50 ¼ 75.51 mM) and dehydroandrographolide (IC50 ¼ 77.80 mM). The similar inhibitory effects of four monomers were further confirmed in Caco-2 cell model. ATPase activity assay implied that emodin and dehydroandrographolide were the substrates of P-gp, with the Km and Vmax values of 48.61 8.85 mM, 71.29 5.46 nmol/min/mg protein and 29.09 8.41 mM, 38.45 3.46 nmol/min/mg protein, respectively. Unlike P-gp substrates, 18b-GA and 20(S)-GF1 inhibited both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Furthermore, the molecular docking study (CDOCKER) suggested that several amino acid residues in the active site of P-gp including Phe974, Phe728, Ser975, Gln721, and Gln191 may be responsible for the binding of herbal inhibitors. After coadministrationof digoxin with emodin and 18b-GA, the AUC(0–t) and Cmax of digoxin were increased by approximately 151% and 58%, respectively, in SD rats. In addition, 18b-GA, dehydroandrographolide and 20(S)-GF1 at 10 mM were found to inhibit CYP3A4/5 activity in human liver microsomes, while emodin showed 35% activation on catalytic reaction mediated

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by CYP3A4/5. In conclusion, four monometers in 25 herbal medicines showed the inhibition of Pgp and /or CYP3A4/5 in certain extents in vitro or in vivo. Our results will provide the useful information for the prediction of transporter mediated herb-drug interaction in clinical, especially important for thecoadministration of drugs with narrow therapeutic and safety window.

P457. DEVELOPMENT OF 96-WELL AUTOMATED OATP1B1 UPTAKE RATE ASSAY FOR LIVER TARGETING


Bo Feng, Hui Zhang, Michael West, Renato Scialis, Rui Li, Avijit Ghosh and Larry Tremaine Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Inc., Groton, CT OATP1B1 is a liver specific uptake transporter, and it is crucial for drug (e.g., statins) uptake into liver for its PD effects. Liver targeting through OATP1B1 is a valid approach to enhance therapeutic index where toxicity is extra-hepatic in nature. Additionally, active uptake can also be rate determining to overall systemic clearance. Previous studies focused on measuring active uptake and passive clearance using sandwich cultured human hepatocytes (SCHH). To complement this approach, a 96well, automated uptake rate assay using OATP1B1 transfected HEK293 cells has been successfully developed to de-convolute active uptake and passive rate. Using statins as substrates, OATP1B1-mediated uptake into cells at multiple time points is measured. Active uptake rate and passive rate are estimated using dynamic fitting. The automated OATP1B1 uptake rate assay has been validated using multiple literature compounds, including pravastatin, rousvastain, cerivastatin, fluvastatin, valsartan, repaglinide, and bosentan, and the passive and active rate are 0.4 and 6.5, 0.5 and 8.2, 37.3 and 19.6, 24.1 and 45.2, 2.3 and 23.2, 43.0 and 7.3, 28.2 and 22.9 cm/sec*10^-6, respectively. The active uptake and passive rate have been incorporated into PBPK modeling to predict hepatic clearance. The automated OATP1B1 uptake rate assay will be very valuable to direct SAR for liver targeting programs.

P458. DRUG ACCUMULATION IN ALVEOLAR MACROPHAGES: ASSESSMENT OF UPTAKE AND LYSOSOMAL DISTRIBUTION IN VITRO Ayse Ufuk1, Michael Gertz1, Jonathan Plumb2, Graham I. Somers3, J. Brian Houston1 and Aleksandra Galetin1 1 School of Pharmacy and Pharmaceutical Sciences, Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, United Kingdom, 2NIHR South Manchester Respiratory and Allergy Clinical Research Facility, University of Manchester, Manchester, United Kingdom, 3GlaxoSmithKline, Stevenage, United Kingdom Alveolar macrophages (AMs) may play an important role in accumulation of inhaled drugs. This trend is particularly evident for cationic amphiphilic drugs, mainly due to their increased ionisation in lysosomes, which are generally more acidic compared to cytosol; this process is often referred to as lysosomal trapping. This study characterised the uptake of 10 compounds (mainly respiratory drugs) in NR8383 (rat AM cell line) and human AMs over 10 minutes at 5 mM substrate concentration at 37 and 4 C using plated cells [1]. In addition, the potential for lysosomal trapping was investigated at 5 mM substrate concentration in NR8383 in the presence of 20 mM ammonium chloride (NH4Cl). Cell-to-media concentration ratios (Kp values) in the presence and absence of NH4Cl were compared to the values predicted by an in silico mechanistic cell model developed in Matlab. Drug physicochemical properties (logP, pKa) and cell properties (e.g., lysosomal and mitochondrial volume, membrane thickness) were used to predict the extent of lysosomal trapping based on pH differences between media, cytosol and lysosome, as well as electrostatic interactions [2]. In NR8383, total uptake clearance ranged from 51 mL/min/106 cells for tiotropium bromide, fenoterol and terbutaline up to 415 mL/min/106 cells for imipramine. Following the resolution of active and passive contributions, the role of the latter ranged from 1% to 460% for clarithromycin and budesonide, respectively. Comparable trends were observed between rat and human AMs for most of the drugs investigated. The reduction in uptake clearance by NH4Cl ranged from 60-80% in the case of fenoterol, imipramine and clarithromycin, whereas 535% was seen in the case of formoterol and terbutaline. The predicted Kp values under control conditions ranged between 3 and 117 for ciprofloxacin and tiotropium bromide, respectively. While predicted Kps were in agreement for fenoterol and budesonide, they were under-predicted for imipramine, clarithromycin and formoterol, and over-predicted for terbutaline and tiotropium bromide. Nevertheless, lysosomal trapping was predicted reasonably well for basic, neutral and zwitterionic drugs. The developed mechanistic cell model can be a useful tool to screen compounds for lysosomal trapping potential based on their physicochemical properties. Such in silico tools, when used together with in vitro data, can aid compound design and selection in early drug discovery.

References 1. Menochet, K., K.E. Kenworthy, J.B. Houston, and A. Galetin, Simultaneous assessment of uptake and metabolism in rat hepatocytes: a comprehensive mechanistic model. J Pharmacol Exp Ther, 2012.341(1): 2–15.

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2. Trapp, S., G.R. Rosania, R.W. Horobin, and J. Kornhuber, Quantitative modeling of selective lysosomal targeting for drug design. Eur Biophys J, 2008. 37(8): 1317–1328.

P459. EFFECTS OF MORPHINE AND ITS METABOLITES ON THE EXPRESSION OF AQP3 IN THE COLON


Risako Kon, Nobutomo Ikarashi, Ayako Mimura, Tomohiko Iizasa, Kiyomi Suenaga, Noriko Suzuki, Reiko Hiruma, Yoshiki Kusunoki, Makoto Ishii, Wataru Ochiai and Kiyoshi Sugiyama Dept of Clin Pharmacokinet, Hoshi Univ, Shinagawa-ku, Japan Morphine is the most frequently used narcotic analgesic in the treatment of cancer pain. Constipation is a serious side effect that frequently occurs soon after morphine administration. Morphine is metabolized in the liver by UDP-glucuronosyltransferase (UGT) into morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G). However, the mechanism of constipation during administration of morphine remains unknown. We have shown that the water channel aquaporin-3 (AQP3) expressed in mucosal epithelial cells in the colon plays an important role in constipation. In this study, we examined the role of morphine and its metabolites in the constipation. Morphine was orally administered to rats, and the relationship between the onset of constipation and the expression levels of colon AQP3 mRNA and proteins were analyzed by real-time RT-PCR and western blotting, respectively. In addition, morphine, M-3-G, or M-6-G was added to HT-29 human colon cancer cells, and the effect of these compounds on the AQP3 mRNA expression level was analyzed by real-time RT-PCR. The water content in feces 5 hours after morphine administration was significantly lower than in the control group, showing the onset of constipation. AQP3 mRNA and protein expression levels in the colon increased significantly, to approximately twice that of the control group. In contrast, no change was observed in the AQP3 mRNA expression level when morphine or M-3-G was added to HT-29 cells, while the AQP3 mRNA expression level increased concentration-dependently when M-6-G was added. Morphine-induced constipation has been considered to be caused by the suppression of peristaltic movement via the m opioid receptor in the intestine. The present study shows that increased AQP3 expression in the colon might be one of the causes of morphine-induced constipation. In addition, it was shown that this increase in the AQP3 expression level was caused by M-6-G.

P460. EFFECT OF RENAL IMPAIRMENT ON THE PHARMACOKINETICS OF MORINIDAZOLE: ALTERED UPTAKE TRANSPORTER-MEDIATED RENAL CLEARANCE? Xiaoyan Chen, Cen Xie, Kong Zhong, Xiuli Li and Dafang Zhong Center for Drug Metabolism and Pharmacokinetics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Morinidazole is a novel 5-nitroimidazole class antimicrobial agent that is currently under phase III clinical development in China. Previous metabolism study showed that after an intravenous administration, morinidazole underwent extensive metabolism in humans via Nþ-glucuronidation (M8-1 and M8-2) and sulfation (M7), and approximately 70% of the dose was excreted in the urine. The objective of the present study was to assess the effect of renal impairment on the pharmacokinetics of morinidazole and its major metabolites (M7, M8-1, and M8-2) following a single 500 mg intravenous infusion dose. Ten subjects with severe renal impairment and ten healthy controls were enrolled in this open-label study. Serial plasma and urine samples were collected up to 48 h after administration for analysis of the pharmacokinetic parameters and renal clearance of morinidazole and its major metabolites. Compared with those obtained from healthy subjects, the values of area under the plasma concentration-time curve up to the last sampling time (AUC0t) in subjects with severe renal impairment for morinidazole, M7, M8-1, and M8-2 were increased by 1.5, 13.0, 18.8, and 10.8-fold, respectively, and the values of half-life (t1/2) were prolonged by 1.3, 1.7, 5.2, and 6.3-fold, respectively. No significant difference was observed in total urinary excretion between two groups, but the renal clearance for M7, M8-1, and M8-2 were 85.3, 92.5, and 92.2% lower in renal impairment group, respectively, than those in healthy controls. These findings implied that the renal elimination, rather than the overall metabolism of morinidazole, is significantly affected by renal impairment. Moreover, renal impairment had greater effects on the pharmacokinetics of the metabolites than that of the parent morinidazole. Based on the observations, we supposed that the effect of renal impairment may be linked to the alterations of renal uptake transporters. The uptake of morinidazole and its metabolites (M7, M8-1, and M8-2) was thus determined in HEK293 cells individually expressing human OAT1, OAT3, and OCT2. Among all tested transporters, none showed significant accumulation of morinidazole compared with vector-HEK. OAT1 and OAT3 mediated the renal uptake of M7, with transfected-HEK/vector-HEK ratios of 42.0 and 45.8, respectively. OCT2 was responsible for the renal uptake of M8-1 and M8-2, with transfected-HEK/vector-HEK ratios of 117 and 47.7, respectively. The downregulation of the renal basolateral OAT1, OAT3, and OCT2 uptake transporters can partially explain the elevated plasma exposure and reduced renal clearance for morinidazole sulfate (M7) and glucuronides (M8-1 and M8-2) in severe renal impairment subjects. Results from this study demonstrated that, compared with subjects with normal renal function, systematic exposure to morinidazole and its conjugated metabolites increased in subjects with severe renal impairment possibly due to the decreased activities of OAT1, OAT3, and OCT2.

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P461. ACTIVATION OF XANTHINE OXIDASE SUPPRESSES DISULFIDE BOND FORMATION OF BREAST CANCER RESISTANCE PROTEIN IN CACO-2 CELLS


Jiro Ogura, Shunichi Sasaki, Kaori Kuwayama, Chihiro Kaneko, Takahiro Koizumi, Keisuke Yabe, Hiroaki Yamaguchi and Ken Iseki Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Gout prevalence is increasing all over the world and is increasing in age. However, the mechanisms of the increasing gout prevalence in age are not clear. It has well known that oxidative stress causes age-related disease. Age-related oxidative stress is caused by the activation of xanthine oxidase. Recently, it shows that breast cancer resistance protein (BCRP) is a high-capacity urate efflux transporter. BCRP is highly expressed in the small intestine and contributes to efflux of urate to the intestinal lumen. In this study, we investigated the effect of activation of xanthine oxidase on BCRP expression. Caco-2 cells were used for a model of the intestinal epithelium in this study. 5,6-Diaminouracil (DAU) used as a xanthine oxidase activator. Messenger RNA level was assessed by real-time RT-PCR. Total protein expression and homodimer level were assessed by Western blot analysis under reducing and non-reducing condition, respectively. The band intensity of Western blot analysis was determined by densitometry using Scion image program. Disulfide (S-S) bond formation was estimated by the ratio of homodimer level/total protein expression level. It was found that BCRP homodimer level was decreased by DAU, which is a xanthine oxidase activator, though BCRP mRNA level and total protein expression was not altered. It has known that nicotinamide adenine dinucleotide phosphate (NADP) provides electron transfer, and works as a co-factor of proteins catalyze S-S bond formation such as thioredoxin and glutaredoxin. Next, we investigated the effect of NADP on DAU-induced suppression of BCRP S-S bond formation. Since NADP has poor membrane permeability, we used nicotinic acid, which is converted to NADP in cells. Although nicotinic acid had no effect on the BCRP mRNA and total protein expression, nicotinic acid ameliorated the DAUinduced suppression of BCRP S-S bond formation. These results suggest that NADP plays an important role for BCRP S-S bond formation. In conclusion, the activation of xanthine oxidase suppresses BCRP S-S bond formation. Nicotinic acid ameliorated the suppression of BCRP S-S bond formation.

P462. IN VITRO FUNCTIONAL ANALYSIS OF NOVEL SINGLE NUCLEOTIDE POLYMORPHISMS IN OATP1B1 Peter Z. Yin and Richard B Kim Physiology and Pharmacology, Western University, London, ON, Canada The goal of our study is to functionally characterize six nonsynonymous single nucleotide polymorphisms (SNPs) in human organic anion transporting polypeptide 1B1 (OATP1B1) using in vitro transporter expression technologies. OATP1B1 is a hepatic transporter responsible for the uptake of many xenobiotics in use today. We hypothesize these SNPs will result in a decrease in transport activity compared to wild-type. OATP1B1 cDNA packaged in pEF6/V5-His TOPO was used as template, and 6 SNPs — 298G4A (rs144508550), 419C4T (rs147450830), 463C4A (rs11045819), 1007C4G (rs72559747), 1463G4C (rs59502379), and 1738C4T (rs71581941) — were introduced separately to wild-type templates using QuikChange site-directed mutagenesis. OATP1B1 variant cDNAs were transferred to pAd/CMV/V5-DEST Gateway vector for further propagation as OATP1B1 variants expressed in adenovirus. We will determine the optimal titre of the OATP1B1-variant adenovirus for transporter expression using a monolayer cell lines such as HeLa. Subsequently, we will use prototypical substrates of OATP1B1 such as estrone sulfate, as well as drugs such as atorvastatin and rosuvastatin to determine the relative affinity (Km) and transport capacity (Vmax), followed by quantification of relative cell surface trafficking by cell-surface biotinylation. Identification of new functional SNPs in OATP1B1 will give us further insights to genetic heterogeneity in OATP1B1 as a contributor to inter-subject variation in response to OATP1B1 substrate drug therapy. Research is funded by the Canadian Institute for Health Research.

P463. STUDY OF TRANSPORT OF TENOFOVIR AND TENOFOVIR DISOPROXIL FUMARATE ACROSS THE RAT TERM PLACENTA Zuzana Neumanova, Lukas Cerveny and Frantisek Staud Department of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor that is administered in the form of a prodrug, tenofovir disoproxil fumarate (TDF). TDF-containing regimens are nowadays recommended as the first line therapy in HIV infected patients and are also used in pregnant women to moderate mother-to-child-transmission of HIV. To evaluate safety of this treatment, it is inevitable to have comprehensive knowledge on transplacental transport of TDF/TFV across the placenta. Among factors affecting fetal drug exposure, placental ABC drug efflux transporters such as P-glycoprotein (ABCB1), breast cancer resistance

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protein (ABCG2) and multidrug resistance protein 2 (ABCC2) are the most important ones actively protecting the fetus against drugs in the maternal circulation. In this study we employed the method of dually perfused rat term placenta to investigate the transplacental transport of TFV and TDF. Using open-circuit perfusion system, transport of TFV or TDF was studied in both fetal-to-maternal and maternal-to-fetal directions. We observed low transplacental clearance of TFV without involvement of a capacity-limited mechanism. In contrast, at low (50 nM) TDF concentration, significant asymmetry in transplacental clearance was detected in favor of fetal-to-maternal direction. Increasing TDF concentration (500 mM) annulled this asymmetry, confirming saturation of transporter(s). Employing closed-circuit perfusion system, we also observed active transport of TDF across the placenta. Moreover, application of a common ABCB1 and ABCG2 inhibitor, GF120908, significantly reduced TDF transport from fetus to mother, whereas co-administration of ABCC inhibitor, indomethacin, had no effect. In conclusion, our findings suggest an important role of Abcb1 and Abcg2 in transport of TDF across the rat placenta. This work was supported by Czech Science Foundation (GACR 170/53/25303), Grant Agency of Charles University (GAUK 695912/C/2012) and Zentiva Group, a.s.


P464. DEVELOPMENT OF A HIGH-THROUGHPUT CELL-BASED FLUORESCENCE ASSAY TO STUDY REGULATORY AUTHORITY RECOMMENDED SLC TRANSPORTERS Jie Wang, Na Li, Joanne Bourgea, Kirsten Cooper, Charles L. Crespi and Christopher J. Patten Corning Life Sciences, Bedford, MA SLC Transporters are membrane-bound proteins that facilitate the vertical movement of endogenous or exogenous substrates across biological membranes. They have been shown to play a critical role in the pharmacokinetics, safety and efficacy profiles of drugs, and are of emerging clinical importance in the absorption and disposition of drugs. A single-use cell based transporter model has been developed to study drug interactions with SLC transporters. This new model has many advantages over the traditional cell-based assay systems such as ease of use, high uptake ratio, and batch-to-batch consistency. In this study, we have developed a high-throughput fluorescence assay to study SLC transporters using the newly developed transporter model. A minimum of one fluorescent substrate has been identified and validated for each of six regulatory authority (FDA, EMA) recommended SLC transporters: Organic Anion-Transporting Polypeptide 1B1 and 1B3 (OATP1B1 OATP1B3), Organic Anion Transporters 1 and 3 (OAT1, OAT3), Organic Cation Transporters (OCT1 and OCT2). The assay was validated with known inhibitors, and the IC50 generated with this fluorescence assay was compared with the IC50 generated with radioactive and LC-MS methods. This fluorescence assay is highly sensitive and less labor intensive, compared to radioactive or LC-MS assays, and is widely accessible because it does not require radioactive compounds nor specialized instrumentation.

P465. DEVELOPMENT AND VALIDATION OF A NOVEL TRANSPORTER MODEL TO STUDY REGULATORY AUTHORITY RECOMMENDED SLC TRANSPORTERS Jie Wang, Joanne Bourgea, Kirsten Cooper, Charles L. Crespi, Christopher J. Patten and Na Li Corning Life Sciences, Bedford, MA SLC transporters are membrane-bound proteins that facilitate the vertical movement of endogenous or exogenous substrates across biological membranes. They have been shown to play a critical role in the pharmacokinetics, safety and efficacy profiles of drugs, and are of emerging clinical importance in the absorption and disposition of drugs. Currently, cell-based assay systems, such as polarized cell lines, primary cells, and stable cell lines, are the main in vitro methods suitable for studying drug interactions with SLC transporters. All these methods have disadvantages such as high cost, intensive cell culture maintenance, or lab-to-lab, donor-to-donor inconsistency. We have developed and validated a new transporter model for studying uptake clearance and drug-drug interaction (DDI) with SLC transporters. The complete portfolio includes all the regulatory authority (FDA, EMA) and industry recommended SLC transporters: Organic Anion-Transporting Polypeptide 1B1 and 1B3 (OATP1B1 OATP1B3), Organic Anion Transporters 1 and 3 (OAT1, OAT3), Organic Cation Transporters 1 and 2 (OCT1 and OCT2). The studies demonstrate that the novel cell-based system has many advantages over the current cell-based assay systems including high uptake activity, high signal-to-noise ratio, ease of use, batch-to-batch consistency and flexibility. We have optimized assay conditions with 96-well as well as 24-well plates for the high-throughput method.

P466. THRESHOLD CONCENTRATION OF EXCIPIENTS TO KEEP THE CACO-2 CELLS VIABLE FOR PERMEABILITY AND EFFLUX TRANSPORTER ACTIVITY ASSESSMENTS Qing Zhu, Bei-Ching Chuang, Haiyan Xia, Cindy Q. Xia, Suresh K. Balani and Mingxiang Liao Dmpk, Millennium Pharmaceuticals, Cambridge, MA In vitro permeability studies using human colorectal adenocarcinoma Caco-2 cells is a valuable model for the evaluation of absorption, disposition and drug-drug interactions (DDI). However, the utility of this model can be hampered by low aqueous

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solubility of the new chemical entity. A common solution to this problem is to employ various solubility-enhancing excipients. While excipients have been observed to cause alterations in permeability and transporter activity in vitro and in vivo, a thorough assessment for the possible effects of various types and concentrations of excipients in Caco-2 cells is needed. In the present study, the apparent permeability coefficient (Papp) of mannitol (a paracellular permeability marker), propranolol (a transcellular permeability marker), digoxin (a probe substrate of P-glycoprotein [P-gp]), and estrone sulfate (E3S, a substrate for breast cancer resistance protein [BCRP]) was assessed in the presence or absence of various types of excipients. Multiple concentrations of commonly used excipients, hydroxypropyl-b-cyclodextrin (HPbCD), sulfobutyl ether b-cyclodextrin (Captisol), dimethylacetamide (DMAC), dimethylformamide (DMF), propylene glycol (PPG), polysorbate 80 (Tween-80), labrasol, and d-alpha tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) were used to assess their effects on tight junctions, Papp, as well as P-gp and BCRP efflux rates in the Caco-2 cell model. Overall, a concentration-dependent disruption of cell integrity and a loss in efflux transporter functions was observed for all excipients tested in the Caco-2 cell model. The safe (no-effect) concentrations (v/v) determined for each excipient were 0.04%, 5%, 5%, 2.5%, 0.002%, 0.01%, 0.04%, and 0.0025% for HPbCD, DMAC, DMF, PPG, Tween-80, labrasol, capitisol, and vitamin E TPGS, respectively. Taken together, it is recommended that the concentration of excipients should not exceed the abovementioned safe (no-effect) threshold levels in Caco-2 permeability assays.

P467. THE MECHANISM OF BCRP INHIBITION BY CYCLOHEXANONE ANALOGUES OF CURCUMIN Jezrael L. Revalde1, Yan Li2, Lesley Larsen3, Rhonda J. Rosengren4 and James W. Paxton1 1 Pharmacology and Clinical Pharmacology, The University of Auckland, Auckland, New Zealand, 2School of of Interprofessional Health Studies, Auckland University of Technology, Auckland, New Zealand, 3New Zealand Institute for Plant and Food Research, Dunedin, New Zealand, 4Dept of Pharmacol, Univ of Otago Med Sch, Dunedin, New Zealand Using flow cytometry and cytotoxicity assays, we have previously demonstrated that the cyclohexanone analogues of the phytochemical curcumin (CUR): 2,6-bis((E)-2,5-dimethoxybenzylidene)cyclohexanone (A12); 2,6-bis((E)-4–hydroxyl–3methoxybenzylidene)cyclohexanone (A13); and 3,5-bis((E)-2-fluoro-4,5-dimethoxybenzylidene)-1-methylpiperidin-4-one (B11) inhibit the transport activity of breast cancer resistance protein (BCRP) (1). To clarify the mechanism of decreased transport, we investigated whether the analogues directly inhibit BCRP, or downregulate protein expression. Aim: To determine the mechanism of BCRP inhibition by curcumin analogues. Methods: Membrane vesicle uptake: Inside-out membrane vesicles from BCRP-transfected Sf9 insect cells were incubated for 20 min at 37 C with increasing concentrations of the analogues and radiolabeled methotrexate ([3H]MTX) in transport buffer. The reaction was stopped and the [3H]MTX uptake measured with a scintillation counter. Western blot: MDCKII/BCRP cells were incubated with noncytotoxic concentrations of the analogues and incubated for 72 h. Samples were subjected to denaturing SDS-PAGE, transferred to a PVDF membrane and detected with BXP-21 antibody and HRP-linked secondary antibody. Surface staining: MDCKII/BCRP cells were incubated for 24 h and 72 h with the analogues (5 mM A12/A13; 0.5 mM B11; 10 mM curcumin) or the PI3K inhibitors, LY294002 and wortmannin. Short-term (2 h) incubations at 20 mM were also conducted in Bewo and MDCKII/BCRP cells. Cells were then stained with biotin-conjugated 5D3 antibody and streptavidin-PE. Samples were resuspended and read with a flow cytometer. Results: The membrane vesicle assay showed direct inhibition of BCRPmediated [3H]MTX uptake by CUR, A12, A13 and B11 with IC50 values (nM) of 512.8 38.3, 324.3 59.1, 502.2 44.6 and 614.6 61.0, respectively. Using the western blot assay, neither CUR, nor its analogues caused any significant change in the total BCRP protein expression after the 72 h incubation. However, a significant 17.0 2.3% decrease in BCRP cell surface expression was observed with 0.5 mM B11 treatment over the same period (72 h but not 24 h). A short term exposure (2 h) to the analogues at high concentrations (20 mM) led to greater downregulation of BCRP surface expression. CUR and A13 decreased expression in MDCKII/BCRP cells by 21.0 3.5% and 36 2.1%, respectively; greater than that observed with 50 mM LY294002 (16.9 0.7%). In addition, A13 also significantly decreased surface expression in Bewo cells by 44.1 6.4%. Conclusions: Cyclohexanone analogues directly inhibit BCRP transport in cell-free inside-out membrane vesicles. The analogues do not change total protein expression of BCRP but can decrease cell surface expression in long term (72 h) and short term (2 h) exposure studies. Inhibition of BCRP transport by curcumin analogues likely result from direct inhibition with a possible contribution from decreased BCRP expression at the cell surface with increasing inhibitor concentrations.

Reference 1. Revalde J, Li Y, Rosengren R, Larsen L, Paxton J. The Modulatory Effects of Novel Curcumin Analogues on Xenobiotic ABC Transporters [abstract]. In: Proceedings of the 19th MDO and 12th European Regional ISSX Meeting; 2012 Jun 17–21; Noordwijk Aan Zee, the Netherlands. Abstract nr P271.

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P468. IMPACT OF OATP2B1 EXPRESSION ON CELL PROLIFERATION IN ER-POSITIVE BREAST CANCER


Jun Matsumoto1, Noritaka Ariyoshi2, Masahiro Sakakibara3, Takeo Nakanishi4, Ikumi Tamai4, Takeshi Nagashima3 and Itsuko Ishii2 1 Pharmaceutical Sciences, International University of Health and Welfare, Tochigi, Japan, 2Division of Pharmacy, Chiba University Hospital, Chiba, Japan, 3Department of General Surgery, Chiba University Graduate School of Medicine, Chiba, Japan, 4Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan Purpose: About two-thirds of cell population in breast tumors proliferate in estrogen-dependent manner. Estrone-3-sulfate (E3S) is thought to be a major estrogen precursor. Since E3S is a highly hydrophilic compound, the uptake of E3S into breast cancer cells is probably mediated by transporters, such as organic anion transporting polypeptide (OATP, SLCO) family members, which recognize and transport E3S into cells in vitro. Among those, OATP2B1 has been reported to be expressed in both normal and malignant breast tissues. The purpose of this study is to investigate the relationship between the expression of OATP2B1 and cell proliferation in estrogen receptor (ER)-positive breast cancer. Methods: To investigate difference in the expression level of SLCO2B1 mRNA between normal breast and breast cancer tissues, and associations of the mRNA expression with clinicopathological parameters, normal breast and cancer tissues from 10 same individuals and ER-positive breast cancer tissues from 35 individuals were collected from Chiba University Hospital. Cell-based assays were conducted in ER-positive human breast cancer-derived MCF-7 cells both with and without being OATP2B1 overexpressed. Stimulation of ER was studied using a reporter plasmid containing estrogen responsive element, whereas E3S uptake in each cell was determined with tritium-labeled E3S. This study was approved by the ethics committee of Graduate School of Medicine, Chiba University (Approved No. 100).Results and Discussion: Expression level of SLCO2B1 mRNA in breast cancer tissues was significantly higher than that in normal breast tissues obtained from the same individuals (p50.01). The expression level of SLCO2B1 mRNA was significantly correlated with Ki-67 labeling index (p50.01), which is utilized as a marker of proliferation activity of breast cancer cells in a clinical treatment. The expression of steroid sulfatase mRNA tended to be positively correlated with that of SLCO2B1 mRNA (p ¼ 0.058). Addition of E3S to culture medium caused stimulations of cell proliferation and ER was observed in MCF-7 cells. These stimulatory effects observed in MCF-7 cells overexpressing OATP2B1 were significantly higher than those in control cells. Similarly, E3S uptake in MCF-7 cells overexpressing OATP2B1 was also significantly higher than that in control cells. These data suggest that ER-positive breast cancer cells can uptake E3S, and OATP2B1 may play an important role in cell proliferation in ER-positive breast cancer. OATP2B1 expression in breast cancer tissues should be confirmed in further study. Conclusions: The present study suggests that OATP2B1 is involved in cell proliferation of ER-positive breast cancer via increasing amount of estrogen in cancer cells.

P469. RESCUE OF MISFOLDED MRP1 MUTANTS INVOLVING THE HIGHLY CONSERVED CHARGED AMINO ACIDS ARG1202 AND GLU1204 BY HETEROLOGOUS EXPRESSION IN INSECT CELLS Chunying Gao, Gwenae¨lle Conseil and Susan P.C. Cole Division of Cancer Biology & Genetics, Queen’s University, Kingston, ON, Canada The 190-kDa human Multidrug Resistance Protein 1 (MRP1) is a multifunctional ATP-binding cassette (ABC) transporter that mediates the cellular efflux of many drugs and organic anions, as well as reduced and oxidized glutathione (GSH). MRP1 has 17 transmembrane (TM) helices and two nucleotide binding domains (NBDs). Levels of MRP1 are reduced in mammalian (HEK) cells by 480% when the highly conserved Arg1202 and Glu1204 at the cytosolic interface of TM16 are replaced with oppositely charged amino acids, while substitutions with same charge or neutral amino acids have no effect.[1] On the other hand, transport activity is altered by neutral substitutions of Glu1204, but not Arg1202.[1] It appears that opposite charge mutations of R1202D and E1204K cause severe misfolding so that MRP1 is targeted for proteosome-mediated degradation. However, unlike many misfolded membrane proteins, R1202D and E1204K are resistant to rescue by exposure of HEK cells to subphysiological temperatures or chemical chaperones. Therefore, we used a heterologous Sf9 insect cell/baculovirus system to determine if the expression and function of R1202D and E1204K could be rescued in this more permissive system. We found that both R1202D and E1204K could be expressed in Sf9 cells at levels comparable to wild-type MRP1 (77% 23% and 90% 20%, respectively). In addition, both heterologously expressed mutants could be photolabeled by the prototypic MRP1 substrate [3H]leukotriene C4 (LTC4) and could transport LTC4 with a somewhat higher affinity (Km) than wild-type MRP1, although Vmaxvalues were reduced by 40–50%. Furthermore, an altered conformation of both NBDs in the R1202D and E1204K mutants was inferred from changes in fragmentation patterns after serial immunoblotting of limited trypsin digests of Sf9 membranes with Mabs against 5 different MRP1 epitopes. In conclusion, despite their resistance to rescue by conventional techniques in mammalian cells, the expression and function of R1202D and E1204K could be largely rescued in Sf9 insect cells. These and previous data[1] indicate that the amino acid requirements for position 1204 are more stringent than those for position 1202 to ensure proper folding and wild-type function of MRP1. Supported by CIHR 10519.

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Reference [1] Situ D, Haimeur A, Conseil G, et al. (2004) J Biol Chem 279, 38871–80

P470. THE INVOLVEMENT OF OATP1B-MEDIATED TRANSPORT IN THE OVERALL HEPATIC CLEARANCE OF HIV PROTEASE INHIBITORS IN MAN


Tom De Bruyn1, Bruno Stieger2, Patrick Augustijns1 and Pieter Annaert1 1 Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium, 2Department of Medicine, University Hospital, Zu¨rich, Switzerland This study aimed to elucidate the contribution of OATP1B transport proteins in the hepatic clearance of HIV protease inhibitors in humans. We measured the uptake of HIV protease inhibitors in OATP1B1/1B3-expressing CHO cells and extrapolated these to uptake clearance values in human hepatocytes by applying a relative activity factor between OATP-transfected cells and human hepatocytes. Metabolic clearance values of all HIV protease inhibitors were determined in pooled human liver microsomes. Based on the individual processes or on the combination of both uptake and metabolism, the in vivo hepatic clearance was calculated. Subsequently, we compared the calculated and published in vivo clearance values. Our results revealed an improved in vitro – in vivo correlation between predicted and reported clearance values when only uptake clearance values were used (r2 ¼ 0.71) as compared to prediction based on only metabolism (r2 ¼ 0.44). When both processes were taken into account, further improvement of the in vitro – in vivo correlation (r2 ¼ 0.76) was observed. Our data support the role of OATP1B mediated transport in the overall hepatic clearance of HIV protease inhibitors in human.



Author Index Aarons, Leon, P393 Aatsinki, S.M., P210 Abd-Rashid, Badrul Amini, P96 Acosta, Leonor, P403 Adegbola, Rachael A., P180 Adeogun, Aina O., P177, P180, P182 Adewuyi, G. O., P180 Agbulut, Onnick, P181 Agustsson, Ingolfur, P173 Ahami, Ahmed, P179 Ahlawat, Sarita, P68 Ahn, Li Young, P362 Ahn, Sung-Hoon, P147 Ai, Chunzhi, P252, P253 Aichholz, Reiner, P300 Aillon, Daniel, P137 Ajani, Olumide S., P183 Akimoto, Izumi, P164 Al Ali, Ahmad, P69, P277 Alelyunas, Yun W., P151 Aleo, Michael, S28 Alexander, Emma, P198 Alfadly, Waleed, P141 Allen, Christine, P419 Almstetter, Martin, P2, P11 Althurwi, Hassan, P98 Amaral, Adam, P85 Amberntsson, Sara, P297 Amini Shakib, Pouyan, P31 Andersen, Melvin, P233 Anderson, Caroline, P294 Andersson, Tommy B., SC3.1, P266 Ando, Hirotaka, P22 Andrews, Sarah, P198 Anjum, Fozia, P407 Annaert, Pieter, P54, P263, P441, P470 Anneveld, Bart, P21 Antenos, Monica, P77 Anwar, Haseeb, P407 Aquino, Giancarlo, P200 Arakawa, Hiroshi, P174 Argikar, Aneesh Arvind, P195, P255 Argikar, Upendra A., P85 Ariyoshi, Noritaka, P468 Arnold, Samuel L.M., P99 Arora, Vinod, P372 Arsenault, Brendan, P133

Arslan, Thao, P266 Ashraf, Tamima, P45 Augustijns, Patrick, P54, P263, P441, P470 Aungst, Bruce, P144, P145, P155 Aurell Holmberg, Ann, P153 Ausch, Christoph, P418 Avery, Lindsay B., P315 Aw, Chiu Cheong, P228, P281 Azadi, Behnam, P23 Aziziyeh, Adel, P26 Azzaoui, Fatima-Zahra, P179 Badal, Simone, P239 Badolo, Lassina, P412 Bae, Jin Kyung, P303, P366, P370 Bae, Myung Ae, P147 Baek, MyoungKi, P87 Bagrodia, Shubha, P378 Bai, April, P150 Baillou, Claude, P184 Bajna, Erika, P418 Balani, Suresh K., P466 Bandiera, Stelvio, P97 Bandzara, Temo, P246 Banerjee, Mayukh, P420 Banerjee, Nilasha, P419 Barbara, Joanna E., P190, P292 Barbier, Olivier, S49 Ba´rtı´kova´, Hana, P111, P237 Baumann, Marcus, P84 Baumgardner, Matthew, P371 Beaton, Melanie, P211 Beaune, Philippe, P69, P184, P277, P345 Bee´ry, Erzse´bet, P444, P445 Behnia, Kamelia, P272 Behnke, Dirk, P300 Belledant, Anaı¨s, P56 Bellemare, Judith, A6 Benade, Vijay, P129, P156 Bendayan, Reina, SC2.5, P45, P419 Ben Haddou, Souade, P1 Benoki, Satoshi, P396 Bentley, Mark C., P2 Bentley, Mark Charles, P11 Berry, Loren, P112

Bershas, David A., P313 Berthiaume, Luc G., P64 Bertho, Gildas, P69, P277 Betschart, Claudia, P300 Bhavar, Ganesh, P240 Bhoopathy, Sid, P230 Bhutani, Priyadeep, P110 Bhyrapuneni, Gopinadh, P156, P301 Bien-Moeller, Sandra, P400 Bigo, Cyril, S49 Bing, Yuntao, P94 Bing xin, Xiao, P95 Bin Jardan, Yousef A., P368 Bjorneboe, Lars H., P244 Bjo¨rquist, Petter, S56 Blackburn, Anneke, P408 Blanz, Joachim, P300 Blo¨meke, Brunhilde, P105 Blomme, Eric A.G., P284 Board, Philip, P408 Bode, Chris, P230 Boehme, Kathleen, P316, P374 Bogaards, Jan JP, P229 Bogers, Jan, P.A.M, P113 Boggavarapu, Rajesh Kumar, P129, P301 Bois, Frederic Y., SC3.2 Bolaji, Oluseye O., P367 Bolgar, Mark, P298 Bolleddula, Jayaprakasam, P314 Bolopion, Anne, P69, P277 Bonnel, David, P5, P377 Bordeaux, Kirk, P122 Boriss, Hinnerk, P188, P267, P316, P374 Borzillo, Gary, P378 Bosgra, Sieto, P264 Boudreau, Chantal, P384 Boukouvala, Sotiria, P65, P176 Bourdet, David L., P314 Bourgea, Joanne, P434, P435, P464, P465 Bousamra, Michael, SC1.2 Boysen, Gunnar, P107 Bracken, William, P312 Brantley, Eileen, P239 Bra¨sen, Jan H., P84

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Braspenning, Joris, P171 Bratton, Stacie M., P160, P276 Breitholtz, Katarina, P278 Brenneman, Debbie, P48 Brenner, Stefan, P417, P418 Bretz, Angela, P122 Bridson, Gary, P308, P309 Briolloti, Philippe, P218 Briolotti, Philippe, S58 Brock, Barry, P298 Brocks, Dion R, P359, P368 Brogin, Giandomenico, P373 Brooks, Bryan, P119 Brouwer, Kenneth R., P442 Brown, Caitlin, P187, P334 Brown, Karin D., P28 Browne, Edward, P228, P281 Bruchmueller, Henrike, P84 Bru¨ck, Susanne, P71 Bruel, Sandrine, P307 Buckley, David B., P75, P186, P201, P224, P292, P331, P428, P431 Budde, Thomas, P400 Bukhari, Shazia Anwer, P407 Buler, M., P210 Bumpus, Namandje´ N., P118, P287 P306, P315, P406 Bundgaard, Christoffer, P412 Burckart, Gilbert J., P361 Burdette, Doug, P293 Bures, Jan, P341, P343 Burk, Oliver, P236 Burkhardt, Britta, P269 Burrell, Richard, P324 Bushee, Jennifer, P85 Butcher, Darci, P453 Butcher, Neville J., S3 Butler, Phil, P302, P333 Buxhofer-Ausch, Veronika, P418 Byeon, Ji-Yeong, P349, P350, P351, P353, P354, P355, P357, P363 Cabanãs, Maria, P397 Cabanãs-Cortes, Maria, P225 Cabaton, Nicolas J., P320 Cai, Lining, P161 Cai, Yongming, P390 Cai, Youyou, P7 Calamia, Justina C., A7 Calderon, Emma S., P403 Cali, James J., P89, P202, P340 Callendrello, Alanna L, P185, P194 Canlet, Cecile, P320 Cantrill, Carina, P422 Capello, Astrid, P369 Cardinal, Kristen, P437 Cardozo, Pelaez, Fernando, P430 Carew, Michael W., P420, P454 Caron, Patrick, A3, P60 Carr, Robert A., P175

Cascorbi, Ingolf, P84 Cashman, John, P139 Cassella, James V., P117 Castellino, Stephen, S54, P313 Castillo-Tong, Dan Cacsire, P417 Cavaco, Isa, P347, P352, P364 Cavallari, Larisa H., S11 Ceckova, Martina, P446 Cen, Hanjing, P358 Cenacchi, Valentina, P373 Cerveny, Lukas, P429, P463 Cesari, Nicola, P373 Chai, Yifeng, P90 Chambers, Howard W., P338 Chambers, Janice E., P338 Chambliss, Kevin, P119 Chan, Chun Yip, A2 Chan, Connie, P389 Chan, Eric Chun Yong, A2, P228, P281 Chan, Jessalyn Mei Xuan, P281 Chan, Paul KS, P375 Chan, Thomas YK, P375 Chander, M.Trilok, P240 Chang, Qi, P18 Chang, Thomas K. H., P97 Chapman, Chiara M., P173 Charnis, Margarita, P173 Charrois, Gregory J., P454 Charron, Dominique, A4 Chatellier, Gilles, P345 Chatur, Shivaji S., P240 Chau, Nuy, P305 Chaudhry, Amarjit S., A7, P361 Chauret, Nathalie, P384 Chen, Emile, P123 Chen, Fu-Shih, P128 Chen, Jacob, P40 Chen, Jin, P122 Chen, Shujuan, S49 Chen, Sigeng, P139 Chen, Sylvia, P346 Chen, Taosheng, P402 Chen, Woei Shin, P228, P281 Chen, Xiaoyan, P460 Chen, Yong, P170 Chen, Yuan-Tsong, S23 Cheng, Longmei, P121 Cheong, Jonathan, P371 Chernov, Nikolay N., P209 Chi, Yong Ha, P158 Chiba, Kan, A5, P39, P416, P451 Chiba, Masato, P109 Chibale, Kelly, P134 Chigr, Fatiha, P179 Chillakur, Muddukrishna, P156 Chin, Young-Won, P303, P366, P370 Chladek, Jenni, P101 Cho, Joo-Youn, P20, P51, P295, P362, P448 Cho, Sun-Ah, P81

Cho, Sung Kweon, P448 Cho, Woon-ki, P147 Choi, Chang-Ik, P349, P363 Choi, Hyuk Joon, P158 Choi, Jae Ho, P329 Choi, Jong Min, P295 Choi, Man Ho, P51 Choi, Young Hee, P303, P366, P370 Chorghade, Mukund S., P173 Chou, Bilin, P40 Chouchana, Laurent, P345 Chow, Edwin C.Y., P114 Chow, Moses S.S., P375 Chowdhury, Swapan, P127 Christopher, L. J., P324 Chuang, Bei-Ching, P127, P466 Chugger, Sandeep, P382 Chukwuka, Azubuike V., P182 Chung, Jae-Yong, P362, P448 Chung, Moon-Koo, P392 Chung, Sophie, P168 Ci, Xiaoyan, P17, P142, P415 Cidlina, Antonin, P141 Cihalova, Daniela, P446 Cinatl, Jindrich, P402 Ciurlionis, Rita, P284 Clark, Robert J., P185 Clemens, Pamela. L., P324 Clewell, Harvey J., S39 Cobb, Shelby C., P404 Cohen, Lawrence H., P169 Cohrs, Christopher, P105 Cole, Susan, P.C., P469 Colizza, Kevin, P92 Coller, Janet K., A1 Colletti, Adria, P112 Colson, Pierre-Jean, P314 Commandeur, Jan, P113 Connors, Kristin, P119 Conseil, Gwenae¨lle, P469 Coons, Alison, P428, P431 Cooper, Kirsten, P434, P435, P464, P465 Couture, Fe´lix, A4, P346 Cox, Philip M., P406 Crespi, Charles L., P434, P435, P464, P465 Crittenden, Frank, P59 Croft, David, P24 Crouau-Roy, Brigitte, P65 Cruz-Hurtado, M., P38 Cui, Julia Yue, P327 Cummings-Lorbetskie, Cathy, P178 Currow, David, A1 Cvijic, Mary Ellen, P439 Czerwinski, Maciej, P224 D’Elia-Eckbold, Jaclyn, P144 D’Eustachio, Peter, P24 D’Hondt, Rebecca, P68


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Dahlinger, Dominik, P197 Dahlstrom, Jane, P408 Dai, Ziru, P252, P253 Dairou, Julien, P181 Dalton, James T., P437 Dalton, Rachel, P430 Damm, Georg, A12, P106, P266, P269 Damzal, Myrtha, P85 Daripelli, Saivishal, P129 Dates, Centdrika, P160, P276 Daujat-Chavanieu, Martine, S58, P218 Davies, Britton, P176 Davis, Willie, P239 Dayer, Jerome, P300 Dean, Brian, P6 De Avila, Manuela, P437 Debrick, Audrey, P2, P11 De Bruyn, Tom, P263, P441, P470 DeGroot, Jeroen, P21, P229 Deguchi, Tsuneo, P66 De´le´monte´, Thierry, P300 Delgoda, Rupika, P239 De Morais, Sonia, P46, P91, P175, P247 Deng, Yuzhong, P6, P432 Dennell, Stephen, P205 Depew, Kris, P133 Desaulniers, Daniel, P178 Deshmukh, Gauri, P40, P41 Desion, Kelly, P247 De Suza, Georges, P250 De Waziers, Isabelle, P69, P184, P277 Di, Li, P267 Diallo, Mohamed, P384 Dick, Ryan A., P334 Dickinson, Gemma, P393 Dickmann, Leslie J., P99 Ding, Caroline, P157 Ding, Xiao, P41 Ding, Xinxin, P47, P68, P74, P213 Dinwiddie, Darrell, P344 Dirven, Hubert, P317 Diry, Monique, P69, P277 Djuv, Ane, P241 Do, Minh Truong, P50 Dodd, Peter R., P103 Doktor, Stella, P247 Dolo, Laurence, P320 Dong, Guicheng, P94 Dong, Shiqi, P142 Donnelly, Danielle J., P23 Doshi, Utkarsh, P249, P332 Dovrtelova, Gabriela, P80, P102 Dragovic, Sanja, P113 Dragovich, Peter S., P389 Driscoll, James, P334 Drozdzik, Marek, P71 Du, Bowen, P119 Du, Feifei, P325, P381 Duche´, Daniel, P206 Duchoslav, Eva, P131

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Du¨chting, Sabrina, P197 Dueker, Stephen, P317 Duellman, Sarah, P340 Du, Plessis, J.B., P14 Dupret, Jean-Marie, P181 Duquet, Marie-Claude, P384 Duret, Cedric, S58, P218 Duriga, Nicole, P379 Dutertre, Quentin, P2, P11 Dutheil, Mary, P69, P277 Dvorak, Zdenek, P399 Dwyer, Mary, P139 Earnhardt, Nicole, P379 Ebeshi, Benjamin U., P367 Edgar, Kyle, P371 Edvardsen, Hege, P347, P352 Eguchi, Hiromi, P425 Eickholt, Emily, P430 Eilstein, Joan, P206 Einhorn, Shannon, P279, P286 Einolf, Heidi, P122 Eismin, Ryan, P293 Ejtehadi, Samad, P31 Ekdahl, Anja, P153 Ekins, Sean, P236 El-Kadi, Ayman O.S., P72, P73, P79, P98 El-Sherbeni, Ahmed A., P79 Elizondo, Guillermo, P225, P397 Elkin, Lisa L., P433, P439 Ellens, Harma, P123 Eloraby, Ghada, P55 Elshenawy, Osama H., P72, P73, P98 Emmen, Harry, P369 Eng, Heather, P267 Ertel, Christoph, P266 Escobar-Wilches, D.C., P38 Evanchik, Marc, P334 Evans, David C., P114 Fabre, Jean-Michel, S58 Fahmi, Odette A., P186 Fahmi, Tariq, P160, P276 Faivre, Jamila, S58 Fakis, Giannoulis, P65, P176 Falany, Charles N., P59, P67, P191 Fallon, John K., P57, P60 Fan, Huirong, P17, P142 Fan, Teresa W-M, SC1.2 Fan, Ying, S17 Fan, Yiping, S50, P361 Fang, Cheng, P74 Fang Rui, Cao, P95 Fanshawe, Bruce, S50 Farooq, Mohsin, P407 Farrow, Emily G., P344 Faustman, Elaine, P226 Faustman, Elaine M., P36 Fekete, Zsolt, P444

Feng, Bo, P438, P457 Feng, Hao, P35 Feng, Shan, P242 Ferguson, James, P131 Ferreira, Suzie, P85 Ferron, Pierre-Jean, P250 Fessard, Vale´rie, P282 Filion, Alain, A4 Fioni, Alessandro, P373 Fischer, Volker, P175 Fisher, Ciaran, P., P254 Fitch, William, P314 Fitzsimmons, Patrick, P119 Flores, Alegre, P397 Fok, B., P375 Fok, Benny, P330 Fontaine, Fabien, P130 Ford, Gill, P124, P299 Ford, Kevin, P104 Foster, Nicholas W., P25 Fox, Katie, P302, P333 Fox, Lisa G, P185 Fradet, Yves, A3 Francis, Sheena, P239 Francis Devaraj, FeAna, P160, P276 Franke, Andre, P84 Franklin, Ronald B., P28 Frechen, Sebastian, SC3.3, P197, P391 Fuhr, Uwe, P197, P391 Fujimoto, Mami, P52 Fukada, Yu, S50 Fukushima, Keizo, P159 Furihata, Tomomi, P39, P416, P451 Gabr, Raniah Q, P359, P368 Gaedigk, Andrea, P344 Gagne, Gerard D., P284 Galetin, Aleksandra, S38, P19, P335, P393, P447, P458 Galgamuwa, Ramindhu, P408 Gallegos, Richard, P309 Gan, Jinping, P324 Ganti, Vaishnavi, P394 Gao, Chunying, P469 Gao, Mian, P433 Gao, Weizhen, P76 Garefalaki, Vasiliki, P176 Garmer, Mats, P412 Garrigues-Mazert, Alexia, P206 Ge, Guang-Bo, P61, P62, P63, P204 Ge, Guangbo, P252 Geissler, Klaus, P219 Geoffroy, Stephanie, P382 George, Tony, P., P26 Gerbal-Chaloin, Sabine, S58, P218 Gerst, Nicolas, P424 Gertsch, Werner, P300 Gertz, Michael, P447, P458 Gesenberg, Christoph, P372 Ghosh, Avijit, P457

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Ghosh, Kaushik, P110 Gianella-Borradori, Athos, P317 Gien, Brad, P137, P386 Gil, Jose´, Pedro, P364 Gil Berglund, Eva, S42 Gilbert, Isabelle, P56 Gilbert, Jon, P268, P273 Gilibili, Ravindranath Reddy, P97 Gillam, Elizabeth M.J., P103 Giovanelli, Michael, P379 Glazier, Anthony, P198 Glenn, Anthony E., P176 Goebel, Carsten, P262 Gohdes, Mark, P133 Gomez-Ortega, Rocio, P356 Gonzalez, Frank J., S48 Gonzalez-Barbosa, Emmanuel, P225 Goodell, Ken, P201 Goodlett, David R., A2 Goosen, Theunis C., P57, P86 Gootzen, Arjen HG, P229 Gorycki, Peter D., P313 Gotoh, Saki, P223 Graham, Phil, P308 Grant, Denis M., P48, P339 Greer, Aleksandra K., P160, P276 Gremaud, Julia, P430 Greving, Matthew, P246 Grewal, Thomas, P138 Griffin, Robert J., P127, P169 Grillo, Mark, P326 Grimm, Scott, P207 Groër, Christian, P71 Groom, Hilary, P192 Groothuis, Geny, P113 Groseclose, M. Reid, S54 Grossi, Irma M., P101 Grossouw, Dimitri, P21 Grosveld, Gerard, S50 Groundwater, Paul, P138 Grubb, Mary, P231 Grube, Markus, P400 Gruensteidel, Johann, P417 Gu, Chungang, P133 Gu, Dan, S33 Gu, Dongxia, P154 Gu, Jingkai, P13, P70, P121, P136 Gu, Xiaomei, P324 Gu, Yi, P154 Gu, Yuan, P7, P15 Gu, Zheming, P35 Guan, Emily, P127 Guantai, Anastasia N., P367 Guengerich, F., Peter, P361 Guerrette, Zachariah, P36 Guillemette, Chantal, A3, A4, A6, P56, P60, P346 Gundert-Remy, Ursula, S40 Gunduz, Mithat, P92 Gunturi, Sitarama, P49, P251

Guo, Jian, P207 Guo, Lining, S47 Guo, Wei, P13 Guo, Yingying, P259 Gurau, Gabriela, P127 Ha, DongJin, P87 Hadi, Mackenzie, P113 Hakkola, J., P210 Halan, Vivek, P110 Hall, Stephen, P393 Hall, Stephen D., P259 Halladay, Jason, P104 Halme, Mia, P9 Halpert, James R., S20 Hals, Petter-Arnt, P317 Hamada, Jun-ichiro, P33 Hamilton, Gerhard, P219, P418 Hamm, Gregory, P5, P377 Hamrick, Michele, P437 Han, Hwa Jeong, P220 Han, HyeKyung, P20 Han, Seong Yon, P366 Han, Songhee, P452 Han, Yifan, P413 Han, Yong-Hae, P439 Hanapi, Nur Aziah, P243 Han Hsu, Helen, P35 Hanna, Daniel, P48 Hanna, Imad, P122 Hannan, Nicholas RF, S55 Harbeson, Scott, P309 Hargreaves, Elyse J., P78 Harper, Patricia A., P453 Harriman, Shawn, P92 Harris, Jennifer, P86 Harris, Stephen, P382 Harteneck, Christian, P236 Hartman, Jessica H., P107 Harvey, Mario, P346 Hashimoto, Mari, A5 Haskova, Pavlina, P140, P141, P341, P343 Hassen, Wafa, S58 Hatley, Oliver J. D., P19 Hatsis, Panos, P85 Haun, Randy, P276 Haw, Robin, P24 Hayakawa, Yoshitaka, P108 Hays, Amanda, P428, P431 He, Guiyuan, P63 He, Kan, P161 He, LS, P413 He, Qun, P136 He, Xiao-Xi, P18 He, Xin, P242 He, Xuehui, P245 He, Zheng, P289 Heeley, Thomas, P198 Heemann, Uwe, P84

Heffron, Timothy, P., P371 Hellum, Bent H., P203, P241, P244 Hemmrich-Stanisak, Georg, P84 Henderson, Colin, P115 Hendriks, Bart, S36 Hennen, Jennifer, P105 Henson, Claire, P382 Hermjakob, Henning, P24 Heron, Alain, P5, P377 Hersman, Elisabeth M., P118 Hewitt, Nicola J., P171 Heyward, Scott, P187, P205 Hibbs, David Edward, P138 Hidalgo, Ismael J., P230 Higashi, Richard M, SC1.2 Himmelfarb, Jonathan, P265 Hingorani, Gary, P., P28 Hintermann, Samuel, P300 Hirano, Mai, P166 Hirayama, Masamichi, P383, P395 Hiruma, Reiko, P459 Hisaka, Akihiro, P22, P167 Hisamichi, Kanehiko, P409 Hisanaga, Noriko, P440 Ho, Cy, P413 Ho, Thuy, P185, P194 Hochart, Guillaume, P377 Hoffman, James M., S10 Hofkens, Johan, P441 Hofman, Jakub, P221, P319 Hofmann, Ute, P106 Hogeveen, Kevin N., P282 Hollenberg, Paul F., S2 Hon, Susana, P114 Hong, Zhanying, P90 Hopper, David, P386 Hoque, Tozammel, P45 Horabik, Kris, P437 Horie, Toru, P52 Horiguchi, Aya, P52 Horton, Timothy M., P404 Horvat, Reinhard, P417 Hoshino, Motohiro, P166 Hou, Jie, P61, P62, P204 Hou, Wen-Bin, P388 Houk, Brett, P378 Houston, J. Brian, P335, P422, P447, P458 Hovington, He´le`ne, P56 Hsiao, Peng, P436 Hu, Jinping, P456 Hu, Lihong, P325 Hu, Steven, P82 Hu, Xin, P4, P12 Huang, George, P239 Huang, Jane, P423, P443 Huang, Min, P358 Huang, Yong, P423, P443 Huang, Yuxing, P325 Huband, Michael, P207

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Hubert, Cassandra L., P442 Hui, Xiong, P293 Huie, Keith, P117 Hunter, Sarah R., P214 Hurley, Jeremy, P433 Husain, Mainul, P178 Hutchinson, Mark R., A1 Hwang, Mi-Sun, P365 Iankova, Irena, S58 Ibor, Oju R., P182 Iida, Junichi, P164 Iikura, Minami, P383, P395 Iizasa, Tomohiko, P459 Ikarashi, Nobutomo, P100, P108, P411, P459 Ikeda, Toshihiko, P128 Ikenaga, Miho, P260 Ikenaka, Yoshinori, P116 Illes, Peter, P399 Im, So Hee, P147 Imahori, Yukari, P100 Imaizumi, Takahiro, P128 Imawaka, Haruo, P143 Impey, Gary, P131 Inaba, Asumi, A5 Ingelman-Sundberg, Magnus, P347, P352 Inoue, Hiroyuki, P270 Inoue, Katsuhisa, P450 Inoue, Tomoko, P449 Inoue, Toshio, P328 Inskeep, Philip, P311 Inui, Ken-Ichi, P167 Iseki, Ken, P461 Ishiguro, Akihiro, P166, P167 Ishii, Daisuke, P8 Ishii, Itsuko, P468 Ishii, Makoto, P100, P108, P459 Ishii, Seiya, P416 Ishii, Yuji, P109 Ishikawa, Masaki, P27, P37 Ishizuka, Mayumi, P116 Isin, Emre M., P278, P297 Ismail, Rusli, P96 Ismail, Sabariah, P243 Ismail, Zakiah, P96 Isoherranen, Nina, SC3.4, A7, P99 Isringhausen, Caleb, P428, P431 Ito, Katsuaki, P8 Ito, Kiyomi, P164, P167 Ito, Ryo, P39 Ito, Shinya, P453 Ito, Sumito, P450 Ito, Yukako, P34, P159, P376 Iwase, Yumiko, P128 Iwata, Daisuke, P166 Iwata, Yuko T., P270 Iwatsubo, Takafumi, P336 Iyer, Rama, P324

Drug Metab Rev, 2013; 45(S1): 270–282

Iyer, Ramaswamy A., P298 Izumi, Takashi, P66 Jackson, Kate, A1 Jacobs, Helen, P239 Jaeger, Walter, P417, P418 Ja¨ger, Walter, P219 Jahic, Mirza, P423, P443 James, Margaret O., P191, P310 Jamin, Emilien L., P320 Jang, Choon-Gon, P350, P351, P357 Jang, Himchan, P172 Jang, In-Jin, P20, P362 Jani, Ma´rton, P444, P445 Jansova, Hana, P341, P343 Jarrar, Yazun B., P81 Jarry, Gerard, P282 Jasmine, Lin, P112 Javed, Sadia, P407 Jenkins, Susan, P372 Jeong, Eun Ju, P392 Jeong, Hye-Eun, P365 Jeong, Hye Gwang, P50, P220, P329 Jeong, HyunJi, P87 Jeong, Hyunyoung A11 Jeong, Tae Cheon, P220 Jesadakultavee, Chatsiri, P271 Ji, Sang Chun, P20 Jia, Weiwei, P325 Jia, Xiaowei, P126 Jia, Yan, P252, P253 Jiang, Wenlei, P45 Jiao, Jian-Jie, P76 Jin, Sun Woo, P329 Jingzhou, Liu, P112 Jirasko, Robert, P111 John, Andrea, P105 John, Brian A, P120 Johnghar, Susan, P272 Johnson, Eric F., S17 Jones, Amanda, P437 Jones, Christopher, P19 Jones, David R., P3 Jonker, Derek J., P346 Josephy, P. David, P192 Juha´sz, Vikto´ria, P445 Jung, Bonnie, P437 Jung, Byung Hwa, P295 Jurica, Jan, P80, P102 Jurva, Ulrik, P278 Kadar, Eugene, P379 Kaddurah-Daouk, Rima, S46 Kajiwara, Sayuri, P411 Kalinina, Elena, P209 Kallipatti, Sanjith, P110 Kalvass, J. Cory, P46 Kamel, Amin, P92 Kamiichi, Atsuko, P39, P416 Kamijo, Takehiko, P416

Kanamarlapudi, Vijaya Bhargava, P301 Kanamori, Tatsuyuki, P270 Kandel, Benjamin, A12 Kandel, Benjamin A., P106, P236, S8 Kandel, Sylvie, P292 Kandikere, Vishwottam, P129, P156, P301 Kandilci, Ayten, S50 Kaneko, Chihiro, P461 Kaneko, Yosuke, P42 Kang, Jong Soon, P385 Kang-Jye, Chou, P389 Kapelyukh, Yury, P115 Karin, Michael, S49 Karlinsey, Molly Z., P313 Kasahara, Yoshinori, P8 Kato, Yukio, P167 Katori, Noriko, P222 Kaufman, Ilene, P423 Kawabata, Yoshihiro, P383, P395 Kawai, Yusuke K., P116 Kawamatsu, Shinya, P39 Kawamura, Akio, P52 Kawata, Minami, P116 Kaya, Yuuka, P128 Kazmi, Faraz, P201, P257, P258 Kazuki, Yasuhiro, A5, P66 Kegel, Victoria, P269 Keilholz, Laurie, P101 Kelley, Shana, S35 Kevin, Hogeveen, P250 Khairallah, Joelle, P23 Khan, Imran, P129 Khanal, Tilak, P329 Khera, Smriti, P326 Khetani, Salman, P233 Khojasteh, Cyrus, P104, P152 Kierzek, Andrzej M., P254 Kikuchi, Ryota, P46 Kim, Dong-Hyun, P81, P348 Kim, Donghak, P452 Kim, Eun Ji, P385 Kim, Ho Sook, P348, P365 Kim, Hyung Gyun, P50, P220 Kim, In Su, P146 Kim, Jae-Eun, P348 Kim, Je Hak, P158 Kim, Kyungbo, P452 Kim, Mi-Jung, P349, P350, P351, P357, P363 Kim, Min-Jung, P162 Kim, Min Hye, P348 Kim, Minyoung, P146 Kim, Mirim, P146 Kim, Richard B., A10, P211, P414, P462 Kim, Sang Kyum, A8, P172, P385 Kim, Sangsoo Daniel, P78 Kim, Se Hyung, P349, P350, P351, P353, P355, P357, P363 Kim, Seokuee, P448

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Kim, Woo Young, P81, P365 Kim, You Jin, P303, P366, P370 Kim-Kang, Heasook, P311 Kimoto, Emi, P232 Kingsmore, Stephen F., P344 Kirby, Brian, P201 Kirby, Gordon M., P77, P78 Kis, Emese, P444 Kishi, Tatsuo, P166, P167 Kishida, Satoshi, P39 Kitamura, Shigeyuki, P52, P328 Kitaoka, Satoshi, P411 Kitoh, Tomoko, P450 Klaassen, Curtis, S14, P327 Klameth, Lukas, P219, P418 Klarskov, Klaus, P55 Klein, Kathrin, A1, A12, P60, P360, S8 Klein, Marcus, P106 Klimko, Peter, P92 Knauer, Michael J., P414 Knipe, Jay, P372 Knorr, Arno, P2, P11 Knott, Katie, P107 Kobayashi, Kaoru, A5, P451 Kobuchi, Shinji, P34, P159, P376 Kodama, Susumu, P396 Koh, Kwi Hye, A11 Koh, Siew Kwan, A2 Koizumi, Takahiro, P461 Koizumi, Tomoko, P222 Kolur, Anil, P259 Komaba, Junji, P143 Komori, Hisakazu, P174 Komori, Kikuo, P234 Kon, Risako, P108, P411, P459 Konagaya, Akihiko, P387 Kondo, Tetsu, P143 Kondo, Yasuteru, P409 Kong, Ling-Ti, P18 Kontomina, Evanthia, P176 Koo, Tae-Sung, P392 Korfmacher, Walter A., SC4.1 Korprasertthaworn, Porntipa, P304 Korsten, John, P369 Kosa, Rachel E., P86 Kotseli, Barbara, P176 Kovalchuk, Nataliia, P74 Kovarikova, Petra, P140, P341, P343 Krajcsi, Peter, P444, P445 Krishnamurthy, Prasad, P110 Kristensen, Vessela, P347, P352 Kroemer, Heyo K., P400 Kroetz, Deanna, P148 Krol, Aurelie J., P69, P277 Krolikowski, Paul, P112 Kruzich, Paul, P386 Krzyzewski, Stacy, P233 Ku, Han Chen, P228 Kubota, Akira, P174 Kubow, Stan, P23

Kudo, Toshiyuki, P164 Kuitunen, Tapio, P9 Kulkarni, Ashutosh, P149, P150 Kumagai, Yuji, P27, P37 Kumar, Gondi, P170 Kume, Toshiyuki, P167 Kurihara, Atsushi, P66 Kurose, Kouichi, P222 Kurtz, Stacey, P386 Kusano, Kazutomi, P125 Kusuhara, Hiroyuki, P125, P387, P450 Kusunoki, Yoshiki, P108, P411, P459 Kuwayama, Kaori, P461 Kuwayama, Kenji, P270 Kuze, Jiro, P189 Kvita, Vojtech, P237 Kwak, Eun-Young, P147 Lacher, Sarah E., P430 Lacombe, Louis, A3, P56 Lade, Julie M., P306 Lai, Yurong, P232 Lalancette, Marc, A4 Lam, Zamas, P144, P145, P155 La Marca, Margherita, P238 LaMarr, William A., P248 Lambert, Diana Li, P224 Lamka, Jirı´, P111 Lanceleur, Rachelle, P282 Lane, Andrew N., SC1.2 Langaee, Taimour, P310 Langlois, Eric, P384 Lankester, David, P198, P294 Lapham, Kimberly, P57 Larsen, Lesley, P467 Larson, Bradley R., P202 Lash, Lawrence Harold, S30 Lau, Yau Yi, P91, P247 Laue, Grit, P300 Lauffenburger, Douglas A., S1 Laverdiere, Isabelle, A3, A4, P346 Le, Hoa, P371 Le, Jian, P90 Le, X. Chris, P420 Leal, Mauricio, P379 LeBlanc, Andre´, P1 LeCluyse, Edward, P233, P279 LeCluyse, Edward L., P286 Lee, Anthony J., P312 Lee, Bang-sub, P99 Lee, Chang Woo, P385 Lee, Choon-myung, P99 Lee, Chunja, P213 Lee, Howard, P362, P448 Lee, Hye-In, P349, P350, P351, P354, P357, P363 Lee, Ji-Yoon, A8, P172 Lee, Jieon, P20 Lee, Ji Young, P334 Lee, Jong-Hwa, P392

Lee, Joo-Yeon, P350, P351, P353, P354, P355, P357, P363 Lee, Joo Han, P158 Lee, Jung-Eun, P350, P351, P353, P354, P355, P357, P363 Lee, Kiho, P385 Lee, KiHo, A8 Lee, Kyeong, P385 Lee, Mey Y., P163 Lee, Moses, P192 Lee, Sang-Joon, P392 Lee, Sang Yoon, A8, P172 Lee, Seok-Yong, P349, P350, P351, P353, P354, P355, P357, P363 Lee, Su-Jun, P81, P361, P365 Lee, Vincent H.L., P330, P375 Lee, Wooin, P452 Lee, Yuan Kun, P228, P281 Lee, Yu Kyung, P303 Leech, Hayley, P124 Leeder, J. Steven, P344 Lefeldt, Stefanie, P84 Legouffe, Raphael, P5, P377 Leingartner, Karen, P178 Lekhooa, Makhotso Rose, P14 Le Marchand, Loı¨c, S33 Lemoine, François M., P184 Lenehan, John, A10 Leng, Xiuyu, P358 Lepeigneux, Ce´line, P69, P277 Lereaux, Guillaume, P206 Lerman, Caryn, P26 Lescaille, Ge´raldine, P184 Leslie, Elaine M., P64, P420, P454 Leuner, Kristina, P236 Leung, Louis, P57 Le´vesque, E´ric, A3, A4, P56, P346 Le´vesque, Jean-François, P384 Li, Albert, P., P212, P249, P332 Li, Chuan, S24, P325, P381 Li, Jie, P94 Li, Ke, P35 Li, Kexin, P4, P12 Li, Lei, P47 Li, Li, P325, P381 Li, Ling, P196 Li, Min, P4 Li, Na, P434, P435, P464, P465 LI, Qin, P76 Li, Quansheng, P17, P142 Li, Rubin, P4 Li, Rui, P457 Li, Wei, P17 Li, Xiuli, P460 Li, Xue, P456 Li, Yan, P456, P467 Li, Yang, P4, P43 Li, Yazhuo, P17, P142 Li, Yibai, A1 Li, Zheng, P455


276

Liang, Guangyi, P17, P142 Liang, Xiaorong, P389 Liao, Cangsong, P280 Liao, Mingxiang, P127, P466 Liao, Yong-Hong, P18 Licari, Giovanni, P16 Liederer, Bianca, P6, P40, P41 Liederer, Bianca M., P152, P165, P389, P432 Liguori, Michael J., P284 Lim, Jae Soo, P158 Lim, Kyoung Soo, P20, P362 Lim, Su Jeong, P348 Lin, Ge, S25, P280, P288, P321, P322 Lin, Jian, P57, P267 Lin, Yvonne S., P36, P226 Lindon, John C., SC1.1 Ling ti, Kong, P95 Lin hu, Ye, P95 Linnet, Kristian, P318 Litlwiler, Kevin S., P28 Liu, Chang-xiao, P17, P142, P388 Liu, Changxiao, P15, P242, P390, P415 Liu, Fei, P455 Liu, Houfu, P43 Liu, Jingrui, P13 Liu, Jinrong, P175 Liu, Junhua, P325 Liu, Lian, P32 Liu, Ning, P175 Liu, Shengmin, P245 Liu, Wenbin, P145, P155 Liu, Xiao-dong, P44 Liu, Xin-Min, P18 Liu, Xingrong, P41 Liu, Xinwei, P325 LIU, Yanxia, P76 Liu, Yingmei, P148 Liu, Yong, P253, P261 Liu, Zhao-Ming, P204 Liu-Kreyche, Peggy, P324 Lobach, Alexandra R., P290 Loberg, Lise, P312 Loewen, Greg, P428, P431 Loiseau, Pascale, A4 Longo, Vincenzo, P238 Lo´pez-Gonza´lez, M.L., P38 Lordi, Alfred, P144 Lorello, Leslie, P379 Loriot, Marie-Anne, P345 Lou, Jianshi, P76 Louie, Steven W., P436 Lu, Hong, P327 Lu, Jingtao, P279, P286 Lu, Rong, P15 Lu, Sijie, P43 Lu, Xiaodan, P148 Lu, Xiufen, P420 Lu, Yasong, P438 Lu¨bberstedt, Marc, P266

Drug Metab Rev, 2013; 45(S1): 270–282

Lubicka, Danuta, P85 Ludovic, Le Hegarat, P250 Luna, Ana Laura, P403 Luo, Gang, P437 Luu, Kenneth, P378 Luzietti, Rick, P86 Lv, Xia, P62 Ly, Justin Q., P389 Ly, Van T., P298 Lyon, Kevin C., P224, P428, P431 Ma, Bin, P321 Ma, Dongping, P89, P202 Ma, Jin Yeul, P172 Ma, Liang-peng, P289 Ma, Shuguang, P152 Ma, Xiao-Chi, P61, P62 Machacek, Miloslav, P141, P341 Mackenzie, Peter I., P58, P305 Mackova, Eliska, P343 Mackova -, Potuckova, Eliska, P140 Maeda, Hiroshi, S34 Maeda, Kazuya, P167, P387, P440 Maejima, Takahiro, P409 Maekawa, Keiko, P27, P37, P167 Maganã-Aquino, Martin, P356 Magnan, Remi, P444, P445 Mahendran, Ratha, P228, P281 Maher, Jonathan M., P284 Mair, Stuart, P299, P317 Majumdar, Tapan, P122 Mak, Joon Wah, P96 Mak, Marvin, P413 Makhseed, Saad, P141 Malcekova, Beata, P221, P319 Mamaril-Fishman, Donna B., P313 Man, Huizi, P252, P253 Mandlekar, Sandhya, P110 Mann, David A., P279, P286 Mano, Nariyasu, P409 Manoharan, Arun kumar, P129 Mansor, Sharif Mahsufi, P243 Mantis, Nicholas J., P68 Manwaring, John D., P262 Marathe, Punit, P272 Marcek, John, P379 Marensi, Vanessa, P64 Margaillan, Guillaume, P60 Ma´rialigeti, Ka´roly, P176 Marion, Tracy L., P442 Mark, Hannah, P29 Marsh, Kennan, P175, P312 Ma˚rtensson, Andreas, P364 Martin, Elyette, P2, P11 Martinez-Guzman, Carmen, P225 Marting, Zechariah, P138 Maruf, Abdullah Al, P285 Masters, Andrea R., P3 Masters, Jeffrey N., P424 Matheson, Rommel, P117

Matouskova, Petra, P221 Matsabisa, M.G., P14 Matsui, Hitoshi, P234 Matsui, Satomi, P449 Matsumoto, Ichiro, P143 Matsumoto, Jun, P468 Matsumoto, Shogo, P451 Matsumura, Yasuhiro, P222 Matsunaga, Norikazu, P260 Matsushima, Eiji, P193 Matsuura, Masaki, P409 Matthews, Jason, S5 Matthieu, Cećile, P181 Maurel, Patrick, S58, P218 Maw, Holly, P405 McDonald, Thomas S, P267 McDonough, Caitrin W., P310 McDuff, François-Olivier, P55 McEwen, Andrew, P124, P299, P382 McKenzie, Donald, P437 McLachlan, Andrew J., P304 McLaughlin, Lesley A., P115 McMahon, Mike, P115 McNally, Jim, P379 McPhee, Fiona, P372 McVay, Michael, P233 Medellıń-Garibay, Susanna, P356 Medina-Bolivar, Fabricio, P160, P276 Meech, Robyn, P58 Meek, Edward C., P338 Megaraj, Vandana, P47, P74 Meireles, Maria-Helena, P320 Meisenheimer, Poncho, P89, P340 Mejia-Garcia, Alejandro, P225 Meńard, Vincent, P60 Meng, Lin, P76 Mera, Yukinori, P383, P395 Mercola, Mark, P139 Metushi, Imir G., P30, P286 Metzger, Ute, P106 Meyers, Charles, P122 Meyer zu Schwabedissen, Henriette, P400 Mezine, Igor, P230 Michaelis, Martin, P402 Milacic, Marija, P24 Milan-Segovia, Rosa del Carmen, P356 Miller, David S., S15 Miller, Grover, P., P107, P404 Miller, Naomi, P419 Miller, Neil A., P344 Miller, Vaughn, P., P248, P249 Millham, Robert, P378 Mimura, Ayako, P459 Minchin, Rodney F., S3 Minekus, Mans, P21 Miners, John O., P304, P305 Mitsuoka, Toshinari, P167 Miura, Shin-ichi, P167 Miyashita, Aiji, P336

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Mizuguchi, Hiroyuki, S57 Mizuguchi, Misa, P451 Mizuno, Hideaki, P441 Mjambili, Faith, P134 Mochizuki, Tsutomu, P8 Moeller, Timothy, P187 Moeller, Timothy A., P202 Moghaddam, Mehran, P149, P150 Mohamed, Ahmed N., P3 Mohamed, Tarek M. S., A9 Mohemmad, Qurish, P296 Mohutsky, Michael A., P259 Molna´r, E´va, P445 Mommeja-Marin, Herve, P101 Momose, Yasunori, P128 Monzel, Judith V., P400 Moon, Kyoung-Sik, P392 Moore, J. Bernadette, P254 Moore, Simon A, P120 Morettoni, Luca, P130 Morgan, Adam, P308, P309 Morgan, Edward T., P99 Morgan, Larry J., P77 Mori, Takanori, P174 Moriyama, Yoshinori, P450 Mostafa Zadeh, Amrollah, P31 Mosure, Kathleen W., P433 Mosure, Kathy, P439 Motohashi, Shinichiro, P416 Mourot, Annick, P282 Muddana, Nageswara Rao, P156 Mukadam, Sophie, P104, P165 Mukoyoshi, Masanobu, P8 Mulgaonkar, Aditi, P168 Mungenast, Felicitas, P417 Mu¨nst, Bernhard, P171 Muppidi, Akhil Babu, P156 Murata, Yukiko, P174 Murayama, Mayumi, P37 Murayama, Norie, P10, P93, P401 Musib, Luna, P6 Musmade, Kranti, P., P240 Musmade, Prashant B., P240 Myhren, Finn, P317 N’Go, Pacoˆme Kouadio, P179 Nafarzadeh, Shimae, P31 Nagai, Miki, P416 Nagai, Naomi, P166, P167 Nagai, Shinjiro, S50 Nagar, Swati, P394 Nagar, Swati V., P255 Nagashima, Takeshi, P468 Nagaya, Yoko, P125 Nagoya, Chika, P411 Najimi, Mohamed, P179 Nakada, Mitsutoshi, P33 Nakada, Tomohisa, P440 Nakamura, Masato, P93, P401 Nakanishi, Takeo, P174, P260, P468

Nakashima, Emi, P421, P425, P426, P427 Nakayama, Kazuki, P143 Nakayama, Shouta M. M., P116 Naranmandura, Hua, P420 Narasimhan, Narayana, P296 Narayanan, Ramamurthi, P49 Nardi, Regina, P405 Naritomi, Yoichi, P52, P135 Narjoz, Ce´line, P345 Natsui, Kiyoshi, P449 Ndubaku, Chudi O., P371 Neale, Geoff, S50 Negishi, Masahiko, P223 Nehmer, Amy, P437 Neubert, Hendrik, P57 Neumanova, Zuzana, P429, P463 Ng, Ricky, P114 Ng, Winnie, P274 Ngai, Lk, P375 Ngan, MT, P375 Nguyen, Dung, P195 Nguyen, Nghia, S49 Nguyen, Sinh, P148 Nguyen, Sophia, P308, P309 Ni, Liang, P154 Nichols, John, P119 Nicolaı¨, Johan, P54 Nilsen, Odd Georg, P203, P241, P244 Ning, Jing, P61, P62, P204 Niosi, Mark, P57 Nirogi, Ramakrishna, P129, P156, P301 Nishi, Koji, P128 Nishimura, Sachika, P425 Nishimura, Tomohiro, P421, P425, P426, P427 Nishimuta, Haruka, P335 Nitta, Shin-ichiro, A5 Niu, Wei, P381 Njoroge, Mathew, P134 Nobuyuki, Sugioka, P159 Nogova, Lucia, P391 Noij, Theo, P369 Nouraldeen, Amr, P196 Novakova, Veronika, P141 Novalen, Maria, P26 Noveń, Anna, P297 Novichkova, Maria, P209 Novotna, Eva, P221, P319 Nozaki, Yoshitane, P125 Nun˜ez-Ruiz, Aleida, P356 Nunn, Brook L., A2 Nu¨ssler, Andreas K., P269 O’Brien, Peter J., P285 O’Brien, Zhihong, P149, P150 O’Connell, Jonathan, P439 Obach, R. Scott, P86 Obringer, Cindy M., P262 Obuchi, Wataru, P33

Ochiai, Wataru, P100, P108, P411, P459 Oda, Kenji, P426 Ofner, Silvio, P300 Ogawa, Kei, P440 Ogawa, Mikio, P143, P260 Ogilvie, Brian, P101 Ogilvie, Brian W., P190, P201 Ogimura, Eiichiro, P449 Ogunshe, Adenike O., P180 Ogura, Jiro, P461 Oh, Haejin, P448 Oh, Kyung-Suk, P81 Oh, Kyung Hoon, P348 Oh, Soo Jin, A8, P172, P385 Ohmori, Shigeru, P10 Ohno, Yasuo, P167 Ohta, Shigeru, P52, P328 Ohtsuki, Sumio, P33, P42 Okaniwa, Takehiro, P100 Okawa, Hoshimi, P409 Okolotowicz, Karl, P139 Okubo, Maho, P10 Okuda, Haruhiro, P222 Ola-Davies, Olufunke E., P177, P183 Olivero, Alan G., P371 Oloyede, Omotade I., P342 Olsen, Jacob A., P199 Olson, Jean, P148 Olson, Loren, P131 Olszewski, Ulrike, P219 Oluka, Margaret, P367 Omogbemi, D. E., P180 Ong, Chin Eng, P96 Opasina, Oladayo F., P177 Orozco, Christine C., P57 Osada, Tomoharu, P234 Oshimura, Mitsuo, A5, P66 Oswald, Stefan, P71, P400 Otterson, Linda, P207 Otwell, Clayton J.M., P75, P331 Ouellet, Daniele, P313 Owen, Albert J., P230 Oyeyemi, Matthew O., P183 Ozaki, Mayuko, P425 Ozawa, Shogo, P167 Ozbal, Can, P248 Paik, Soo Heui, P158 Palacharla, Raghava Chaudary, P301 Palamar, Safet, P122 Paliwal, Pankaj, P156 Pamelard, Fabien, P5 Pan, Rui-Le, P18 Pan, Xian, A11 Pan, Yan, P96 Pang, Jodie, P371 Pang, K. Sandy, P114 Pappan, Kirk, P27 Parant, John, P59 Parikh, Nikunj, P249


278

Park, B. Kevin, S31 Park, Jihye, P146 Park, Kyung-Jin, P323 Park, So-Young, P349, P350, P351, P357, P363 Parkinson, Andrew, P190, P257, P258 Pasanen, Markku, P9 Pascual, Xavier, P130 Pascussi, Jean-Marc, P218 Patillo, Peter, P137 Patten, Christopher J., P434, P435, P464, P465 Patterson, Sarah, P144 Pavek, Petr, P398 Paxton, James W., P467 Pearson, David, P300 Pelkonen, Olavi R., S27 Peng, Lai, P216 Peng, Lana, P122 Peng, Lijuan, S33 Penski, Jette, P71 Perdu, Elisabeth F., P307 Perkins, John, P6 Perry Jr, Martin D., P404 Pesonen, Maija, P9 Peters, Scott, P137 Phillips, Brian, P36 Pin, Christopher, P211 Pineau, Brigitte, P345 Ping, Jie, P32, P289 Piquette-Miller, Micheline, S16 Pirmohamed, Munir, S9 Plant, Nick J., P254, P256, P410 Plise, Emile, P389 Plise, Emile G., P371, P432 Plumb, Jonathan, P458 Polasek, Thomas M., P304 Ponnamaneni, Ranjith kumar, P129 Pope, Chad, P216 Portales-Perez, Diana, Patricia, P356 Porter, James, P133 Prakash, Satya, P23 Prather, Paul L., P160, P276 Prentiss, Pilar L., P186 Press, Randall, P133 Prueksaritanont, Thomayant, S43 Puccini, Paola, P200, P373 Purcell, Chad, P29 Pyrek, Sebastian, P160, P276 Qazi, Sohail S., P64 Qi, Chang, P95 Qian, Shuai, P413 Quinney, Sara K., P3 R, Narayanan, P251 Radominska-Pandya, Anna, P160, P276 Raghavan, Nirmala, P324 Ragueneau-Majlessi, Isabelle, P168 Rahatgaonkar, Anjali M., P173

Drug Metab Rev, 2013; 45(S1): 270–282

Rahavendran, Ravi, P57, P378 Rainville, Paul D., P151 Rajaraman, Ganesh, P91 Rajnai, Zsuzsanna, P444 Ramachandran, Sarada Devi, P171 Ramagiri, Suma, P131 Ramanathan, Ragu, SC4.2, P131 Ramarao, Manjunath, P110 Rami, Bhadresh, P110 Ramos, Jeanne, S58 Ramstein, Philippe, P300 Rasmussen, Henrik B., P318 Raughley, Bethany, P230 Raulet, Edith, P218 Rautio, Jari, P188 Ray, Tapan, P122 Rehmel, Jessica F., P259 Reiner-Concin, Angelika, P418 Ren, Hongcan, P154 Renaud, Helen J., P327 Renders, Lutz, P84 Rettie, Allan E., A7 Revalde, Jezrael L., P467 Rhamani, Roger, P250 Riccardi, Benedetta, P200 Richards-Peterson, Lauren E., P313 Richardson, Des R., P140 Richardson, Samantha, P150 Richter, Marco, P266 Riddick, David S., P213, P214, P217 Rieger, Jessica K., A12 Riha, Juliane, P417, P418 Rizvi, Syed A. A., P127 Roberge, Joannie, A6, P56 Roberts, Ellen, P247 Roche, Denis, P345 Rode, Anja, P115 Roden, Dan M., S12 Rodes, Laetitia, P23 Rodrigues, David, P272, P439 Rodrigues-Lima, Fernando, P181 Rodriguez-Pinal, Christian, P356 Rogers, Anne, P58 Rogers, Robin D., P127 Roggenbeck, Barbara A., P454 Roh, Jaroslav, P140 Rohde-Johnson, Candace, P137 Rohn-Glowacki, Katie Jo, P191 Romano-Moreno, Silvia, P356 Roos, Marcel, P84 Rose, Kelly, P233 Rosengren, Rhonda J., P467 Rostami-Hodjegan, Amin, SC2.4, P19, P393 Rothe, Helga, P262 Rothfels, Karen, P24 Rotter, Charles J., P438 Rouleau, Me´lanie, A3, A6 Rowan-Carroll, Andrea, P178 Rowland, Andrew, P304

Roy, Rene´, P1 Ruan, Jianqing, P280, P288, P322 Ruan, Jinxiu, P455 Rumak, Vladimir S., P275 Sabally, Kebba, P23 Sabbagh, Audrey, P65 Sabesan, Vijay, P314 Sabova, Michaela, P80, P102 Sadeghi Ekbatan, Shima, P23 Safr, Miroslav, P221 Sahi, Jasminder, P43 Sai, Kimie, P222 Sai, Yang, P154 Saijo, Nagahiro, P222 Saito, Kaku, P193 Saito, Kosuke, P27, P37 Saito, Masayo, P411 Saito, Yoshiro, P27, P37, P167, P222 Sakai, Yasuyuki, P234 Sakakibara, Masahiro, P468 Sakuma, Takeo, P132 Sakurai, Kaori, P215 Sakurazawa, Ai, P128 Salazar-Gonza´lez, Raul, P356 Sa¨ll, Carolina, P447 Sallustio, Benedetta C., P16 Salminen, William, P443 Salo, Heta, P9 Salphati, Laurent, P371, P432 Salvadori, Michela, P200 Sampathkumar, Aparna, P281 Sana, Theodore, SC1.3 Sano, Yuichiro, P427 Sanoh, Seigo, P52 Sanseau, Philippe, S22 Santone, Elizabeth A, P194 Santone, Elizabeth A., P185 Santone, Kenneth, P372 Saruta, Kanako, P100 Sasaki, Shunichi, P461 Sathirakul, Korbtham, P271 Sato, Koya, P52 Sato, Masanobu, P166, P167 Sawada, Jun-ichi, P222 Scheer, Nico, P115 Scheffler, Matthias, P391 Schlicht, Kari E., P248, P249 Schmarderer, Christoph, P84 Schneider, Christin, P266 Schoelm, Madeleine, P418 Schubert, Ernest M., P313 Schuetz, Erin G., A7, P361 Schuetz, John D., S50 Schultz, Rachel NV, P205 Schulz, Antje, P266 Schulz, Theresa, P269 Schulze, Franziska, P266 Schwab, Matthias, P360 Scialis, Renato, P457

279


DOI: 10.3109/03602532.2013.868114

Scola, Paul, P372 Scott, Emily E., S4 Seehofer, Daniel, P269 Seeto, Dena, P29 Segall, Matthew D., P25 Segre, Gino, P88, P246 Seguin, Johanne, P184 Seidel, Albrecht, P105 Sekiya, Keisuke, P174 Selvakumar, Sindhuja, P110 Selvam, Sabariya, P110 Sempels, Wouter, P441 Senda, Naoto, A5 Senn, Tauri, P226 Senoo, Yuya, P37 Seo, Hyewon, P147 Seo, Ji Eun, P348 Seo, Won Sik, P158 Serra, Blanca, P130 Sestak, Vit, P140 Seto, Carmai, P132 Shackman, Holly, P298 Shadid, Mohammad, P127 Shahzadi, Andleeb, P407 Shan, Qi, P388 Shang, Jianing, P76 Shaw, Iain, P299, P317 Shaw, Joanne, P302 Shebley, Mohamad, P91 Shen, Ding Ren, P439 Shen, Helen L., P144, P145, P155 Shen, Wenjin, P390 Sherwin, Catherine M.T., P430 Shi, Aixin, P4, P12 Shi, Meiyun, P121, P136 Shi, Qiang, P443 Shi, Qin, P161 Shiao, Tze Chieh, P1 Shibakawa, Kimio, P143 Shim, Eon-Jeong, P81 Shim, Hyun Joo, P323 Shimada, Miki, P409 Shimizu, Makiko, P401 Shimosegawa, Tooru, P409 Shimozato, Osamu, P416 Shin, Beom Soo, P158 Shin, Dongseong, P362 Shin, Ho-Jung, P348 Shin, Jae-Gook, P81, P348, P365 Shin, Kwang-Hee, P20, P51 Shingaki, Tomotaka, S52 Shinohara, Marie, P234 Shirasaka, Yoshiyuki, A7 Shivasami, Aravind, P58 Shizu, Ryota, P396 Shou, Wilson, P439 Shroads, Albert L., P310 Shu, Pan, P35 Shu, Yue-Zhong, P231 Shultz, John, P340

Shur, Irina N., P275 Si, Duanyun, P7, P15, P17, P142, P415 Siegmund, Werner, P71 Sier, Joanna H., P256, P410 Sierra-Santoyo, A., P38 Silva, Jose, P114 Sim, Edith, S18, P65, P176 Sim, Sarah, P347, P352 Simunek, Tomas, P140, P141, P341, P343 Singh, Vijay, P49, P251 Sinz, Michael, P372 Siu, Y. Amy, P208 Skagen, Kasse, P430 Ska´lova´, Lenka, P111, P237 Skarka, Adam, P319 Skarydova, Lucka, P221 Skordos, Konstantine W., P195 Slatter, J. Greg, S44 Sleno, Lekha, P1, P23 Slocum, Kelly, P133 Slon, Barry, A1 Smalley, James, P272 Smiles, Jonathan, P408 Smith, Philip C., P57, P60 Smith, Sherri, P133 Smith, William R., P442 Smutny, Tomas, P398 Snoeys, J., P441 Soars, Matthew, SC2.1, P439 Soars, Matthew G., P433 Socie´, Ge´rard, A4 Sodhi, Jasleen, P104 Somers, Graham I., P458 Somogyi, Andrew A., A1 Song, Jin Sook, P147 Song, Zihui, P390 Sorrentino, Brian, P., S50 Sota, Shigeto, P401 Speiser, Paul, P417 Spilker, Mary, P378 Spriggs, Franklin, P379 Squires, Jessica, P137 St. Claire III, Robert L., P442 Staben, Steven T., P371 Stacpoole, Peter W., P283, P291, P310 Stambergova, Hana, P221 Stanley, Forrest A., P292 Stariat, Jan, P140 Stauber, Jonathan, P5, P377 Staud, Frantisek, P429, P446, P463 Ste-Marie, Line, P384 Stein, Lincoln, P24 Steubel, Dominik, P84 Stevens, Lloyd, P299, P317 Stieger, Bruno, P470 Stout, C. David, S17 Stratton, Tim, P157 Stresser, David M., P185, P194 Strom, Stephen C., P361

Stuchlikova, Lucie, P111, P237 Su, Weiguo, P154 Subramanian, Murali, P110 Subramanian, Ramkumar, P156 Suemizu, Hiroshi, P93, P401 Suenaga, Kiyomi, P459 Sugamori, Kim S., P48, P339 Sugden, Kent, P430 Sugihara, Kazumi, P52 Sugiyama, Kiyoshi, P100, P108, P411, P459 Sugiyama, Yuichi, P387, P440, P450 Suhar, Tom, P247 Suhara, Tetsuya, S53 Sukrutharaj, Sunil, P110 Sulcova, Alexandra, P80, P102 Suleiman, Ahmed A., P391 Sultan, Sirag, P417 Sun, Hao, S19 Sun, Heping, P121 Sun, Jun, P91 Sun, Kefeng, P41 Sun, Xiao-Yu, P63 Sun, Xiaobo, P245 Sun, Yantong, P13, P121, P136 Sun, Yuchen, P416 Surapaneni, Sekhar, P170 Suzuki, Hiroshi, P22, P167 Suzuki, Noriko, P459 Suzuki, Yuki, P451 Svoboda, Martin, P219 Swanson, Sue, P247 Sweet, Suzanne, P317 Sydow, Konrad, P197 Szota´kova´, Barbora, P111, P237 Tabata, Kenji, P135, P424 Tachikawa, Masanori, P33, P42 Taguchi, Rina, P164 Tai, Guoying, P123 Taimi, Mohammed, P268, P273 Tajima, Masataka, P100 Tajima, Yoko, P37 Takada, Hidenori, P414 Takada, Kanji, P34, P159, P376 Takahashi, Lori H., P117 Takahashi, Miki, P396 Takahashi, Ryan, P152 Takano, Ryohji, P401 Takashima, Tadayuki, S52 Takehara, Shoko, A5, P66 Takenaka, Osamu, P125 Takeuchi, Yuichi, P164 Takusagawa, Shin, P336 Takwi, Apana Agha L., P402 Tamai, Ikumi, P174, P260, P468 Tamouza, Ryad, A4 Tamura, Kouichi, P424 Tamura, Tomohide, P222 Tang, Huaping, P439


280

Tang, Hui, P114 Tang, Xianli, P154 Tang, Yang, P149, P150 Tarantini, Adeline, P282 Ta¨rning, Joel, P364 Tartour, Eric, P184 Tateno, Chise, P52 Tawara, Tomohiro, P425 Teffera, Yohannes, P112 Teft, Wendy A., A10 Templin, Markus, P106 Teramura, Toshio, P135, P424 Terasaki, Tetsuya, P33, P42 Tetsuka, Kazuhiro, P424 Teˆtu, Bernard, A3, P56 Thakkar, Nilay, P452 Thalhammer, Theresia, P219, P417, P418 Thasler, Wolfgang E., P236 Theodoratos, Angelo, P408 Thomas, Holly, P59 Thomas, Maria, P106, S8 Thomsen, Ragnar, P318 Thorsell, Annika, P278 Thu, Ole Kristian F., P203 Thummel, Kenneth, P265, P361 Thummel, Kenneth E., A7 Thumser, Alfred E., P410 Thu¨rmel, Klaus, P84 Tian, Dandan, P325 Tian, Luyang, P408 Tibbets, Garrett, P428, P431 Tibbs, Zachary E., P67 Tillman, Patrick, P29 Tippin, Tim, P101 Tipping, Iain, P198 Tirona, Rommel G., SC2.3, P211, P414 Toda, Ryoko, P383, P395 Toda, Takahiro, P100 Tomi, Masatoshi, P421, P425, P426, P427 Tomlinson, Brian, P330, P375 Tong, Zeen, P170 Torres, Nallely A., P403 Toselli, Francesca, P103 To´th, Bea´ta, P444 Touati, Walid, P184 Traeger, Sarah C., P298 Tran, Phi, P122 Tran, Thi, P184 Tran, Thuy, P326 Tremaine, Larry, P457 Tremblay-Franco, Marie, P320 Tremmel, Roman, P360 Trost, Lawrence, P101 Tsamandouras, Nikolaos, P393 Tseng, Elaine, P86 Tsirka, Theodora, P65 Tsujikawa, Kenji, P270 Tsukada, Ai, P401

Drug Metab Rev, 2013; 45(S1): 270–282

Tsyrlov, Ilya B., P275 Tu, Yaping, P380 Tucker, Geoffrey T., P304 Tugbobo, Oladimeji S., P342 Tukey, Robert H., S49 Tunquist, Brian J., P28 Turcotte, Ve´ronique, A3, P60 Turesky, Robert, S33 Tweedie, Donald, P405 Twist, Greyson, P., P344 Tyndale, Rachel F., P26 Tzeng, Tzuen-RongJeremy, P239 Uchida, Yasuo, P33, P42 Udupa, Nayanabhirama, P240 Uehara, Shotaro, P336 Uetrecht, Jack, S29, P290 Uetrecht, Jack, P., P30, P274, P286 Ufuk, Ayse, P458 Ukairo, Okechukwu, P233 Umemura, Shin-ichiro, P128 Unadkat, Jashvant, P201 Unadkat, Jashvant D., P436 Unger, Steve, SC4.4 Upthagrove, Alana, P122 Uramaru, Naoto, P328 Urata, Masayo, P27, P37 Urban, Thomas J., S21 Urtti, Arto O., S37 Usui, Takashi, P336 Usuki, Etsuko, P101 Utoh, Masahiro, P93 Uttamsingh, Vinita, P308, P309 Vaes, Wouter HJ, P229 Va¨ha¨kangas, Kirsi, P9 Vala´t, Martin, P111 Vale´rie, Fessard, P250 Valle, Nicole, P41 van Adrichem, Anita M, P229 VanAusdall, Jennifer, P287 van der Straaten, Tahar, P113 van de Steeg, Evita, P21, P229, P264 Vanninen, Paula, P9 Van Sickle, Kathy, P101 Van Veldhoven, Paul, P54 Vapurcuyan, Arpine, P122 Varma, Manthena, P232, P438 Vaskova, Lucie, P237 Vath, James, P311 Veiteberg, Tone, P317 Venkatarangan, Lata, P145 Verhoeckx, Kitty CM, P229, P264 Vermeulen, Nico, P113 Verwei, Miriam, P21, P229, P264 Vidugiris, Gediminis, P340 Vigoren, Eric, P36, P226 Viitala, P., P210 Villeneuve, Lyne, P60 Vishwanathan, Karthick, P207

Vlaming, Marijn LH, P264 Voellinger, Jenna, P235 Vogl, A. Wayne, P97 Vokra´l, Ivan, P111 Vollmerhaus, Pauline, P132 Volta, Roberta, P373 Vonk, Alex, P217 von Richter, Oliver, P374 Vos, Karin, P364 Vredevoogd, Melinda, P36, P226 Wada, Sho, P260 Wagner, Andrew, P439 Wagner, Richard, P55 Wakamiya, Kumiko, P8 Walker, Duncan H., P28 Walker, Ellen, P394 Wallin, Jeff, P371 Walsky, Robert L., P86 Walubo, Andrew, P14 Wan, Huayin, P4 Wang, Can, P121 Wang, Fengqing, P325, P381 Wang, Hao, P13 Wang, Hongbing, S6 Wang, Hui, P32, P89, P289, P340 Wang, Jian, P154 Wang, Jie, P434, P435, P464, P465 Wang, Jing, P88, P246 Wang, Julia, P155 Wang, Kepeng, S49 Wang, Lai, P122 Wang, Leslie, P389 Wang, Qi, P272 Wang, Qian, P18 Wang, Shuang, P339 Wang, Stephen W.J., P169 Wang, Tingting, P136 Wang, Yang, P35 Wang, Yao, S50 Wang, Yue-Ming, P402 Wang, Zhican, P265 Ware, Joseph A., S13 Warner, Yvette, P144 Warrack, Bethanne M., P298 Warren, Mark, P423, P443 Washington, Carla, P314 Wassenaar, Catherine A., P26 Watanabe, Hiroshi, P167 Watanabe, Keisuke, P215 Watanabe, Kensuke, P., P116 Watanabe, Mio, A5 Watanabe, Takao, P449 Watanabe, Yasuyoshi, S51, S52 Watanabe, Yoko, P328 Watkins, Paul B., S32, P279, P286 Watts, Richard W., P16 Webb, Heather, P148 Webber, Colin, P120 Webborn, Peter J.H., SC2.2

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DOI: 10.3109/03602532.2013.868114

Wei, Guangli, P7, P15, P17, P142 Wei, Yuan, P47 Weidolf, Lars, P153 Weksberg, Rosanna, P453 Welch, Stephen, A10 Wen, Jun, P90 Wenker, Mira, P369 Wentland, JoAnn, P379 Werk, Anneke N., P84 West, Michael, P457 Westerhout, Joost, P21 Westley, Ian S., P16 Wetter, Jill, P312 White, Kami K., S33 Widmer, Joel, P428, P431 Wiegand, Catherine, P186 Wilce, Peter A., P103 Wilde, Thomas C., P313 Wilkens, Lynne R., S33 Wilkinson, David, P198 Williams, Andrew, P178 Willig, Laurel K., P344 Wilson, Alan GE, P196 Wilson, Cherylle, P187 Wilson, Karen, P124 Winter, Stefan, A12, P360 Wishart, David, SC1.4 Witmer, Mark, P272 Wo, Siu kwan, P330, P375 Wolf, C.Roland, P115 Wolf, Ju¨rgen, P391 Wolff, Andrea, P428, P431 Wollenberg, Lance A., P28 Wong, Simon G., P163 Wong, Susan, P104, P371 Wong, Timothy, A7 Wong, TV, P375 Woo, Charles, P275 Wood, Stuart G., P124, P299, P382 Woodahl, Erica L., P430 Woodward, Caroline, P337 Woodworth, Zell, P75, P331 Woolsey, Sarah J., P211 Worboys, Philip, P314 Worrall, Simon, P103 Wortelboer, Heleen M., P21, P229, P264 Worzella, Tracy, P202 Wrona, Mark D., P151 Wsol, Vladimir, P221, P319 Wu, Alex, P453 Wu, Chiping, P45 Wu, Dong-mei, P289 Wu, Guanming, P24 Wu, Jing, P402 Wu, Jing-Tao, P127 Wu, Jingjing, P261 Wu, Lijun, P309 Wu, Linan, P15 Wu, Sean Xiang, P88

Wu, Xiaoyun, P35 Wu, Yiwen, P43 Xia, Cindy Q., P169, P466 Xia, Haiyan, P466 Xia, Yang-Liu, P63 Xia, Yuanyuan, P142, P415 Xiao, Bin-Xin, P18 Xiao, Deqing, P194 Xiao, Gong-hua, P178 Xiao, Wenjin, P234 Xie, Cen, P460 Xie, Dianlin, P433 Xie, Fang, P68 Xie, Wen, S7 Xu, Fang, P325, P381 Xu, Mingkai, P390 Xu, Xiaoyan, P154 Xu, Ximing, P181 Xue, Wei, P4 Yabe, Keisuke, P461 Yabuki, Masashi, P449 Yajima, Kanako, P93, P336 Yamada, Harumi, P100 Yamada, Hideyuki, P109 Yamaguchi, Hiroaki, P461 Yamaguchi, Takashi, P383 Yamamiya, Ikuo, P109 Yamamoto, Noboru, P222 Yamamuro, Tadashi, P270 Yamaori, Satoshi, P10 Yamaura, Yoshiyuki, P265 Yamazaki, Hiroshi, P10, P93, P401 Yamazoe, Yasushi, P215, P396 Yan, Zhu, P325 Yang, Cuiping, P455 Yang, Dan, P321 Yang, Haibin, P154 Yang, Junling, P325, P381 Yang, Ling, S26, P61, P62, P63, P204, P252, P253, P261 Yang, Lulu X., P389 Yang, MaryAnn, P161 Yang, Mengbi, P288, P322 Yang, Mingdong, P453 Yang, Qian, P212, P249 Yang, Xia, P361 Yang, Yan, P13, P121, P136 Yang, Zhihong, P245 Yanni, Souzan B., P53 Yap, Megan, P64 Yatsu, Masami, P164 Yatsuno, Yoshiyuki, P164 Yauk, Carole, P178 Ye, Lin-Hu, P18 Ye, Yang, P280, P288 Ye, Yihua Emily, P296 Yee, Brendan, P187 Yerino, Phyllis, P201, P257, P258, P331

Yerramilli, Usha, P170 Yeung, Pollen K. F., P29 Yi, Xiulin, P17, P142, P415 Yi, Yijun, P311 Yin, Lei, P136 Yin, Peter Z., P462 Yip, Lian Yee, P228, P281 Yonemori, Kim M., S33 Yoon, Ji Eun, P385 Yoshida, Ken-ichiro, P193 Yoshida, Kenta, P387 Yoshii, Kazuyoshi, P383, P395 Yoshimura, Tsutomu, P125 Yoshinari, Kouichi, P215, P396 Yoshino, Ichiro, P416 Yoshisue, Kunihiro, P109, P193 You, Yuyang, P245 Yu, Ai-Ming, A11 Yu, Hongbin, P405 Yu, Kyung-Sang, P20, P362 Yu, Zhen-Wen, P61 Yuasa, Hiroaki, P450 Yue, Jiang, P94 Yueh, Mei-Fei, S49 Yumita, Nagahiko, P128 Yuska, Brad, P155 Zalko, Daniel, P307, P320 Zamora, Ismael, P130 Zander, Thomas, P391 Zanger, Ulrich M., S8, A1, A12, P60, P106, P236, P360 Zeijdner, Evelijn E, P21 Zeilinger, Katrin, P266 Zeillinger, Robert, P417 Zendulka, Ondrej, P80, P102 Zeng, Yong, P142, P415 Zhang, Chunyuan, P321 Zhang, Hui, P457 Zhang, Huiwen, P390 Zhang, J. George, P185, P194 Zhang, Lei, P168, S41 Zhang, Li, P154 Zhang, Lingling, P35 Zhang, Mengliang, P121 Zhang, Miaoyun, P7 Zhang, Minli, P293 Zhang, Qing-Yu, P68, P74 Zhang, Tianhong, P455 Zhang, Weihan, P154 Zhang, Weijun, P154 Zhang, Xiaolin, P371 Zhang, Xuexiang, P423 Zhang, Yinying, P90 Zhang, Yunhui, P121, P136 Zhang, Zhengqing, P455 Zhang, Zongpeng, P390 Zhao, Jingjing, P154 Zhao, Jinyi, P242 Zhao, Li-zi, P358

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Zhao, Ping, P168 Zhao, Sylvia, P35 Zheng, Qishan, P358 Zheng, Yuxin, P126 Zheng, Zhi, P147 Zhiyang, Zhao, P112 Zhong, Dafang, P460 Zhong, Kong, P460

Drug Metab Rev, 2013; 45(S1): 270–282

Zhong, Peiru, P242 Zhong, Xiao-bo, P216, P327 Zhou, Fu-Jun, P388 Zhou, Julia, P196 Zhou, Lei, A2 Zhou, Wenhui, P340 Zhou, Xiang, P388 Zhu, Bing, P122

Zhu, Liang-Liang, P63 Zhu, Qing, P466 Zhu, Siying, P94 Zhu, Yi, P68, P74 Zientek, Michael A., P86 Zimcik, Petr, P141 Zou, Chao, P204 Zuo, Zhong, P330, P375, P413



Keyword Index ADME, P106 ADP-RIBOSYLATION, S5 ATP, P29 ABSORPTION, P321, P288 ACETYLBOSWELLIC ACID, P415 ACONITINE, P455 ADRENAL; CAFFEINE; CHOLESTEROL; SCAVENGER RECEPTOR CLASS B TYPE I; DNA METHYLATION, P289 ADVERSE DRUG REACTIONS, S21 ALDEHYDE OXIDASE, P334 ANALYTICAL, SC1.1, SC4.1, SC4.2, SC4.4, S33, S54, A2, P1, P2, P3, P4, P5, P6, P7, P8, P70, P111, P120, P129, P130, P131, P132, P133, P134, P158, P190, P226, P230, P246, P247, P248, P292, P293, P294, P297, P298, P299, P300, P366, P367, P368, P369, P370, P410 ANIMAL MODELS OF IDIOSYNCRATIC DRUG REACTIONS, P274 ANTIBODY-DRUG CONJUGATE, P379 ANTI-CANCER AGENT, P139 ANTIOXIDANT, P407 AQUAPORIN, P411, P459 ARSENIC, P73, P420 ARYLAMINE N-ACETYLTRANSFERASE, S18 ARYLHYDROCARBON RECEPTOR, P105 ASSAY AND WORK FLOW, P247 BCRP, P412, P428, P467 BDDCS, P46 BEPOTASTINE BESILATE, P12 BILE DUCT CANNULATED RATS, P144 BILIARY EXCRETION, P424 BIOAVAILABILITY, SC4.4, S34, P15, P16, P17, P18, P71, P121, P127, P135, P136, P137, P159, P240, P251, P265, P371, P372, P373, P374, P375, P412, P413, P414, P415 BIOINFORMATICS, S10, S14, S22, P24, P138, P344, P345, P366, P275 BIOMARKERS, SC1.1, S23, S25, S32, S33, S47, A3, A4, A9, P1, P9, P24, P26, P27, P28, P29, P30, P31, P32, P33, P55, P129, P206, P346, P347, P376, P377, P378, P416, P417, P418, P419 BIOTECHNOLOGY/NANOTECHNOLOGY, S34, P120, P176, P266, P379 BIOTHERAPEUTICS, P135 BLOOD-BRAIN BARRIER, SC2.5, S15, P39, P42, P43, P44, P45, P121, P125, P156, P415, P430, P449 BREAST CANCER, P347, P419

CTNNB1, P218 CYP2A6, P253 CYP2D6 GENOTYPE, P349 CYP3A, A5 CYPS ENDOGENOUS FUNCTIONS, S4, S55, P35, P76, P77, P78, P79, P80, P210 CACO-2, P18 CARBOXYL ESTERASES, P335 CARBOXYLESTERASE, P318 CARCINOGENESIS/MUTAGENESIS, S33, P47, P48, P56, P72, P73, P74, P126, P139, P140, P141, P177, P209, P339, P396, P417, P418, P420 CATALPOL, P15 CELL PROLIFERATION, P397 CHANNELS, P42 CHEMOSYNTHETIC LIVERS, P173 CHEMOTHERAPY, P33 CILOSTAZOL, P350, P351, P357 CLEARANCE PREDICTION, S19, P19, P34, P51, P52, P53, P57, P135, P231, P257, P258, P259, P301, P302, P331, P371, P372, P376, P421, P422, P423 COMPUTATIONAL MODELING, SC32 COMPUTATIONAL SYSTEMS BIOLOGY, P254, P256 CONJUGATION REACTIONS AND ENZYMES, SC3.2, S18, A6, P2, P57, P58, P59, P60, P61, P62, P63, P64, P71, P75, P112, P113, P122, P160, P176, P191, P192, P206, P260, P276, P297, P303, P304, P305, P348, P424 CYTOCHROME P450, SC3.1, SC3.3, S2, S4, S6, S8, S16, S17, S19, S20, S43, S55, S56, S57, A1, A5, A7, A11, A12, P1, P2, P3, P10, P11, P20, P26, P35, P36, P47, P51, P68, P69, P70, P71, P72, P73, P75, P76, P77, P78, P79, P81, P82, P84, P85, P86, P87, P88, P89, P90, P91, P92, P93, P94, P95, P96, P97, P98, P114, P115, P116, P123, P142, P159, P161, P162, P163, P164, P165, P178, P184, P185, P186, P187, P190, P193, P194, P195, P196, P197, P198, P199, P200, P201, P202, P203, P210, P211, P212, P213, P214, P215, P216, P217, P218, P231, P240, P241, P242, P243, P244, P246, P249, P250, P252, P253, P257, P258, P261, P277, P278, P279, P280, P292, P301, P303, P304, P306, P307, P308, P309, P344, P349, P350, P351, P352, P353, P354, P355, P366, P367, P398, P401, P404, P405, P406 CROCIN, P102 DEUTERIUM ISOTOPE EFFECTS, P308, P309 DGAT1 INHIBITOR, P147 DNA DAMAGE AND REPAIR, S33, P59, P126, P282


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DEVELOPMENTAL NEUROTOXICITY, P179 DICHLOROACETATE, GSTZ1, TYROSIN METABOLISM, LINKAGE DISEQUILIBRIUM, P310 DIFFERENCES IN METABOLISM (SPECIES, GENDER, AGE, DISEASES), SC1.2, S16, S30, S34, A8, P26, P27, P37, P52, P76, P84, P99, P100, P111, P112, P114, P115, P116, P117, P118, P143, P179, P211, P275, P281, P306, P307, P311, P312, P332, P333, P345, P356 DISPOSITION, SC2.3, S16, S27, S44, S54, P53, P85, P114, P120, P123, P135, P136, P143, P144, P145, P207, P257, P258, P267, P281, P305, P311, P313, P314, P368, P373, P380, P381, P382, P383, P410, P412, P420, P421, P422, P424, P425, P426, P427, P428, P429, P430, P431 DISULFIDE BOND FORMATION, P461 DRONEDARONE, RAT, P368 DRUG ALLERGY, P328 DRUG DELIVERY, S15, S34, S36, S37, S51, P117, P120, P121, P124, P127, P136, P137, P384, P390 DRUG DISCOVERY AND DEVELOPMENT, SC1.2, SC2.2, SC4.1, SC4.2, S1, S4, S13, S18, S19, S22, S28, S31, S36, S39, S43, S44, S48, S51, S54, S56, P4, P5, P6, P12, P21, P28, P29, P35, P40, P41, P42, P49, P52, P65, P66, P75, P86, P87, P88, P101, P115, P120, P121, P124, P125, P127, P129, P130, P131, P132, P133, P135, P138, P139, P146, P147, P148, P149, P150, P151, P152, P153, P154, P155, P156, P160, P166, P167, P168, P204, P212, P246, P247, P248, P251, P252, P265, P293, P297, P298, P299, P302, P303, P308, P311, P315, P316, P317, P331, P332, P334, P340, P366, P369, P370, P371, P372, P374, P377, P379, P380, P384, P385, P386, P413, P432, P433, P434, P435, P436, P437, P438, P439, P341 DRUG INTERACTION, SC3.1, SC3.2, SC3.3, SC3.4, S2, S30, S38, S41, S42, S43, S48, S51, S52, A11, P10, P15, P16, P24, P35, P57, P71, P86, P87, P89, P101, P102, P115, P123, P158, P159, P161, P162, P163, P164, P165, P166, P167, P169, P170, P185, P186, P188, P190, P193, P194, P195, P196, P197, P198, P199, P200, P201, P219, P232, P236, P240, P241, P242, P249, P261, P268, P276, P315, P318, P319, P387, P421, P428, P429, P432, P433, P434, P435, P436, P437, P438, P440, P441, P442, P443, P444, P445, P446, P447, P448, P449, P450, DRUG RESISTANCE, P319 DRUG TARGET, S3 DRUG TARGETING, S37 ENDOCRINE DISRUPTING CHEMICALS, P180 ENVIRONMENTAL TOXICOLOGY, S58, P47, P116, P178, P179, P181, P213, P214, P226, P275, P339, P403, P420, P430, ENZYME ACTIVATION, P10 ENZYME INDUCTION, SC3.1, S6, S8, S42, P35, P51, P76, P77, P89, P90, P91, P92, P93, P102, P103, P161, P171, P185, P186, P187, P188, P193, P210, P215, P216, P217, P236, P237, P398 ENZYME INHIBITION/INACTIVATION, S2, S3, S4, S42, P10, P51, P76, P84, P86, P87, P90, P94, P95, P96, P104, P148, P161, P162, P163, P164, P171, P190, P193, P194, P195, P196, P197, P198, P202, P204, P213, P237, P241, P243, P249, P318, P432

Drug Metab Rev, 2013; 45(S1): 283–286

ENZYME PROTEIN CONCENTRATION; SUBSTRATE CONCENTRATION; SAMPLING TIME, P261 EPOXYEICOSATRIENOIC ACIDS, P98 ESTERASES, P337 EX VIVO PROTEIN BINDING, P145 EXCRETION, P323 EXTRAHEPATIC METABOLISM, S29, P19, P26, P68, P74, P97, P103, P105, P123, P206, P313, P333, P335, P336 FORMALDEHYDE, P126 GENE EXPRESSION AND REGULATION, S5, S6, S7, S8, S14, S16, S48, S58, A5, A12, P50, P56, P59, P60, P69, P78, P94, P97, P99, P103, P106, P178, P188, P209, P211, P212, P215, P218, P220, P221, P222, P223, P236, P276, P277, P396, P398, P414, P451, P452, P453 GENOMICS/METABONOMICS/PROTEOMICS, SC1.1, S10, S14, S22, A3, A6, P20, P24, P36, P37, P60, P69, P178, P216, P277, P278, P283, P320, P344, P414 GLUCOSIDATION, P305 GLUTATHIONE, P192 GONADAL HISTOLOGY, OVO-TESTIS, STEROID HORMONES, TILAPIAS, ENDOCRINE DISRUPTING CHEMICALS (EDCS), OGUN RIVER, P182 HER2, P50 HEPATOCYTES, SC2.1, SC2.2, S8, S28, S48, S57, S56, A11, A12, P30, P77, P78, P91, P106, P161, P171, P185, P186, P194, P195, P196, P199, P200, P202, P205, P210, P212, P216, P218, P231, P232, P233, P236, P238, P249, P250, P254, P257, P258, P260, P262, P263, P266, P269, P270, P279, P284, P285, P286, P287, P302, P306, P309, P315, P320, P332, P335, P410, P422, P424, P440, P441, P442, P454 HERB-DRUG INTERACTIONS, S20, S27, P95, P96, P102, P162, P172, P203, P236, P238, P240, P243, P245, P276, P443, P456 HERB-INDUCED HEPATOTOXICITY, S25 HERBAL MEDICINE-ACTIVE COMPOUNDS, S25, S27, P7, P61, P62, P90, P183, P238, P239, P243, P244, P271, P407, P455, HIGH THROUGHPUT TECHNIQUES, SC4.2, S18, S28, S55, P11, P13, P27, P49, P60, P88, P89, P106, P130, P131, P163, P188, P197, P202, P246, P247, P249, P250, P282, P316, P340, P374, P433, P439, P457 HUMAN EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 1, P451 HUMAN KIDNEY, S30 HUMANIZED MOUSE, P66 HYDROLYSIS; PARAOXONASE; ALBUMIN, P336 HYPOXIA-INDUCIBLE FACTOR-1A (HIF-1A) INHIBITOR, LW6, P385 IMMUNE MEDIATED IDIOSYNCRATIC DRUG TOXICITY, P286 IN SILICO, S19, S20, S22, S36, P22, P24, P25, P49, P59, P165, P253, P254, P255, P256, P275, P384, P404, P455


DOI: 10.3109/03602532.2013.868114

IN VITRO TECHNIQUES, SC2.1, SC2.3, SC2.5, SC3.1, S27, S28, S30, S56, A1, A2, A8, P2, P21, P28, P31, P43, P54, P61, P62, P63, P65, P75, P87, P89, P91, P96, P101, P104, P107, P122, P124, P140, P171, P173, P186, P190, P197, P198, P199, P200, P201, P202, P204, P205, P224, P233, P234, P235, P241, P243, P244, P247, P248, P257, P258, P259, P266, P269, P271, P272, P279, P284, P285, P288, P292, P297, P301, P305, P307, P321, P322, P331, P332, P340, P422, P423, P424, P428, P431, P434, P435, P439, P440, P441, P442, P444, P445, P452, P455, P458 IN VITRO TRANSPORTER ASSAY, P457 IN VITRO/IN VIVO EXTRAPOLATION, SC2.1, SC2.2, SC3.4, S39, S41, P28, P46, P53, P54, P57, P91, P107, P122, P127, P171, P185, P194, P198, P199, P201, P224, P231, P259, P260, P261, P262, P263, P275, P279, P302, P304, P371, P428, P431, P442 INDUCED PLURIPOTENT STEM CELLS, P279 INFLAMMATION, P269 INFLAMMATION, HIV-1 GP120, P45 INTERINDIVIDUAL DIFFERENCE, A7 INTRAUTERINE GROWTH RESTRICTION; EPIGENETICS; DNA METHYLATION; PERIPHERAL BLOOD, P32 IRIDOID GLYCOSIDES, P7 IRREVERSIBLE INHIBITORS, P150, P149 JUVENILE, P117 KUPFFER CELLS, P224 LIPID PROFILING, P295 LIPIDOMICS, P37 LOSARTAN, P363 LUNG PHARMACOKINETIC, P373 LYSOSOMAL TRAPPING, P458 MIST, S44, P153 MASS SPECTROMETRY, SC41 MECHANISMS OF XENOBIOTIC TOXICITIES, S25, S47, S48,S54, A2, P1, P47, P69, P92, P107, P113, P134, P151, P179, P181, P184, P200, P206, P228, P233, P250, P278, P280, P282, P283, P284, P286, P289, P290, P291, P312, P320, P322, P339, P340, P403 MALARIA, P364 MASS BALANCE, P324 METABOLIC PROFILING, SC1.1, S44, S47, P27, P38, P51, P63, P79, P85, P92, P98, P113, P130, P134, P136, P152, P157, P292, P293, P294, P295, P306, P308, P309, P311, P313, P314, P316, P320, P323, P324, P325, P345, P372, P377, P388, METABOLIC SITE; MECHANISM-BASED PREDICTION; ACTIVATION ENERGY, P252 METABOLISM, SC1.2, SC3.2, SC3.3, SC3.4, SC4.2, S2, S6, S7, S17, S19, S26, S27, S29, S33, S41, S44, A1, A7, A8, A10, A11, P1, P4, P9, P10, P11, P12, P17, P29, P36, P38, P52, P54, P56, P61, P62, P63, P66, P67, P72, P73, P74, P75, P79, P80, P84, P85, P88, P92, P93, P98, P101, P106, P111, P112, P113, P114, P115, P118, P122, P130, P131, P132, P133, P134, P140, P142, P143, P148, P151, P152, P153, P157, P160, P164, P166, P167, P168, P173,

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P184, P195, P206, P207, P211, P234, P253, P254, P255, P256, P259, P262, P263, P265, P268, P270, P273, P278, P280, P283, P290, P292, P293, P295, P300, P301, P308, P309, P312, P313, P314, P315, P316, P318, P320, P323, P324, P325, P326, P327, P328, P329, P330, P331, P333, P334, P337, P338, P339, P345, P356, P357, P358, P370, P372, P385, P388, P401, P404, P405, P410, P459, P460 METABOLITE IDENTIFICATION, P157 METAL TOXICITY, P72, P73, P140, P177, P183, P341, P342, P408, P420 METFORMIN, RAT, RENAL CLEARANCE, HYPERLIPIDEMIA, OCT-1, PHARMACOKINETICS, P359 MICRORNA, CONSTITUTIVE ANDROSTANE RECEPTOR, MIR-137, MDR1, DOXORUBICIN, P402 MICRODIALYSIS, P125 MODELING, P260 MOLECULAR IMAGING, S53 MONOAMINES, P129 MORINDA CITRIFOLIA, P177 MULTIDRUG RESISTANCE, P209 NANOMATERIALS, P282 NEW DRUG APPLICATIONS, P168 NEXT GENERATION SEQUENCING, P344 NON-P450 PHASE I ENZYMES, P36, P187, P193, P204, P207, P208, P231, P304, P312, P318, P332, P337, P405 NON-ALCOHOLIC STEATOHEPATITIS, P329 NONLINEAR PHARMACOKINETICS, P380 NOVEL TECHNOLOGIES, P188 NUTRITION AND CHEMOPREVENTION, P177, P183, P237 OATP, P431 OATP1B1/BCRP, P423 OATP1B3, P452 OTAP, P468 OBESITY DRUG, P311 ORAL LICHEN PLANUS, SMAD 3, TRANSFORMING ¯ , P31 GROWTH FACTOR A ORGANIC CATION TRANSPORTER, P450 OROSOMUCOID, P222 OSELTAMIVIR, P375 OTHER RELATED TOPICS, P224, P287, P384 OXIDATIVE STRESS, S15, A6, P55, P64, P141, P209, P237, P238, P282, P285, P339, P343, P408, P461 P450 STRUCTURE, S20 PBPK, P371 PBPK MODEL, P22 PBPK MODELING, S38, P123, P401, P447 PET IMAGING, S36 PARACETAMOL, P330 PATHWAYS, P287 PERMEABILITY, P196 PHARMACOGENETICS/PHARMACOEPIGENETICS, S10, S11, S12, S18, S22, S23, A1, A4, A7, A9, A10, P60, P65, P205, P216, P291, P344, P349, P350, P351, P352, P353, P354, P355, P356, P357, P358, P360, P361, P362, P363, P364, P365, P367, P423, P452, P462


286

PHARMACOGENOMICS/PHARMACOGENETICS, S9 PHARMACOKINETICS, S24 PHARMACOKINETICS AND PHARMACODYNAMICS, SC2.2, SC2.3, SC2.4, SC3.2, SC4.2, SC4.4, S34, S36, S37, S39, S40, S51, S54, A10, P3, P5, P6, P13, P16, P21, P28, P29, P34, P38, P49, P57, P70, P80, P88, P100, P108, P116, P121, P122, P127, P131, P136, P137, P142, P143, P154, P158, P165, P201, P240, P241, P242, P246, P251, P255, P256, P263, P272, P291, P303, P316, P317, P319, P330, P348, P350, P351, P357, P366, P367, P368, P370, P374, P375, P377, P378, P379, P380, P381, P382, P386, P388, P389, P390, P391, P392, P401, P413, P421, P429, P436, P446, P447, P458, P460, P463 PHARMACOMETABOLOMICS, S46 PHASE II ENZYMES, P187 PHOTODYNAMIC THERAPY, P141 PH REGULATION, P219 PHYSIOLOGICALLY-BASED PHARMACOKINETIC MODELING, P393 PICROSIDE, P388 PIPERINE MDCK PERMEABILITY, P271 PIPERPHENTONAMINE, P4 PLASMA PROTEIN BINDING, DISSOCIATION RATE CONSTANT, OFF RATE, P272 PLASMA PROTEIN BINDING, P155, P374 POLYCHLORINATED BIPHENYLS, P178 POLYPHENOLS, BIOTRANSFORMATION, MICROFLORA, DIGESTION, GASTROINTESTINAL MODEL, P23 POST-TRANSLATIONAL REGULATION, P99 PROTEIN MODIFICATION, P55 PROTEIN STRUCTURE, P469 PROTEIN STRUCTURE-FUNCTION, P404 PROTEOMICS, P8 RACLOPRIDE, P80 REACTIVE METABOLITE(S), P2, P30, P278 REACTIVE METABOLITE TRAPPING, P230 RECEPTORS/NUCLEAR RECEPTORS, S5, S6, S7, S8, S14, S15, A11, A12, P33, P78, P94, P108, P214, P215, P217, P218, P223, P225, P276, P396 REGULATED BIOANALYSIS, SC44 REGULATION, S13 REGULATORY, S42 REGULATORY GUIDANCE, S43 REGULATORY SCIENCE, P166, P167 RENAL IMPAIRMENT, P460 RHODIOLA ROSEA, P244, P203 RISK ASSESSMENT, S25, S41, A3, A4, P116, P124, P153, P163, P165, P224, P346, P401, P431, P433

Drug Metab Rev, 2013; 45(S1): 283–286

ROSUVASTATIN, P362 SIMCYP, P384 SIRNA/MICRORNA, SC2.1, A12, P126, P235 SKIN RASH, SULFATION, S29 SORAFENIB, ADVERSE EFFECT, PHARRMACOKINETICS, P409 SPHEROID, P270 SPONDIAS MOMBIN, SPERMATOZOA CHARACTERISTICS, P183 STABLE ISOTOPE RESOLVED METABOLOMICS; SYSTEMS BIOCHEMISTRY, SC12 STEM CELL RESEARCH, S28, S55, S56, S57, S58, P235, P286, P403 STEREOSELECTIVE METABOLISM, P13, P90, P109, P405, P406 TAMOXIFEN, P352 THERAPY, P33, P137, P160, P177, P184, P219, P283, P291, P338, P347, P356, P358, P383, P391, P407, P408, P409, P429 THYROID HORMONE, THYROID RECEPTOR, CELL LINE, REPORTER ASSAY, P399 TISSUE DISTRIBUTION, P382 TOLPERISONE, P353 TOXICITY, S49 TOXICOKINETICS, P392 TOXICOLOGY, P48 TRANSCYTOSIS, P39 TRANSPORTERS, SC2.1, SC2.2, SC2.3, SC2.5, SC3.1, S13, S14, S15, S16, S30, S38, S41, S42, S43, S51, S52, A9, P17, P18, P42, P43, P44, P45, P46, P114, P125, P156, P159, P166, P167, P168, P170, P174, P187, P212, P219, P229, P232, P254, P255, P256, P260, P263, P264, P266, P325, P347, P348, P363, P364, P387, P400, P410, P412, P413, P415, P419, P422, P423, P429, P430, P433, P434, P435, P436, P437, P438, P440, P441, P442, P443, P444, P445, P448, P449, P450, P452, P453, P454, P455, P456, P457, P460, P461, P462, P464, P465, P466, P467, P468, P469 TUBERCULOSIS, P356 UGT, P259 UGT2B15, P365 VELUSETRAG, P314 WARFARIN, P358

PXR,

Y101, P17, P142

POR,

PHARMACOGENETICS,

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