Meeting Highlights

13th Annual Meeting of the Safety Pharmacology Society: focus on novel technologies and safety pharmacology frontiers




Plenary sessions’ presentations


Drug target-related safety

Icilio Cavero


Expanding safety

Paris, France

pharmacology frontiers

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Expert opinion

Introduction: The 13th Annual Meeting of the Safety Pharmacology (SP) Society discussed novel therapeutic areas, recent regulatory developments, emerging biology technologies and non-pharmaceutical dairy products that may need SP evaluations for ensuring their human safety. Areas covered: The meeting honored Willem Einthoven, the father of electrocardiography. The Comprehensive in vitro Proarrhythmia Assay (CiPA) is an under-discussion proposal for replacing the International Conference on Hamonization (ICH) S7B guideline strategy. Drugs targeting epigenetic mechanisms (e.g., histone deacetylase inhibitors) have the potential to produce proarrhythmic safety liabilities by dysregulating the synthesis of cardiac ion channel proteins as well as the intracellular machinery, moving them to sarcolemmal residence. Novel frontiers of regulatory SP investigations are functional food and probiotic (microorganisms) preparations. Expert opinion: The CiPA initiative is a unique opportunity for concerned stakeholders to drive SP into the adoption of 21st century safety assessment platforms, which, for discovering and mitigating mechanisms conferring safety risks to drugs, apply chiefly in silico and in vitro rather than traditional in vivo pharmacodynamics assays. The SP evaluation of functional foods and probiotics needs the development of product-tailored investigational approaches. Keywords: anticancer agents, comprehensive in vitro proarrhythmia assay, CiPA, ECG history, epigenetics, functional foods, HDACis, probiotics, safety pharmacology. Expert Opin. Drug Saf. (2014) 13(9):1271-1281



The 13th annual meeting of the Safety Pharmacology Society (SPS), held in Rotterdam in September 2013, offered to the 400 participants cutting-edge information on novel biology technologies, recent regulatory developments, emerging therapeutic areas, non-pharmaceutical areas in need of safety pharmacology (SP) investigations, and novel drugs with the intrinsic potential to cause adverse effects. The few meeting talks detailed in this article were not reviewed in any of the previous eight SPS annual meeting reports [1-8] published by this author in this journal. 2.

Plenary sessions’ presentations

ECG historical talk with emphasis on the QT interval. Prof Marc Vos, University Medical Center, Division Heart and Lung, Utrecht, The Netherlands


The SP organizers thanked the country hosting the 13th annual meeting of SPS by asking a Dutch scientist to reminisce the living legacy of his countryman, Willem Einthoven (1860 -- 1927), the inventor of string electrocardiograph, a scientific 10.1517/14740338.2014.940310 © 2014 Informa UK, Ltd. ISSN 1474-0338, e-ISSN 1744-764X All rights reserved: reproduction in whole or in part not permitted


I. Cavero

Article highlights. .


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The 13th Annual Meeting of the Safety Pharmacology (SP) Society discussed novel therapeutic areas, recent regulatory developments, emerging technologies and non-pharmaceutical products for human use needing SP evaluations. The Comprehensive in vitro Proarrhythmia Assay initiative envisages the assessment of ventricular ion channel mechanisms conferring proarrhythmic safety liability to drugs. Drugs acting via epigenetic mechanisms require targeted investigational efforts to clear class-specific cardiac liabilities resulting from defective ion channel expression and tethering. Tailored SP assays are needed to ensure the pharmacological safety of dairy functional foods and probiotic microorganisms.

This box summarizes key points contained in the article.

instrument, which, at the early years of the 20th century, enabled the accurate recording of the cardiac electrical activity from electrodes applied to body surface. For this discovery, Prof Einthoven received the 1924 Nobel Prize for Physiology and Medicine. Prof Einthoven’s interest in electrocardiography probably originated in 1889 when, during his participation at the First International Congress of Physiology in Basel, assisted to Prof Augustus Waller experiment on the ‘electromotive changes connected to the beat of the heart’ captured by skin electrodes (on the front and the back of a man’s chest) coupled to the poles of a mercury capillary electrometer [9-11]. The mercury meniscus oscillations were recorded on photographic paper after optic magnification. The intrinsic lack of highfrequency response (due to the natural inertia of liquid mercury) prevented the faithful recording of the rapid electrical waves generated by the heart during a cardiac cycle. Indeed, the ECG profile obtained with a mercury capillary electrometer exhibited damped QRS and T waves (see Figure 3 in [12]). Various improvements of the instrument and the application of mathematical corrections enabled Prof Einthoven to obtain the now familiar waveform profile of the human ECG [12,13], which was subsequently confirmed with the quartz (an ~ 3 µm silver-coated quartz filament traversing a strong magnetic field) galvanometer designed by Einthoven himself. The low intensity and the polarity of cardiac biopotentials produced minuscule deflections of the quartz filament, which were displayed, after magnification with a projecting microscope, on photographic plates moving at 25 mm/s. The galvanometer sensitivity parameter for ECG measurements was set to yield 1 cm deflection for each 10 mV signal by adjusting the tension of the quartz filament. This calibration setup has become an electrocardiography standard. Prof Einthoven reported his clinical ECG results in a 1906 French publication [14] and then, in a 1908 German publication [15], which contain normal and pathological (e.g., right and left 1272

ventricular hypertrophy, ventricular extrasystoles, bigeminy, premature ventricular contractions, atrial flutter, complete heart block, and various types of arrhythmia) ECG patterns. The familiar ECG waveform components designated as P, Q, R, S and T, the traditional three-limb leads or derivations for recording a standard ECG, and the equilateral triangle using leads I, II and III for calculating the electrical axis of the heart are also Einthoven contributions to electrocardiography [10,11,13]. Two other giants of clinical electrocardiography development deserving to be mentioned are Sir Thomas Lewis (1881 -- 1945) and Frank Wilson (1890 -- 1952) [16] who worked, respectively, at University College Hospital in London and Michigan University. Sir Thomas Lewis continued the research initiated by Einthoven and was Prof Wilson mentor. In 1913, Prof Lewis published the first textbook of electrocardiography (Clinical Electrocardiography [17]), which was preceded, in 1911, by the first of three editions of ‘The Mechanism and Graphic Registration of the Heart Beat’ [18] and, in 1912, by ‘Clinical Disorders of the Heart Beat’ [19] for practitioner physicians. These texts detail the then available knowledge about electrocardiography and cardiac arrhythmias. They introduce various novel terms, still presently used, (e.g., pacemaker, sino-auricular node, premature contractions, paroxysmal tachycardia and auricular fibrillation) in the medical dictionary [20]. The ECG is now a routinely applied noninvasive and inexpensive clinical procedure for cardiac evaluations. It is also an essential tool of SP for investigating the cardiac electrophysiological safety of drugs [21,22]. Over the past century, advances in electrocardiography field have been numerous (e.g., introduction of six precordial surface leads; continuous ECG recording for 24 h and over [Holter monitoring]; miniaturization of ECG recording instrumentation; automated ECG interpretations; diagnosis of inherited cardiac diseases from ECG tracings, etc.). Update and feedback on the Cardiac Safety Research Consortium -- The Health and Environmental Sciences Institute (CSRC-HESI)-FDA workshop: “Arrhythmia risk assessment during drug development… without the thorough QT (TQT) study” by Dr Gary Gintant, AbbVie, North Chicago, IL, USA 2.2

This subject, extensively discussed in a recent report by Cavero and Holzgrefe [23], is also briefly reviewed here due to its critical importance for the future of SP. From 2005 on, the pharmaceutical industry has systematically mitigated proarrhythmic liability in any novel drug selected for human investigation in accordance with the recommendations of ICH nonclinical S7B [21] and clinical E14 [22] guidelines. The typical cellular mechanism responsible for potentially lethal drug-induced arrhythmias (Torsades de Pointe [TdP]) is blockade of the rapid delayed rectifier potassium

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channel, coded by the human ether-a-go-go-related gene (hERG or KCNH2). The electrical current (IKr) carried by this channel plays a major role in the repolarization phase (mediating the recovery of resting state by the myocardium) of the ventricular action potential [24]. Recent advances in arrhythmia mechanisms, the emergence of novel screening technologies based on human stemcell--derived cardiomyocytes (hSC-CMs) and the high cost of the E14 recommended TQT study [22] are major drivers behind the recent think-tank proposal by the CSRC, HESI and FDA [25] for rejuvenating the S7B strategy [21]. The ICH S7B guideline [21] recommends the performance of a functional in vitro assay to evaluate the effects of novel drugs on IKr measured in native or heterologously expressed protein encoded by the hERG gene, and an in vivo pharmacodynamic assay to evaluate possible prolongation of QT/QTc interval. The novel strategy, which was named ‘The Comprehensive in vitro Proarrhythmia Assay’ (CiPA), consists of the following in vitro and in silico assays: i) Study of candidate drugs on key cardiac sodium, calcium and potassium ion channels. ii) Introduction of ion channel dataset generated in the latter investigation into computational models of ventricular action potential to determine whether the profile of the reconstructed action potential exhibits proarrhythmic liability signals by itself and in relation to action potential reconstructions obtained with reference proarrhythmic drugs. iii) The third assay of CiPA suite investigates the effects of the candidate drug on the electrical activity (ideally the action potential) in hSC-CM preparations. If the CiPA proposal is adopted, the FDA has indicated to abandon the requirement for a TQT study [22] as a precondition for granting marketing approval to drugs with systemic bioavailability [25,26]. 3.

Drug target-related safety

This session discussed novel mechanisms mediating drug safety liabilities. Safety considerations for epigenetic mechanisms as drug targets. Dr Amy H Yang, Pfizer, San Diego, CA, USA


Epigenetics refers to molecular mechanisms that initiate and maintain changes in gene expression without affecting the genome (DNA nucleotide sequence). Thus, the epigenome is a second layer of information differentially distributed across the genome in the form of chemical markers and switches allowing for nuanced regulation of gene expression. Histone modification, DNA methylation, and noncoding RNAs are epigenetic means for controlling gene expression. This presentation focused on drugs targeting histone functions critical for implementing protein synthesis. Histones are an integral part of the nucleosome, the basic building

chromatin-packaging unit containing all DNA expression instructions. Each nucleosome consists of 146 base pairs of DNA wrapped around a histone octamer core. The Nterminal histone tails protrude from nucleosomes into the nuclear lumen, and can be posttranslationally modified by targeted methylation, acetylation, phosphorylation, sumoylation, S-nitrosylation and ubiquitination. Enzymes mediating chemical histone modifications are referred to as writers (e.g., histone methyltransferases [HMTs] and histone acetyltransferases [HATs]) or erasers (e.g., histone demethylases [HDMs] and histone deacetylases [HDACs]). Lysine (K) acetylation on histone tails by lysine acetyltransferases (KATs) is generally associated with an open chromatin state allowing transcriptional activation, whereas lysine deacetylation by lysine deacetylases (KDACs) results in chromatin folding that signals transcriptional repression. On the other hand, the effects of histone methylation by HMTs on target gene transcription are context-specific depending on the location of methylated residues on histone tails. The epigenetic regulation of gene expression is present at all development, maturation and aging stages and contributes to health and disease processes. Epigenetic mechanisms are involved in numerous pathologies (e.g., neuropsychiatric [e.g., mental retardation, schizophrenia, depression and drug addiction], metabolic [e.g., diabetes, obesity], inflammatory and autoimmune disorders, asthma, and so on [27-31]). In particular, aberrant histone acetylation and abnormal gene expression intervene in causing a broad range of cancers. Histone deacetylase inhibitor medicines exhibit surprising efficacy in the treatment of specific types of cancers since they reduce expression of genes critical for cancer cell survival and proliferation [32]. Vorinostat was the first and romidepsin (natural cyclic peptide) the second HDACi approved by the FDA for specific cancer treatment. Numerous HDACis undergo clinical evaluation (e.g., belinostat, ITF2357, dacinostat [LAQ824], MS-275, panobinostat [LBH589]). HDACis are usually used in combination with traditional chemotherapeutic agents and their use is accompanied by serious adverse effects (nausea, anemia, thrombocytopenia, neutropenia, leukopenia and stem-cell toxicity) [32]. As the interest in epigenetic therapies steadily grows, an emerging critical issue of SP is how to predict and identify their potential toxicities. Information on potential targetmediated liabilities may be derived from phenotypes of human genetic variants and gene knockout or knockdown animal models. For example, heart-specific deletion of HDAC1 and HDAC2 in mice (over 18 distinct mammalian HDACs have been identified [33,34]) causes neonatal lethality accompanied by cardiac arrhythmias and dilated cardiomyopathy [35]. Clinical electrocardiographic alterations produced by HDACis are likely to result from altered cardiac ion channel protein expression (see Section 3.2). It should be noted that many epigenetic proteins have both an enzymatic function and a scaffold-dependent function (e.g., recruitment of protein partners). Whenever a drug discovery program targets a

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I. Cavero

protein enzymatic function, it is, thus, important to investigate potential safety issues by using appropriate chemical tools in relevant tissue and cell lines. Second, gene knockdown/ knockout investigations often can highlight the roles of these proteins in fetal development, which may not have a physiological function in healthy adults. Conversely, these proteins may be essential for a function only in the adult state and, therefore, this needs to be assessed in relevant experimental models. Additional risk mitigation strategies for epigenetic target-mediated toxicities include the assessment of body tissue distribution of the drugs to understand tissue- and target gene-specific effects. Moreover, safety studies of sufficiently long duration are necessary to evidence late-appearing epigenetically mediated risks. Finally, therapeutic index and risk--benefit ratios are critical parameters to consider for determining the impact and relevance of given safety liabilities of a medicine to a given patient population.

HDAC inhibitor-induced alteration of cardiac repolarization (QT) mediated by epigenetic mechanisms. Dr Stan Spence, Novartis Institutes for BioMedical Research Inc., Cambridge, MA, USA


Drug-induced prolongation of cardiac repolarization resulting from direct inhibition of hERG channel function in cardiomyocytes is a well-known safety liability which is nowadays routinely mitigated during the drug research and development process. However, drugs can also impair gene transcription of ion channel synthesis and/or of proteins required for intracellular transport of ion channels from endoplasmic reticulum to Golgi apparatus and ultimately to their residence destination [36]. These mechanisms can reduce the number of expressed hERG channels [36,37] and underlie the QTc prolongation produced, for instance, by pentamidine [38,39] and arsenic trioxide (As2O3) [40]. Panobinostat is a mild hERG blocker (IC50 3.5 µM). In conscious beagle dogs, it did not significantly increase QTcF after a single oral dose administration (1.5 mg/kg) yielding a peak total plasma concentration (Cmax) and free Cmax of, respectively, 0.22 µM (safety ratio [SR] 16) and 0.04 µM (SR 77) within the first hour after dosing. These levels declined by ~ 90% 2 h later. After a second and third intermittent dose (given every other day), QTcF increased by ~ 20 ms from the 6th to 13th hour post-dose although the plasma pharmacokinetic profile of panobinostat did not change with repeated dosing. In humans, oral panobinostat (20 mg every other day/3 weeks) produced a total Cmax of 0.12 µM (SR 30), a free Cmax of 0.012 µM (SR 300) and a mean QTc increase of ~ 10 -- 15 ms from the third to the eighth treatment day. A twofold higher dose produced a maximal mean QTc increase of 30 ms after the fifth day treatment. With continued drug administration, there was a trend toward a paradoxical shortening of the QTc interval, but, in terms of arrhythmia risks, this effect was not considered clinically meaningful. 1274

The cellular mechanisms of HDACi-induced QTc prolongation were investigated by using three pan-HDACis (vorinostat, NVS200, and NVS 400) and a HDACi (mocetinostat, MGCD0103) not inhibiting HDAC6 [34,41]. The expressed hERG channel-blocking activity of these compounds was virtually nil (IC50 > 35 µM). In CHO cells, NVS200, NVS400, mocetinostat and vorinostat (1 -- 30 µM) increased the hERG mRNA levels and a core-glycosylated immature hERG form. Additionally, these compounds altered the molecular weight of fully glycosylated hERG protein. Mocetinostat increased the hERG 135 kDa complex and 155 kDa core-glycosylated form, whereas the ratio immature/mature forms of hERG were consistently increased by the pan-HDACis. This difference may be due to the HDAC6-sparing activity of mocetinostat. However, the four investigated HDACis failed to modify the hERG protein expression in experiments performed in HEK-293 cells. The differential behavior in CHO and HEK-293 cells may reflect differences in HDAC hERG expression promoters in the two engineered cell lines. In this regard, some promoter regions in CHO cells respond to methylation differently than HEK-293 cells. The effects of i.v. administered mocetinostat, NVS200 and vorinostat on QTcF interval, HDAC activity, gene transcription and tissue drug concentration were determined in conscious male beagle dogs (n = 3/compound) necropsied 16 h after a second dose when the maximal QTcF increase occurred. QTcF increased gradually to +40 ms (17% over baseline) from the 13th to the 19th hour after the first NVS200 (0.25 mg/kg/day/2 days, i.v.) dose. During this time interval, NVS200 plasma concentrations were almost nil (the peak occurring within 1 h after dosing and declining by 90% 2 h later). A few hours prior to the peak QTcF, heart rate increased by ~ 50 beats/min over baseline. Vorinostat (50 mg/kg, i.v.) behaved similarly to NVS200: delayed QTcF increase (+20 ms, 8.3% over baseline) between the 13th and the 14th hour following dosing. This effect was preceded by a +40 beats/min increase in heart rate. Similarly, mocetinostat increased heart rate by 35 beats/min but produced a smaller increase in QTcF (15 ms; 6.25% over the baseline) 14 -- 19 h post-dose. Mocetinostat, but not vorinostat, decreased HDAC activity by ~ 50% in cytoplasmic and nuclear extracts from cardiac left ventricular wall and apex tissues. QTcF prolongation correlated with the occurrence of dysregulation of genes intervening in the synthesis of proteins (microtubules, associated transport motors, GTPases, endosomal adaptor proteins, sorting nexins and membrane anchoring proteins) ensuring intracellular trafficking and tethering of ion channels to the sarcolemma. For NVS200, these include CAV3 (responsible for LQT9), which tethers hERG (KCNH2; responsible for LQT2) and ANK3 (responsible for LQT4), which tethers the Na+/Ca++ exchanger to the cell outer membrane. Mocetinostat treatment affected the expression of SCN1B gene, which regulates SCN5A protein

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(Nav1.5 responsible for LQT3) expression and gating. NVS200-induced upregulation of tubulin and dynein genes is likely due to HDAC6-inhibiting activity since mocetinostat, lacking the latter property, did not produce this effect. HDAC6 is a microtubule-associated deacetylase that binds dynein motors and regulates endocytic trafficking. There was also a direct transcriptional dysregulation of other K+ channel genes such as KCNJ8/SUR2 (encoding for the Kir 6.1 channel) and KCNMB4 (Maxi-K channel). Overall, the dog studies indicate that marked QTc prolongation produced by NVS200 and vorinostat but not mocetinostat may be related to inhibition of HDACs, which regulate gene transcription responsible for trafficking and tethering ion channels to the cardiomyocyte sarcolemma. SP approaches to determine the potential adverse effects of epigenetic drugs require targeted in vitro and in vivo approaches. In particular: i) In vitro findings may be cell-line- and/or promoterdependent implying that the cell line selected for human outcome purposes studies should recapitulate mechanisms of adult human cardiomyocyte. ii) Transcriptomics studies in tissues sampled from pharmacodynamic experiments can provide information on cellular mechanisms of action. iii) Maximal in vivo effects (e.g., QTcF) of epigenetic medicines may not correlate with drug plasma Cmax and Tmax. iv) In vivo effects may be treatment duration- dependent. v) The mechanism of QTcF prolongation by HDACis appears to result from reduced expression of functional cardiac (in particular, hERG) ion channels in the cardiomyocyte sarcolemma.


Expanding safety pharmacology frontiers

Functional food safety. Dr Roberta Bradford, Safety & Environmental Assurance Centre Unilever, Bedford, UK


For the International Food Information Council, functional foods are foods or dietary components providing a health benefit beyond basic nutritional properties. For the European Food Safety Authority (EFSA), these products should beneficially affect targeted body functions beyond adequate nutritional characteristics resulting in health improvement, well-being and/or reduction in disease risks. They should not be used in pill or capsule formulations and any claimed beneficial effects should occur with amounts normally consumed in diet. Japan is one of the few countries, which requires a marketing approval by the Ministry of Health Labor and Welfare for functional foods, which are referred to as Foods for Specified Health Use [42]. The functional ingredients of these products should maintain or regulate specific body functions or parameters (e.g., gastrointestinal well-being, blood pressure or cholesterol within physiological levels).

Functional foods are composed of basic (carrots containing the antioxidant b-carotene) or processed foods (oat bran cereal containing b-glucan for control of cholesterol and blood glucose; fruit juices with added vitamins or minerals), foods with naturally enhanced functional components (tomatoes containing high levels of the antioxidant carotenoid lycopene; oat bran with higher b-glucan levels, eggs with omega-3 acids obtained from hens fed with flax [linseed]), and isolated, purified preparations of active food ingredients (omega-3 acids DHA [docosahexaenoic acid] and EPA [eicosapentaenoic acid] from fish oils, phytosterols). Functional food regulations are handled by EFSA in the European Union; FDA ([Everything Added to Food in the US] list) in the USA; Food Standard Code in Australia and New Zealand, and the state Food and Drug Administration, in the Republic of China. The Food and Agriculture Organization of the United Nations (FAO) and WHO have established ‘The Codex Alimentarius Commission’ for the development of global food standards. Regulations concerning functional foods containing intentionally added chemicals require safety demonstration by the manufacturer. Evidence of chemical safety can be provided by experts advising regulatory authorities (e.g., EFSA panels, the Joint FAO/WHO Expert Committee on Food Additives [JECFA]) or the production company. Preapproval or authorization is required for addition (e.g., additives, veterinary drugs) or presence (e.g., pesticides) of certain chemicals. In Europe, nutrition and health claims made on foods require approval by EFSA, which considers foods or ingredients as novel if they were not consumed to a significant degree by humans within the European Union before May 15, 1997. These products must be demonstrated safe and properly labeled to avoid consumer misleading. This is achieved by providing novel food specifications, quantity of appropriate nutritional ingredients (e.g., no excessive salt content), production process, history of the source (if used to produce the novel food), anticipated human intake and use, information on previous human exposure, nutritional, toxicological and microbiological data, guarantees of compatibility with product specifications at the consumption time and established quality control methods. Special information may be requested for foods from genetically modified organisms. In the USA, foods generally recognized as safe (GRAS) are foods relying upon a common and safe use history. GRAS may be FDA-affirmed GRAS (FDA approved and listed in the Federal Registry); self-affirmed GRAS (on data reviewed by qualified experts). However, the later classification can be challenged by the FDA. The FDA--non-approved GRAS may be objected or not by the FDA. Information for GRAS substance evaluation are use history, consumption by USA and non-USA populations, available epidemiology data, product specifications, consumption amounts, product composition and specifications, contaminant identification, analytical procedures for the identification of components, manufacturing

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processes, production flow diagram, GMP compliance, process reproducibility, data on five manufactured batches meeting specifications, safety and toxicology data, pharmacological and clinical studies, calculation of 90th percentile consumption, and comparison of exposure to the NOAEL (No Observed Adverse Effect Level) determined in toxicology studies. From a toxicological perspective, foods may contain a large variety of materials, some of which may confer potential liabilities. A four-step risk assessment paradigm is applied to foods (hazard identification, hazard characterization, exposure assessment, risk characterization). This is designed to ensure that their use is safe and does not present undue risk for consumers [43]. Hazard identification activities initiate with the collection and robustness of pertinent literature data. If nothing satisfactory is found, acute toxicity, allergy (type I), teratogenicity, genotoxicity, carcinogenicity and other toxicological tests (e.g., systemic subchronic and chronic 28- or 90-day toxicity studies, toxicokinetics) conducted in accordance with Organization for Economic Co-operation and Development guidelines are used to characterize potential hazards. Their goal is the determination of the acceptable daily (ADI) and tolerable daily intake (TDI), which estimate, respectively, the amount of beneficial chemicals or contaminants (usually expressed in mg/kg body weight/day) that human subjects can consume daily over a lifetime without appreciable health risk. ADIs and TDIs are established by recognized and independent experts or regulatory agencies (e.g., JECFA, EFSA). Risk characterization is provided by a margin of safety for a food, which is given by the ratio NOAEL/exposure. Generally, this safety parameter for food ingredients or contaminants is set to a value > 100 in order to account for possible variability between the species used for toxicological studies and humans. The margin of exposure for ingredients or contaminants with unknown threshold effects should be set to be > 10,000. The safety assessment of novel whole foods or complex mixtures is generally initiated with a comparison to traditional counterparts (if available). The use of typical safety studies for allergenicity and fate in biological systems may be performed, but they may not be appropriate for whole foods. A historyof-safe-use approach is often more relevant. Human studies should also address the identification and characterization of effects, including claimed efficacy on target populations. Post-launch monitoring may also be used as a component of overall risk assessment [44]. Safety pharmacology and probiotics. Dr Magali Cordaillat-Simmons, Pharmabiotic Research Institute, Aurillac, France


Probiotics are live microorganisms (e.g., Lactobacillus bacteria, Bifidobacterium and Saccharomyces boulardii genera), which, when administered in adequate amounts, confer health 1276

benefits to hosts. They may be used as drugs, foods, food ingredients or dietary supplements. Their doses are expressed in colony-forming units (CFU)/day (e.g., 50 million, 1 trillion CFU/day) [45]. Live viruses, used for vaccine preparations, are not considered probiotics. Human and animal body cavities (e.g., oral, intestinal and vaginal tracts) are colonized by numerous species of bacteria living symbiotically with the host. The main physiological functions of these bacteria are metabolic (e.g., fermentation of non-digestible dietary residues, energy salvaging in short-chain acid form, vitamin K production), trophic (epithelial cell proliferation and differentiation control), immunomodulatory (homeostasis of immune system) and protective against pathogens (barrier effect and antimicrobial substance production). Dysbiosis (alteration of the composition in species types and/or relative quantities of microbiota) can be responsible for serious human pathologies (irritable bowel syndrome, inflammatory bowel disease, colon cancers, vaginitis, allergies, etc.). Probiotics for human use are expected to lack risks to consumers. SP investigations recommended by ICH S7A [46] are not per se designed for probiotic safety assessment. For instance, assays based on mechanism of action are not applicable to probiotics since the mechanism of their positive or negative biological effects is often unknown or poorly clarified. Moreover, the selection of the most appropriate animal models for determining adverse effects is virtually impossible since probiotic composition and function depend on host genetics and local environment, which render probiotic effects host-specific. An additional challenge is the determination of probiotic potency affording beneficial activity from dose--response relationships, as recommended by the S7A for pharmaceutical agents [46]. Indeed, for probiotics, the latter classical pharmacological relationship is generally bell-shaped or shows similar effects for various dose levels above a threshold. Often, the adverse effects observed with probiotic preparations are caused by excipients rather than the probiotic itself. Potential adverse effects of probiotics are those of any microorganism and can be predicted from the probiotic taxonomy, namely, translocation into tissues, development of resistance to human antimicrobial medicines, resistance transfer and presence of virulence factors in the preparations. Hence, standard S7A core battery studies [46] are not tailored to define these potential dangers. It should be emphasized that the majority of probiotic strains used for human consumption are from genera colonizing the human gastrointestinal tract and their long history of use by humans has not been accompanied by noticeable adverse cardiovascular, respiratory and central nervous system effects. If SP evaluations of probiotics have to be performed, assays tailored to the probiotic species should be selected. Translocation has the potential to cause organ infections. For instance, cardiac infection (endocarditis) was observed in a clinical trial of different strains of Lactobacillus but this condition could not be attributed to the

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probiotic investigated. Nevertheless, any new probiotic should be cleared for translocation potential [47,48] by using, for instance, the rat endocarditis model [49], which can detect the ability of probiotic strains to attain the systemic blood circulation from the gastrointestinal tract and adversely affect organ functions. Moreover, since probiotic preparations may per se express antibiotic/antifungal resistance, they should be free of the potential for transference of this property to endogenous commensal bacteria populations. All bacterial products used for food additives must be assessed by using internationally standardized methods for their susceptibility to a range of available antimicrobials of human or veterinary use. Hence, as a basic requirement, the minimum inhibitory concentration, expressed as mg/l or µg/ ml, of ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, quinupristin + dalfopristin, tetracycline and vancomycin [50] against the growth of the investigated probiotic strain should be determined. The potential for transferring virulence needs also investigation. If pathogenicity is limited to particular strains within the species and, if the mechanism underlying the pathogenicity is understood and testable, the taxonomic unit might be eligible for qualified presumption of safety status (QPS). However, such a status would be refused to microorganism groups in which the majority of members produce any form of adverse effect. Worldwide, probiotic strains used as food supplements are generally considered safe by the Agency for Healthcare Research and Quality (AHRQ), which has the mission to improve the quality, safety, efficiency and effectiveness of health care products for all Americans. This agency acknowledges that rare adverse events are of difficult assessment, and despite the substantial number of publications, the current literature cannot provide conclusive answers regarding the absolute safety of probiotic interventions [51]. A current issue for probiotics is the approach used to establish the safety of novel probiotic preparations intended to re-establish deficient microbial function in patients suffering from dysbiosis pathologies. If probiotic preparations are developed to cure, treat or prevent diseases, they should be referred to as medicinal probiotics or pharmabiotics, and, as such, be subjected to the same safety assessment procedures used for human medicines. Future studies on probiotics should specifically attempt to quantify potential adverse events such as infections and treatment failure in order to quantify the risk for participants in intervention studies. The measurement and report of critical outcomes, such as all causes of mortality, should also be integral provisions of primary study protocols. Probiotic safety is of continuous concern for governmental food agencies as indicated by the EFSA Guideline on Probiotics [51] despite their recognized QPS status. New species and strains of probiotic bacteria are constantly identified and developed for human use. However, prior to their incorporation into traditional foods, they should be scrupulously evaluated for efficacy and safety. In particular, these preparations should be

demonstrated to lack infection propensity and absence of potential genetic transmissibility to innocuous members of the commensal microbial community [48]. Finally, marketing companies should make efforts in clearly labeling and indicating the regulatory status of the microbial products (food supplements, food-grade organisms, or medicinal products) for their correct selection by consumers (healthy people or patients) and healthcare providers [45]. 5.

Expert opinion

The leading themes of the 13th annual meeting of the SPS were: i) novel drug mechanisms with a potential to confer safety liabilities to drugs; and ii) dairy products used to improve health state which are generally not systematically subjected to scrutiny for organ safety. In addition to these topics, the meeting offered many interesting highlights among which a presentation on the invention of the modern electrocardiography and on the CiPA initiative were selected for reviewing in this report. Willem Einthoven, the Dutch inventor of the quartz string electrocardiograph, succeeded in the early 20th century and for the first time in the history of medicine, to faithfully record the electrical activity of the human heart from skin electrodes. This innovative technology allowed the rapid collection of such a significant amount of clinical data that, in 1912, Sir Thomas Lewis affirmed that ‘the time is at hand, if it has not already come, when an examination of the heart is incomplete if this new method is neglected’ [52]. We owe Prof Einthoven and his contemporary investigators, Sir Thomas Lewis and Prof Frank Wilson, our deepest recognition for their passionate work on the science of the ECG, which is now a centric diagnostic means to assess cardiac electrophysiological safety of candidate drugs and detect and treat numerous human heart diseases. CiPA is a laudable initiative to be earnestly supported by the drug development community since it offers a unique opportunity for SP investigators to develop and implement 21st century screening platforms allowing a mechanistic assessment of human safety of drugs. Indeed, CiPA envisages the application of in vitro and in silico assays as chief investigational means to identify compounds possessing cardiac ion channel mechanisms that have the potential to cause dangerous proarrhythmic events [53,54]. However, having CiPA operational by July 2015 to replace the current S7B and E14 cardiac safety paradigms [25,26] may be a difficult task since the various components of the novel suite remain to be defined in detail and experimentally validated. For instance, protocols for the functional ionic current assays are still under discussion, and the computational simulation programs for reconstructing the ventricular action potential are not yet selected. Additionally, the currently available hSC-CM cell lines do not exhibit phenotypically homogenous electrophysiology, have not yet been optimized to fully recapitulate the electrophysiological features of adult native healthy human CMs and have been found not to

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I. Cavero

respond as expected to certain established proarrhythmic medicines [55,56]. Hence, for the moment, CiPA appears to be an attractive visionary SP paradigm rather than an impending validated research platform. Nevertheless, this initiative deserves all our efforts for a rapid and rational implementation [23]. A focused theme of the SP society meeting reviewed in this report concerns potential liability mechanisms of epigenetic drugs. Epigenetics is an exciting, large ‘druggable’ biological domain holding bona fide promises for curing numerous pathological conditions in an innovative manner since it aims to normalize altered gene transcription playing a causative and maintenance role in numerous pathological conditions and, in particular, in certain cancer types. Indeed, there are tumors, which respond poorly to conventional treatments, but are successfully treatable by HDACis, as highlighted for romidepsin in the 2012 SP Society annual meeting [8]. However, the use of these drugs targeting epigenetic mechanisms is often accompanied by severe adverse effects (e.g., nausea, anemia, thrombocytopenia, neutropenia and leukopenia), which patients tolerate with courage since no alternative efficacious medication exists for healing their serious and often terminal disease. These adverse effects are likely due to inhibition of protein-expressing mechanisms mediating not only the disease to be treated but ensuring also vital functions in the healthy cells. Thus, the adverse effects of currently used epigenetic drugs are the likely outcome of doses necessary to produce the desirable effect (inhibition of disease-enhanced production of certain proteins [e.g., histone deacetylation upregulation appears to be a common hallmark of numerous neoplasia conditions]). However, at the same time, these therapeutic doses reduce also the expression of gene products necessary to ensure the viability of healthy cell populations. A detailed presentation of the 2013 SP Society meeting (see Section 3.2) provided evidence that HDACis can cause dysregulation of the de novo synthesis and the intracellular tethering process of various cardiac ion channels and thereby significantly prolong cardiac repolarization in pharmacodynamic assays in conscious dogs. The demonstration of the biochemical mechanisms underlying phenotypic effects required the use of rather complicated, nonstandard in vitro SP assays performed in ventricular myocardium sampled in dogs undergoing necropsy after a suitable treatment period. For the moment, routinely used SP assays can only detect phenotypic manifestations, such as QTc prolongation, resulting from drug-altered protein expression. However, this approach is unsuitable for the drug optimization phase during which high-throughput platforms need to be used to select the best compound among numerous ones prepared by medicinal chemists [37]. Therefore, there is an impeding need for developing simple in vitro assays able to detect adverse effects resulting from drug-induced liabilities at the level of the molecular modulators of expression and trafficking mechanisms of the cardiac ion channels. In this perspective, it is germane to ask whether the CiPA paradigm has the theoretical ability to identify the latter liabilities, which have been 1278

proposed to account for the cardiac electrophysiological effects caused by various drugs such as pentamidine [38], As2O3 [40] and HDACi [35]. This issue was briefly examined in a report by Cavero and Holzgrefe [23] who proposed that the core CiPA electrophysiological assay in hSC-CMs could be tailored to identify drug-impaired expression and tethering of cardiac ion channel proteins. Theoretically, this would require either a prolonged exposure (e.g., 24 -- 48 h) of hSC-CMs to the test article or a prolonged passage of time before performing any electrophysiological and/or mechanical measurement if a test article, after a brief exposure, binds irreversibly to one (or more) key element(s) of the channel expression and trafficking chain. However, the canonical electrophysiological CiPA assay will be generally performed only after a short time exposure of hSC-CM to test articles since its main objective is the determination of drug effects on expressed cardiac ion channels [23]. Nevertheless, conscientious SP investigators generally adapt the experimental conditions of safety assays to drug properties. The requirement of a prolonged exposure and follow-up of effects produced by drugs interfering with protein expression and transport appears to be supported by three published experimental studies on pentamidine [57-59]. Indeed, in hSC-CMs, pentamidine (3 µM) reduced beating rate and elicited proarrhythmia from the 20th to the 120th hour after addition of the drug to the preparation [57]. In another study using also hSC-CMs [58], pentamidine (0.3 -- 30 µM) reduced contractile amplitude but not beating rate. However, in embryonic m(ouse)SC-CMs, it decreased beating rate and increased contractile amplitude. Similarly, in the latter preparation, pentamidine (20 µM) reduced markedly beating rate from a normalized baseline value of 100 to 6 and 4 beats per min, respectively, after 900 and 1200 min of incubation [59]. The common finding of these investigations is that the effects of pentamidine in SC-CMs requires prolonged exposure to become manifest. However, the effects of pentamidine in hSC-CMs span from a marked, to no, decrease in beating rate. This may be due to the features of the cell line used for the investigation and/or the experimental conditions used. If this were the case, it is uncertain whether the mechanism of the adverse effects of pemtamidine in healthy or diseased adult human heart is the same as that mediating the described phenotypic effects of pentamidine in presently available hSC-CMs for screening studies. It is regrettable that the majority of the SP studies performed in hSCCMs search for phenotypic drug effects resembling those observed in humans, rather than attempting to elucidate the underlying mechanism(s) mediating such effects. Indeed, true advancement in safety sciences needs the knowledge of liability mechanisms, as a recent excellent paper [60] did for the biochemical mechanisms accounting for the trafficking defects associated to the long QT syndrome of type 2 (LQTS2) modeled in LQTS2 human-induced pluripotent stem-cell cardiomyocytes. The second focused topic of the SP Society meeting concerned functional foods [61] and probiotic [62,63] products used

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13th Annual Meeting of the Safety Pharmacology Society

for health-promoting purposes. The organ safety assessment of these agents requires the use of tailored SP investigations since the traditional studies recommended by the ICH S7A [46] and S7B [21] guidelines were not intended to assess the safety of non-pharmaceutical products. However, traditional SP assays may be appropriate and required by regulatory authorities to assess the organ safety of these products if they are developed as medicinal products. The main take-home messages of the 13th annual meeting of the SPS concerned the novel CiPA paradigm [25] and the potential mechanisms conferring safety liability that characterize the emerging class of epigenetic pharmaceuticals. CiPA should be considered by all concerned stakeholders as an opportunity to move SP from present, predominant, traditional pharmacodynamics approaches to 21st century cuttingedge in silico and in vitro technologies. As a personal take-home message to my fellow SP investigators, I would like to propose the following thought of Abraham Lincoln: ‘Always bear in mind that your own resolution to succeed is more important than any one thing’. If this is done, the CiPA endeavor will not fail to become a SP success.

Acknowledgments The author thanks in particular Mr. H Holzgrefe for carefully reviewing all the manuscripts and the help of Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.






Cavero I, Crumb W. Safety Pharmacology Society: 5th Annual Meeting, 27 -- 29 September, 2005, Mannheim, Germany. Expert Opinion Drug Saf 2006;5:181-5 Cavero I. Safety Pharmacology Society: 6th Annual Meeting 26-28 September, 2006, San Diego, USA. Expert Opinion Drug Saf 2007;6:87-92 Cavero I. Safety Pharmacology Society: 7th Annual Meeting 19-20 September, 2007, Edinburgh, UK. Expert Opinion Drug Saf 2008;7:91-100 Cavero I. Safety Pharmacology Society: 8th Annual Meeting 22-25 September, 2008, Madison, USA. Expert Opinion Drug Saf 2009;8:1-11 Cavero I. Safety Pharmacology Society: 9th Annual Meeting 15-18 September, 2009, Strasbourg, France. Expert Opin Drug Saf 2010;9:365-78 Cavero I. 10th Annual Meeting of the Safety Pharmacology Society:

Drs R Bedford, K Bruse, M Cordaillat-Simmons, G Gintant, L Guo, S Spence, M Vos and A Yang for valued suggestions and material which allowed me improve specific sections of the manuscript. Additionally, I would like to extend my warm thanks, accompanied by my heartfelt excuses, to the speakers of the 2013 annual meeting of the SP Society, P Andersson, R Bialecki, B Rodriguez, H Caplain, MJ Engwall, A Giarola, SE Harding, P Hewitt, H Holzgrefe, DR Jones, GK Massey, K Prasad, D Puppala, G Ravel, W Redfern, J Reynold, A van den Berg, G Waldron, R Wallis, J Wan and R Weiss for generously providing to me their valued slide presentations which unfortunately I could not review in this report due to space limitations.

Declaration of interest The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Additionally, the content of this manuscript reflects exclusively the personal opinion of the author. hundred years ago. Card Electrophysiol Rev 2003;7:99-104

an overview. Expert Opin Drug Saf 2011;10:319-33 7.

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Cavero I. 2012 Annual Meeting of the Safety Pharmacology Society: spotlight on targeted oncology medicines. Expert Opin Drug Saf 2013;12:589-603


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13th Annual Meeting of the Safety Pharmacology Society


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Affiliation Icilio Cavero 54, rue de la Glacie`re, 75013 Paris, France Tel: +33 1 43 37 58 97; E-mail: [email protected]


13th Annual Meeting of the Safety Pharmacology Society: focus on novel technologies and safety pharmacology frontiers.

The 13th Annual Meeting of the Safety Pharmacology (SP) Society discussed novel therapeutic areas, recent regulatory developments, emerging biology te...
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