Future
Editorial
Medicinal Chemistry
For reprint orders, please contact [email protected]
Modeling hepatic drug metabolism and toxicity: where are we heading? “
The need for more ‘personalized’ systems, that is, systems that incorporate both genotype (HLA alleles) and phenotype (disease, inflammation), is critically required if the in vitro detection of idiosyncratic liver toxicity is ever going to become reality.
”
Keywords • bioreactor • drug safety • hepatic • hepatotoxicity • liver • metabolism • microtissue • toxicity
Drug-induced toxicity constitutes a significant health burden. A study from our department suggested that 1 in 16 hospital admissions and 4% of the hospital bed capacity are due to drug side effects [1,2] . Based on these studies, we estimate that adverse drug reactions cost the NHS in England in excess of £637 million annually with other studies estimating the cost at £2 billion/year [3] . Toxicity issues account for approximately 21% drug attrition during drug development and current safety testing strategies require considerable animal use. During the drugdevelopment process, the majority of animal use is during the lead identification and lead optimization phases. Importantly, these phases have embedded within them testing/ screening for drug toxicity. It is estimated that, worldwide, approximately 10–14 million animals are used by the pharmaceutical industry in lead identification and lead optimization [4] . Despite such a profligate use of animals during these phases, over 90% of drug candidates that move into clinical trials and beyond will fail due to toxicity issues [5] . Alternative approaches that supersede animal testing and can meet industry and regulatory requirements are crucially required. Current high-throughput in vitro models to assess hepatotoxic potential of drug candidates suffer from major limitations arising from lack of optimal physiological and drugmetabolizing competence. As a consequence, in vitro–in vivo extrapolation with regards to pharmacokinetics and toxicity is also limited. The development of in vitro models with improved physiological recapitulation
10.4155/FMC.14.31 © 2014 Future Science Ltd
(flow, multiple cell types, cellular zonation and bile clearance) is required if accurate mechanistic drug safety studies are to be performed. Mathematical modeling techniques, if adopted early during the development of novel in vitro models, can enhance research, through directing experimentation to areas of importance or data deficiency and getting more out of each experiment. Drugs and chemical entities cause hepatotoxicity through a variety of mechanisms, including direct toxicity of the parent compound or stable metabolite(s), direct accumulation and organelle interaction, reactive metabolite formation or a combination of the two. An ideal physiologically based pharmacokinetic model coupled with a cellbased in vitro model, which aims to report on the total hepatotoxic potential of chemicals, should encompass the normal physiology of the human liver cell, competence in drug metabolism and relevant susceptibility to drug/metabolite-mediated toxicological processes. Current in vitro models tend to be simple 2D cultures of either primary hepatocytes or hepatocyte cell lines. More recently, hepatic spheroids or microtissues have been developed. These models provide advantages of being technically easier to operate and amenable to high-throughput screening (HTS) of compounds. However, they tend not to possess sufficient physiological and metabolic competence and, therefore, do not faithfully approximate the functioning liver in terms of physiology, drug metabolism, cell adaptation and toxicity outcomes. This leads to a physiological gap between in vitro models and liver
Future Med. Chem. (2014) 6(7), 725–727
Dominic P Williams* *MRC Centre for Drug Safety Science, Department of Molecular & Clinical Pharmacology, Institute of Translational Medicine, Sherrington Building, The University of Liverpool, Ashton Street, Liverpool, L69 3GE, UK Tel.: +44 151 794 5791 Fax: +44 151 794 5540 [email protected]
part of
ISSN 1756-8919
725
Editorial Williams that does not account for integration of adaptation or amplification processes or the inability to assess how minor, yet chronic, chemical stress could lead to major toxicity. In vitro hepatic hollow fiber bioreactors used for the development of bioartificial livers comprise complex chemical engineering technology coupled to physiological recapitulation (3D scaffold, flow, biliary excretion, oxygen, and so forth), have demonstrated far superior hepatic functionality when compared with 2D cell culture [6] . However, bioreactors have drawbacks that include high-complexity and low-throughput nature.
“
In order to close the gap between real physiology and in vitro systems, we need to understand the crucial processes underlying the retention of desired cell functionality.
”
Importantly, comparisons between novel, low-complexity (e.g., microtissues), high-complexity (e.g., bioreactors) in vitro systems and in vivo animal models are required in order to facilitate the understanding of the physiological factors that are required for in vivolike drug metabolism capability and adaptation or toxicity processes. The following section details some features that should be considered when developing in vitro models of hepatic drug metabolism and toxicity. In order to close the gap between real physiology and in vitro systems, we need to understand the crucial processes underlying the retention of desired cell functionality (e.g., drug metabolism, adaptation and mechanistically correct forms of cell death), consequently, which processes play a relatively minor part, and are not necessary for designing into a novel in vitro system. Physiological recapitulation Media flow
The importance of media flow is difficult to evaluate due to the multiple effects this can elicit in vitro. The positive aspects for inclusion of flow include the removal of metabolites and bile, which would otherwise be toxic to hepatocytes [7] . Failure to remove bile, in particular, could lead to the generation of artificially sensitive in vitro models. Flow also allows concentration gradients to be established, such as oxygen, nutrients or hormones, more correctly simulating a sinusoid. These concentration gradients subsequently allow phenotypic alteration of the hepatocytes, termed zonation, where the cells exposed to higher oxygen and nutrients at the inlet have altered drug metabolism functionality compared with the cells nearer the outlet, which are exposed to decreased concentrations. Mathematical models have been established, designed to enhance physiology in bioartificial livers [8,9] . Hepatocyte zonation can be achieved
726
Future Med. Chem. (2014) 6(7)
by setting operating parameters. For in vivo models this is 70 mmHg, which is lower than that required in vitro, because in vivo the red blood cells carry the oxygen so there is effectively controlled release. Zonation has been achieved in vitro [10,11] ; however, taking this to the next level is regulating the size of the zones. Difficulties concerning inclusion of media flow include the requirement for pumps and a sterile flow environment. At this moment, the biggest drawback is that flow systems are not amenable to HTS. The rate of flow is important, as hepatocytes are generally protected from flow or shear stress due to the lining of hepatic endothelial cells. Co-culture
There is a current trend for hepatic cell co-culture either in a 2D or 3D environment. On the most basic level, this consists of hepatocyte and Kupffer cell coculture and demonstration that trovafloxacin causes greater cytotoxicity in the presence of lipopolysaccharide in co-culture versus hepatocytes alone [12] , due to release of inflammatory mediators. Clearly, simply sensitizing cells is very different to understanding in vivo risk factors [13] , and demonstrating increased sensitivity to hepatotoxins does not necessarily mean an improved system. More interestingly, however, is the use of endothelial cells to enhance and prolong hepatocyte function, in particular, the use of liver sinusoidal endothelial cells [14] . Hepatocyte-fibroblast and liver sinusoidal endothelial cells crosstalk, through production of short-range paracrine signals, are able to prolong phenotypic function. Metabolic & toxicological recapitulation Metabolic functionality
The recapitulation of metabolic functionality within a novel in vitro system should be the absolute minimum requirement, with assessment of parent compound clearance and qualitative and quantitative similarity of metabolites produced and definition of bioactivation. This should be irrespective of cytochrome P450 mRNA or protein level – mRNA does not always translate to protein, and protein maybe present but non-functional – these should be seen as red herrings when assessing novel in vitro models. Bioactivation of drugs and chemicals should be assessed through correct endpoints also. Drug bioactivation does not equate to cytotoxicity, hence, bioactivation needs to be assessed through nucleophilic trapping (high resolution mass spectrometry) or covalent binding of radiolabeled drug. Toxicological functionality
This endpoint has been used incorrectly across numerous published articles. The allusion of correct metabo-
future science group
Modeling hepatic drug metabolism & toxicity: where are we heading?
lism and mechanism of toxicity through a quantitative similarity in cytotoxic endpoints (ATP, MTS, MTT or plasma membrane disruption, and so forth) is completely incorrect. These endpoints can be used as an initial assessment of whether toxicity is able to be observed in any novel system, but definitely not for quality assessment. An example of this taken from my own laboratory, while using the human liver cell line, HepG2, and exposing this to acetaminophen (paracetamol), we observed 2% turnover to only the sulphate metabolite after 72 h of incubation (no evidence of bioactivation), yet a ball-park cytotoxicity of 5–10 mM – the metabolic parameters do not match with the toxicological endpoint.
to be balanced by the importance of the information gained. Currently, in vitro systems for drug metabolism and toxicity are developing rapidly. However, the need for more ‘personalized’ systems, that is, systems that incorporate both genotype (HLA alleles) and pheno type (disease, inflammation), is critically required if the in vitro detection of idiosyncratic liver toxicity is ever going to become reality. Financial & competing interests disclosure
Conclusion The balance between recapitulation of hepatic functionality and the requirement for HTS systems has
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 t-estimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
References
8
Davidson AJ, Ellis MJ, Chaudhuri JB. A theoretical method to improve and optimize the design of bioartificial livers. Biotechnol. Bioeng. 106(6), 980–988 (2010).
9
Davies EC, Green CF, Taylor S, Williamson PR, Mottram DR, Pirmohamed M. Adverse drug reactions in hospital in-patients: a prospective analysis of 3695 patient-episodes. PLoS ONE 4(2), e4439 (2009).
Davidson AJ, Ellis MJ, Chaudhuri JB. A theoretical approach to zonation in a bioartificial liver. Biotechnol. Bioeng. 109(1), 234–243 (2012).
10
Boseley S. Adverse drug reactions cost NHS £2 billion. www.theguardian.com/society/2008/apr/03/nhs. drugsandalcohol
Allen JW, Bhatia SN. Formation of steady-state oxygen gradients in vitro: application to liver zonation. Biotechnol. Bioeng. 82(3), 253–262 (2003).
11
Allen JW, Khetani SR, Bhatia SN. In vitro zonation and toxicity in a hepatocyte bioreactor. Toxicol. Sci. 84(1), 110–119 (2005).
12
Messner S, Agarkova I, Moritz W, Kelm JM. Multi-cell type human liver microtissues for hepatotoxicity testing. Arch. Toxicol. 87(1), 209–213 (2013).
13
Ramm S, Mally A. Role of drug-independent stress factors in liver injury associated with diclofenac intake. Toxicology 312, 83–96 (2013).
14
March S, Hui EE, Underhill GH, Khetani S, Bhatia SN. Microenvironmental regulation of the sinusoidal endothelial cell phenotype in vitro. Hepatology 50(3), 920–928 (2009).
1
2
3
4
Pirmohamed M, James S, Meakin S et al. Adverse drug reactions as cause of admission to hospital: prospective analysis of 18 820 patients. BMJ 329(7456), 15–19 (2004).
Thomas S. Physiologically-based pharmacokinetic modelling for the reduction of animal use in the discovery of novel pharmaceuticals. Altern. Lab. Anim. 38(Suppl. 1), 81–85 (2010).
5
Kola I, Landis J. Can the pharmaceutical industry reduce attrition rates? Nat. Rev. Drug Discov. 3(8), 711–715 (2004).
6
Nibourg GA, Chamuleau RA, van Gulik TM, Hoekstra R. Proliferative human cell sources applied as biocomponent in bioartificial livers: a review. Expert Opin. Biol. Ther. 12(7), 905–921 (2012).
7
Editorial
Schulz S, Schmitt S, Wimmer R et al. Progressive stages of mitochondrial destruction caused by cell toxic bile salts. Biochim. Biophys. Acta 1828(9), 2121–2133 (2013).
future science group
www.future-science.com
727
Editorial
Medicinal Chemistry
For reprint orders, please contact [email protected]
Modeling hepatic drug metabolism and toxicity: where are we heading? “
The need for more ‘personalized’ systems, that is, systems that incorporate both genotype (HLA alleles) and phenotype (disease, inflammation), is critically required if the in vitro detection of idiosyncratic liver toxicity is ever going to become reality.
”
Keywords • bioreactor • drug safety • hepatic • hepatotoxicity • liver • metabolism • microtissue • toxicity
Drug-induced toxicity constitutes a significant health burden. A study from our department suggested that 1 in 16 hospital admissions and 4% of the hospital bed capacity are due to drug side effects [1,2] . Based on these studies, we estimate that adverse drug reactions cost the NHS in England in excess of £637 million annually with other studies estimating the cost at £2 billion/year [3] . Toxicity issues account for approximately 21% drug attrition during drug development and current safety testing strategies require considerable animal use. During the drugdevelopment process, the majority of animal use is during the lead identification and lead optimization phases. Importantly, these phases have embedded within them testing/ screening for drug toxicity. It is estimated that, worldwide, approximately 10–14 million animals are used by the pharmaceutical industry in lead identification and lead optimization [4] . Despite such a profligate use of animals during these phases, over 90% of drug candidates that move into clinical trials and beyond will fail due to toxicity issues [5] . Alternative approaches that supersede animal testing and can meet industry and regulatory requirements are crucially required. Current high-throughput in vitro models to assess hepatotoxic potential of drug candidates suffer from major limitations arising from lack of optimal physiological and drugmetabolizing competence. As a consequence, in vitro–in vivo extrapolation with regards to pharmacokinetics and toxicity is also limited. The development of in vitro models with improved physiological recapitulation
10.4155/FMC.14.31 © 2014 Future Science Ltd
(flow, multiple cell types, cellular zonation and bile clearance) is required if accurate mechanistic drug safety studies are to be performed. Mathematical modeling techniques, if adopted early during the development of novel in vitro models, can enhance research, through directing experimentation to areas of importance or data deficiency and getting more out of each experiment. Drugs and chemical entities cause hepatotoxicity through a variety of mechanisms, including direct toxicity of the parent compound or stable metabolite(s), direct accumulation and organelle interaction, reactive metabolite formation or a combination of the two. An ideal physiologically based pharmacokinetic model coupled with a cellbased in vitro model, which aims to report on the total hepatotoxic potential of chemicals, should encompass the normal physiology of the human liver cell, competence in drug metabolism and relevant susceptibility to drug/metabolite-mediated toxicological processes. Current in vitro models tend to be simple 2D cultures of either primary hepatocytes or hepatocyte cell lines. More recently, hepatic spheroids or microtissues have been developed. These models provide advantages of being technically easier to operate and amenable to high-throughput screening (HTS) of compounds. However, they tend not to possess sufficient physiological and metabolic competence and, therefore, do not faithfully approximate the functioning liver in terms of physiology, drug metabolism, cell adaptation and toxicity outcomes. This leads to a physiological gap between in vitro models and liver
Future Med. Chem. (2014) 6(7), 725–727
Dominic P Williams* *MRC Centre for Drug Safety Science, Department of Molecular & Clinical Pharmacology, Institute of Translational Medicine, Sherrington Building, The University of Liverpool, Ashton Street, Liverpool, L69 3GE, UK Tel.: +44 151 794 5791 Fax: +44 151 794 5540 [email protected]
part of
ISSN 1756-8919
725
Editorial Williams that does not account for integration of adaptation or amplification processes or the inability to assess how minor, yet chronic, chemical stress could lead to major toxicity. In vitro hepatic hollow fiber bioreactors used for the development of bioartificial livers comprise complex chemical engineering technology coupled to physiological recapitulation (3D scaffold, flow, biliary excretion, oxygen, and so forth), have demonstrated far superior hepatic functionality when compared with 2D cell culture [6] . However, bioreactors have drawbacks that include high-complexity and low-throughput nature.
“
In order to close the gap between real physiology and in vitro systems, we need to understand the crucial processes underlying the retention of desired cell functionality.
”
Importantly, comparisons between novel, low-complexity (e.g., microtissues), high-complexity (e.g., bioreactors) in vitro systems and in vivo animal models are required in order to facilitate the understanding of the physiological factors that are required for in vivolike drug metabolism capability and adaptation or toxicity processes. The following section details some features that should be considered when developing in vitro models of hepatic drug metabolism and toxicity. In order to close the gap between real physiology and in vitro systems, we need to understand the crucial processes underlying the retention of desired cell functionality (e.g., drug metabolism, adaptation and mechanistically correct forms of cell death), consequently, which processes play a relatively minor part, and are not necessary for designing into a novel in vitro system. Physiological recapitulation Media flow
The importance of media flow is difficult to evaluate due to the multiple effects this can elicit in vitro. The positive aspects for inclusion of flow include the removal of metabolites and bile, which would otherwise be toxic to hepatocytes [7] . Failure to remove bile, in particular, could lead to the generation of artificially sensitive in vitro models. Flow also allows concentration gradients to be established, such as oxygen, nutrients or hormones, more correctly simulating a sinusoid. These concentration gradients subsequently allow phenotypic alteration of the hepatocytes, termed zonation, where the cells exposed to higher oxygen and nutrients at the inlet have altered drug metabolism functionality compared with the cells nearer the outlet, which are exposed to decreased concentrations. Mathematical models have been established, designed to enhance physiology in bioartificial livers [8,9] . Hepatocyte zonation can be achieved
726
Future Med. Chem. (2014) 6(7)
by setting operating parameters. For in vivo models this is 70 mmHg, which is lower than that required in vitro, because in vivo the red blood cells carry the oxygen so there is effectively controlled release. Zonation has been achieved in vitro [10,11] ; however, taking this to the next level is regulating the size of the zones. Difficulties concerning inclusion of media flow include the requirement for pumps and a sterile flow environment. At this moment, the biggest drawback is that flow systems are not amenable to HTS. The rate of flow is important, as hepatocytes are generally protected from flow or shear stress due to the lining of hepatic endothelial cells. Co-culture
There is a current trend for hepatic cell co-culture either in a 2D or 3D environment. On the most basic level, this consists of hepatocyte and Kupffer cell coculture and demonstration that trovafloxacin causes greater cytotoxicity in the presence of lipopolysaccharide in co-culture versus hepatocytes alone [12] , due to release of inflammatory mediators. Clearly, simply sensitizing cells is very different to understanding in vivo risk factors [13] , and demonstrating increased sensitivity to hepatotoxins does not necessarily mean an improved system. More interestingly, however, is the use of endothelial cells to enhance and prolong hepatocyte function, in particular, the use of liver sinusoidal endothelial cells [14] . Hepatocyte-fibroblast and liver sinusoidal endothelial cells crosstalk, through production of short-range paracrine signals, are able to prolong phenotypic function. Metabolic & toxicological recapitulation Metabolic functionality
The recapitulation of metabolic functionality within a novel in vitro system should be the absolute minimum requirement, with assessment of parent compound clearance and qualitative and quantitative similarity of metabolites produced and definition of bioactivation. This should be irrespective of cytochrome P450 mRNA or protein level – mRNA does not always translate to protein, and protein maybe present but non-functional – these should be seen as red herrings when assessing novel in vitro models. Bioactivation of drugs and chemicals should be assessed through correct endpoints also. Drug bioactivation does not equate to cytotoxicity, hence, bioactivation needs to be assessed through nucleophilic trapping (high resolution mass spectrometry) or covalent binding of radiolabeled drug. Toxicological functionality
This endpoint has been used incorrectly across numerous published articles. The allusion of correct metabo-
future science group
Modeling hepatic drug metabolism & toxicity: where are we heading?
lism and mechanism of toxicity through a quantitative similarity in cytotoxic endpoints (ATP, MTS, MTT or plasma membrane disruption, and so forth) is completely incorrect. These endpoints can be used as an initial assessment of whether toxicity is able to be observed in any novel system, but definitely not for quality assessment. An example of this taken from my own laboratory, while using the human liver cell line, HepG2, and exposing this to acetaminophen (paracetamol), we observed 2% turnover to only the sulphate metabolite after 72 h of incubation (no evidence of bioactivation), yet a ball-park cytotoxicity of 5–10 mM – the metabolic parameters do not match with the toxicological endpoint.
to be balanced by the importance of the information gained. Currently, in vitro systems for drug metabolism and toxicity are developing rapidly. However, the need for more ‘personalized’ systems, that is, systems that incorporate both genotype (HLA alleles) and pheno type (disease, inflammation), is critically required if the in vitro detection of idiosyncratic liver toxicity is ever going to become reality. Financial & competing interests disclosure
Conclusion The balance between recapitulation of hepatic functionality and the requirement for HTS systems has
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 t-estimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
References
8
Davidson AJ, Ellis MJ, Chaudhuri JB. A theoretical method to improve and optimize the design of bioartificial livers. Biotechnol. Bioeng. 106(6), 980–988 (2010).
9
Davies EC, Green CF, Taylor S, Williamson PR, Mottram DR, Pirmohamed M. Adverse drug reactions in hospital in-patients: a prospective analysis of 3695 patient-episodes. PLoS ONE 4(2), e4439 (2009).
Davidson AJ, Ellis MJ, Chaudhuri JB. A theoretical approach to zonation in a bioartificial liver. Biotechnol. Bioeng. 109(1), 234–243 (2012).
10
Boseley S. Adverse drug reactions cost NHS £2 billion. www.theguardian.com/society/2008/apr/03/nhs. drugsandalcohol
Allen JW, Bhatia SN. Formation of steady-state oxygen gradients in vitro: application to liver zonation. Biotechnol. Bioeng. 82(3), 253–262 (2003).
11
Allen JW, Khetani SR, Bhatia SN. In vitro zonation and toxicity in a hepatocyte bioreactor. Toxicol. Sci. 84(1), 110–119 (2005).
12
Messner S, Agarkova I, Moritz W, Kelm JM. Multi-cell type human liver microtissues for hepatotoxicity testing. Arch. Toxicol. 87(1), 209–213 (2013).
13
Ramm S, Mally A. Role of drug-independent stress factors in liver injury associated with diclofenac intake. Toxicology 312, 83–96 (2013).
14
March S, Hui EE, Underhill GH, Khetani S, Bhatia SN. Microenvironmental regulation of the sinusoidal endothelial cell phenotype in vitro. Hepatology 50(3), 920–928 (2009).
1
2
3
4
Pirmohamed M, James S, Meakin S et al. Adverse drug reactions as cause of admission to hospital: prospective analysis of 18 820 patients. BMJ 329(7456), 15–19 (2004).
Thomas S. Physiologically-based pharmacokinetic modelling for the reduction of animal use in the discovery of novel pharmaceuticals. Altern. Lab. Anim. 38(Suppl. 1), 81–85 (2010).
5
Kola I, Landis J. Can the pharmaceutical industry reduce attrition rates? Nat. Rev. Drug Discov. 3(8), 711–715 (2004).
6
Nibourg GA, Chamuleau RA, van Gulik TM, Hoekstra R. Proliferative human cell sources applied as biocomponent in bioartificial livers: a review. Expert Opin. Biol. Ther. 12(7), 905–921 (2012).
7
Editorial
Schulz S, Schmitt S, Wimmer R et al. Progressive stages of mitochondrial destruction caused by cell toxic bile salts. Biochim. Biophys. Acta 1828(9), 2121–2133 (2013).
future science group
www.future-science.com
727
