Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion-CYP2B6, diclofenac-CYP2C9, omeprazole-CYP2C19, bufuralol-CYP2D6, and midazolam-CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

Clinically Relevant Cytochrome P450 3A4 Induction Mechanisms and Drug Screening in Three-Dimensional Spheroid Cultures of Primary Human Hepatocytes.

Cytochrome P450 (CYP) 3A4 induction is an important cause of drug-drug interactions, making early identification of drug candidates with CYP3A4 induction liability in drug development a prerequisite. Here, we present three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs) as a novel CYP3A4 induction screening model. Screening of 25 drugs (12 known CYP3A4 inducers in vivo and 13 negative controls) at physiologically relevant concentrations revealed a 100% sensitivity and 100% specificity of the system. Three of the in vivo CYP3A4 inducers displayed much higher CYP3A4 induction capacity in 3D spheroid cultures as compared with in two-dimensional (2D) monolayer cultures. Among those, we identified AZD1208, a proviral integration site for Moloney murine leukemia virus (PIM) kinase inhibitor terminated in phase I of development due to unexpected CYP3A4 autoinduction, as a CYP3A4 inducer only active in 3D spheroids but not in 2D monolayer cultures. Gene knockdown experiments revealed that AZD1208 requires pregnane X receptor (PXR) to induce CYP3A4. Rifampicin requires solely PXR to induce CYP3A4 and CYP2B6, while phenobarbital-mediated induction of these CYPs did not show absolute dependency on either PXR or constitutive androstane receptor (CAR), suggesting its ability to switch nuclear receptor activation. Mechanistic studies into AZD1208 uncovered an involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in CYP3A4 induction that is sensitive to the culture format used, as revealed by its inhibition of ERK1/2 Tyrosine 204 phosphorylation and sensitivity to epidermal growth factor (EGF) pressure. In line, we also identified lapatinib, a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/HER2) inhibitor, as another CYP3A4 inducer only active in 3D spheroid culture. Our findings offer insights into the pathways involved in CYP3A4 induction and suggest PHH spheroids for preclinical CYP3A4 induction screening.

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Managing the Risk of CYP3A Induction in Drug Development: A Strategic Approach.

Induction of cytochrome P450 (P450) can impact the efficacy and safety of drug molecules upon multiple dosing with coadministered drugs. This strategy is focused on CYP3A since the majority of clinically relevant cases of P450 induction are related to these enzymes. However, the in vitro evaluation of induction is applicable to other P450 enzymes; however, the in vivo relevance cannot be assessed because the scarcity of relevant clinical data. In the preclinical phase, compounds are screened using pregnane X receptor reporter gene assay, and if necessary structure-activity relationships (SAR) are developed. When projects progress toward the clinical phase, induction studies in a hepatocyte-derived model using HepaRG cells will generate enough robust data to assess the compound's induction liability in vivo. The sensitive CYP3A biomarker 4ß-hydroxycholesterol is built into the early clinical phase I studies for all candidates since rare cases of in vivo induction have been found without any induction alerts from the currently used in vitro methods. Using this model, the AstraZeneca induction strategy integrates in vitro assays and in vivo studies to make a comprehensive assessment of the induction potential of new chemical entities. Convincing data that support the validity of both the in vitro models and the use of the biomarker can be found in the scientific literature. However, regulatory authorities recommend the use of primary human hepatocytes and do not advise the use of sensitive biomarkers. Therefore, primary human hepatocytes and midazolam studies will be conducted during the clinical program as required for regulatory submission.

Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions.

Long-term chronic toxicity testing using human pluripotent stem cell-derived hepatocytes.

Human pluripotent stem cells (hPSC) have the potential to become important tools for the establishment of new models for in vitro drug testing of, for example, toxicity and pharmacological effects. Late-stage attrition in the pharmaceutical industry is to a large extent caused by selection of drug candidates using nonpredictive preclinical models that are not clinically relevant. The current hepatic in vivo and in vitro models show clear limitations, especially for studies of chronic hepatotoxicity. For these reasons, we evaluated the potential of using hPSC-derived hepatocytes for long-term exposure to toxic drugs. The differentiated hepatocytes were incubated with hepatotoxic compounds for up to 14 days, using a repeated-dose approach. The hPSC-derived hepatocytes became more sensitive to the toxic compounds after extended exposures and, in addition to conventional cytotoxicity, evidence of phospholipidosis and steatosis was also observed in the cells. This is, to the best of our knowledge, the first report of a long-term toxicity study using hPSC-derived hepatocytes, and the observations support further development and validation of hPSC-based toxicity models for evaluating novel drugs, chemicals, and cosmetics.

In vitro evaluation of major in vivo drug metabolic pathways using primary human hepatocytes and HepaRG cells in suspension and a dynamic three-dimensional bioreactor system.

Major human specific metabolites, not detected during in vivo and in vitro preclinical studies, may cause unexpected drug interactions and toxicity in human and delays in clinical programs. Thus, reliable preclinical tools for the detection of major human metabolites are of high importance. The aim of this study was to compare major drug metabolic pathways in HepaRG cells, a human hepatoma cell line, to fresh human hepatocytes, cryopreserved human hepatocytes, and human in vivo data. Furthermore, the maintenance of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) activities in a dynamic three-dimensional (3D) bioreactor were evaluated over time by using HepaRG cells and human hepatocytes. (14)C-diclofenac and a candidate from AstraZeneca's drug development program, (14)C-AZD6610, which are metabolized by P450 and UGT in vivo, were used as model substrates. The proportion of relevant biotransformation pathways of the investigated drug was clearly different in the various cell systems. The hydroxylation route was favored in primary human hepatocytes, whereas the glucuronidation route was favored in HepaRG cells. The human in vivo metabolite profile of AZD6610 was best represented by human hepatocytes, whereas all major diclofenac metabolites were detected in HepaRG cells. Moreover, the metabolite profiles in cryopreserved and fresh human hepatocytes were essentially the same. The liver bioreactor using both fresh human hepatocytes and HepaRG cells retained biotransformation capacity over 1 week. Thus, the incubation time can be increased from a few hours in suspension to several days in 3D cultures, which opens up for detection of metabolites from slowly metabolized drugs.

The HepaRG cell line: a unique in vitro tool for understanding drug metabolism and toxicology in human.

INTRODUCTION: HepaRG is a unique cell line showing a great plasticity, which differentiates to both canaliculae-like and hepatocyte-like cells. The long-term stability of key cell functions, for example, the drug-metabolizing cytochrome P450 (CYP) enzyme activities, in culture is especially useful in drug metabolism, disposition and toxicity studies. AREAS COVERED: This review describes features of the HepaRG cells focusing on drug-metabolizing enzymes and drug transporters, their functionality and regulation. Several applications in drug discovery studies are discussed and the use of HepaRG, as a human relevant predictive in vitro CYP induction model, is described. In addition, promising studies using HepaRG cells for understanding liver toxicity mechanisms by drug compounds are also discussed. EXPERT OPINION: HepaRG cells exhibit features which make them useful as an in vitro model for drug metabolism, disposition and toxicity studies, and could, for many studies, replace the requirement for primary human hepatocytes. Care should be taken since HepaRG cells are of a specific genotype which is reflected in the expression of drug processing proteins. The finding that HepaRG cells form tight junctions provides the basis for formation of functional canalicular structures and this should be investigated further to aid development of human relevant hepatic in vitro 2D and 3D models.

Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines - a report on the Vitrocellomics EU-project.

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.

Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies.

HepaRG cells, a newly developed human hepatoma cell line, differentiate into hepatocyte-like morphology by treatment with dimethyl sulfoxide (DMSO). The expression of cytochrome P450 (P450) enzymes, transporter proteins, and transcription factors was stable in differentiated HepaRG cells over a period of 6 weeks when cultured with DMSO. Compared with human hepatocytes, expression of P450 in HepaRG cells was in general lower with the exception for a considerably higher expression of CYP3A4 and CYP7A1. The expression of P450s generally decreased when DMSO was removed from the medium, whereas transporters and liver-specific factors were unaffected. The relative mRNA content of drug-metabolizing P450s displayed the highest resemblance between human hepatocytes and differentiated HepaRG cells 1 day after removal of DMSO from the medium. The metabolism of midazolam, naloxone, and clozapine in HepaRG cells was similar to human hepatocytes, indicating the function of CYP3A4, CYP1A2, and UDP-glucuronosyltransferase enzymes. However, the metabolism of 7-ethoxycoumarin and dextromethorphan was low, confirming low levels of CYP2E1 and CYP2D6 in HepaRG cells. The P450 probe substrates indicate a decrease in CYP1A2, CYP2B6, CYP2C9, and CYP3A4 activities in HepaRG cells 1 day after removal of DMSO from the medium. The activities were then relatively stable in DMSO-free medium for up to 14 days. Based on the stable expression of liver-specific functions over a long period in culture, the relative mRNA content of drug-metabolizing P450s, and metabolic properties, HepaRG cells provide a valuable in vitro model for human drug metabolism studies.

Evaluation of human liver slices and reporter gene assays as systems for predicting the cytochrome p450 induction potential of drugs in vivo in humans.

PURPOSE: The aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods. METHODS: The CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR). RESULTS: In human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUCtot to PXR EC50 values. CONCLUSIONS: Liver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.

Metabolic stability for drug discovery and development: pharmacokinetic and biochemical challenges.

Metabolic stability refers to the susceptibility of compounds to biotransformation in the context of selecting and/or designing drugs with favourable pharmacokinetic properties. Metabolic stability results are usually reported as measures of intrinsic clearance, from which secondary pharmacokinetic parameters such as bioavailability and half-life can be calculated when other data on volume of distribution and fraction absorbed are available. Since these parameters are very important in defining the pharmacological and toxicological profile of drugs as well as patient compliance, the pharmaceutical industry has a particular interest in optimising for metabolic stability during the drug discovery and development process. In the early phases of drug discovery, new chemical entities cannot be administered to humans; hence, predictions of these properties have to be made from in vivo animal, in vitro cellular/subcellular and computational systems. The utility of these systems to define the metabolic stability of compounds that is predictive of the human situation will be reviewed here. The timing of performing the studies in the discovery process and the impact of recent advances in research on drug absorption, distribution, metabolism and excretion (ADME) will be evaluated with respect to the scope and depth of metabolic stability issues. Quantitative prediction of in vivo clearance from in vitro metabolism data has, for many compounds, been shown to be poor in retrospective studies. One explanation for this may be that there are components used in the equations for scaling that are missing or uncertain and should be an area of more research. For example, as a result of increased biochemical understanding of drug metabolism, old assumptions (e.g. that the liver is the principal site of first-pass metabolism) need revision and new knowledge (e.g. the relationship between transporters and drug metabolising enzymes) needs to be incorporated into in vitro-in vivo correlation models. With ADME parameters increasingly being determined on automated platforms, instead of using results from high throughput screening (HTS) campaigns as simple go/no-go filters, the time saved and the many compounds analysed using the robots should be invested in careful processing of the data. A logical step would be to investigate the potential to construct computational models to understand the factors governing metabolic stability. A rational approach to the use of HTS assays should aim to screen for many properties (e.g. physicochemical parameters, absorption, metabolism, protein binding, pharmacokinetics in animals and pharmacology) in an integrated manner rather than screen against one property on many compounds, since it is likely that the final drug will represent a global average of these properties.