Integrated experimental-computational analysis of a HepaRG liver-islet microphysiological system for human-centric diabetes research.

Microphysiological systems (MPS) are powerful tools for emulating human physiology and replicating disease progression in vitro. MPS could be better predictors of human outcome than current animal models, but mechanistic interpretation and in vivo extrapolation of the experimental results remain significant challenges. Here, we address these challenges using an integrated experimental-computational approach. This approach allows for in silico representation and predictions of glucose metabolism in a previously reported MPS with two organ compartments (liver and pancreas) connected in a closed loop with circulating medium. We developed a computational model describing glucose metabolism over 15 days of culture in the MPS. The model was calibrated on an experiment-specific basis using data from seven experiments, where HepaRG single-liver or liver-islet cultures were exposed to both normal and hyperglycemic conditions resembling high blood glucose levels in diabetes. The calibrated models reproduced the fast (i.e. hourly) variations in glucose and insulin observed in the MPS experiments, as well as the long-term (i.e. over weeks) decline in both glucose tolerance and insulin secretion. We also investigated the behaviour of the system under hypoglycemia by simulating this condition in silico, and the model could correctly predict the glucose and insulin responses measured in new MPS experiments. Last, we used the computational model to translate the experimental results to humans, showing good agreement with published data of the glucose response to a meal in healthy subjects. The integrated experimental-computational framework opens new avenues for future investigations toward disease mechanisms and the development of new therapies for metabolic disorders.

Physiologically Based Pharmacokinetic Model of Itraconazole and Two of Its Metabolites to Improve the Predictions and the Mechanistic Understanding of CYP3A4 Drug-Drug Interactions.

Physiologically based pharmacokinetic (PBPK) modeling for itraconazole using a bottom-up approach is challenging, not only due to complex saturable pharmacokinetics (PK) and the presence of three metabolites exhibiting CYP3A4 inhibition, but also because of discrepancies in reported in vitro data. The overall objective of this study is to provide a comprehensive mechanistic PBPK model for itraconazole in order to increase the confidence in its drug-drug interaction (DDI) predictions. To achieve this, key in vitro and in vivo data for itraconazole and its major metabolites were generated. These data were crucial to developing a novel bottom-up PBPK model in Simcyp (Simcyp Ltd., Certara, Sheffield, United Kingdom) for itraconazole and two of its major metabolites: hydroxy-itraconazole (OH-ITZ) and keto-itraconazole (keto-ITZ). Performance of the model was validated using prespecified acceptance criteria against different dosing regimens, formulations for 29 PK, and DDI studies with midazolam and other CYP3A4 substrates. The main outcome is an accurate PBPK model that simultaneously predicts the PK profiles of itraconazole, OH-ITZ, and keto-ITZ. In addition, itraconazole DDIs with midazolam and other CYP3A4 substrates were successfully predicted within a 2-fold error. Prediction precision and bias of DDI expressed as geometric mean fold error were for the area under the concentration-time curve and peak concentration, 1.06 and 0.96, respectively. To conclude, in this paper a comprehensive data set for itraconazole and its metabolites is provided that enables bottom-up mechanism-based PBPK modeling. The presented model is applicable for studying the contribution from the metabolites and allows improved assessments of itraconazole DDI.

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.

A comparison of 4ß-hydroxycholesterol : cholesterol and 6ß-hydroxycortisol : cortisol as markers of CYP3A4 induction.

AIM: To compare plasma 4ß-hydroxycholesterol : cholesterol with urinary 6ß-hydroxycortisol : cortisol as markers of cytochrome P4503A4 activity before and after treatment with rifampicin for 2 weeks. METHOD: 6ß-hydroxycortisol and cortisol were determined by liquid chromatography tandem mass spectrometry and 4ß-hydroxycholesterol was determined by gas chromatography-mass spectrometry in three groups of healthy volunteers. RESULTS: Induction ratios for 6ß-hydroxycortisol : cortisol were 1.8, 3.9 and 4.5 for 20 mg day(-1) , 100 mg day(-1) or 500 mg day(-1) of rifampicin, respectively. The corresponding ratios for 4ß-hydroxycholesterol : cholesterol were 1.5, 2.4 and 3.8. CONCLUSIONS: Plasma 4ß-hydroxycholesterol : cholesterol gave similar induction ratios to urinary 6ß-hydroxycortisol : cortisol.

Optimized experimental design for the estimation of enzyme kinetic parameters: an experimental evaluation.

A set of compounds (n = 30), including traditional cytochrome P450 substrates and compounds from AstraZeneca's compound library, was used in an experimental evaluation of an optimal design approach (ODA) for the estimation of enzyme kinetic parameters (CL(int), V(max), and K(m)). A depletion method previously shown to provide reliable results, the multiple depletion curves method (MDCM), was used as reference. Experiments were conducted with human liver microsomes, and samples were analyzed using liquid chromatography-tandem mass spectrometry. CL(int) estimated with the ODA were in >90% of the cases within a 2-fold difference compared with MDCM estimates. In addition, good agreement was generally seen for V(max) and K(m) estimates between the two methods as >80% of the estimates were within or almost within a 2-fold difference. The variability in V(max) and K(m) estimates were generally higher than for CL(int) estimates. In addition, decreased substrate turnover considerably increased the variability in V(max) and K(m) estimates, whereas only a modest increase was observed for CL(int) estimates. The experimental design of using multiple starting concentrations for the estimation of enzyme kinetics was shown to be appropriate even when there was a limitation to the number of samples. The method allowed for good estimates of CL(int) and also for V(max) and K(m) in many cases. Hence, this approach is a good alternative for the estimation of enzyme kinetic parameters, especially if enzyme saturation and an assessment of a potential risk for nonlinear metabolism are of interest.

Primary Human Hepatocyte Spheroid Model as a 3D In Vitro Platform for Metabolism Studies.

3D cultures of primary human hepatocytes (PHH) are emerging as a more in vivo-like culture system than previously available hepatic models. This work describes the characterisation of drug metabolism in 3D PHH spheroids. Spheroids were formed from three different donors of PHH and the expression and activities of important cytochrome P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4) were maintained for up to 21 days after seeding. The activity of CYP2B6 and 3A4 decreased, while the activity of CYP2C9 and 2D6 increased over time (P < 0.05). For six test compounds, that are metabolised by multiple enzymes, intrinsic clearance (CLint) values were comparable to standard in vitro hepatic models and successfully predicted in vivo CLint within 3-fold from observed values for low clearance compounds. Remarkably, the metabolic turnover of these low clearance compounds was reproducibly measured using only 1-3 spheroids, each composed of 2000 cells. Importantly, metabolites identified in the spheroid cultures reproduced the major metabolites observed in vivo, both primary and secondary metabolites were captured. In summary, the 3D PHH spheroid model shows promise to be used in drug discovery projects to study drug metabolism, including unknown mechanisms, over an extended period of time.

4beta-hydroxycholesterol as an endogenous marker for CYP3A4/5 activity. Stability and half-life of elimination after induction with rifampicin.

AIMS: The oxysterol 4beta-hydroxycholesterol has been suggested as a marker for CYP3A4/5 activity. We have previously shown that plasma 4beta-hydroxycholesterol continues to increase for several weeks after maximal induction of CYP3A4/5 by carbamazepine at the dose given. In the present study we aimed to determine the time course of the decrease in plasma 4beta-hydroxycholesterol after termination of induction of CYP3A4/5 by rifampicin. An additional aim was to determine the variation in plasma level of 4beta-hydroxycholesterol with time in 12 untreated healthy volunteers. METHODS: Twenty-four healthy subjects were allocated into three study groups of equal sizes. The volunteers were treated with rifampicin (either 20 mg day(-1), 100 mg day(-1) or 500 mg day(-1)) for 2 weeks. Blood samples were taken before, during and after rifampicin treatment. In another group of 12 untreated volunteers blood samples were collected at different time points in order to determine the intraindividual variations in plasma 4beta-hydroxycholesterol concentrations. Plasma levels of 4beta-hydroxycholesterol were determined by isotope-dilution gas chromatography-mass spectrometry. RESULTS: Rifampicin treatment increased plasma 4beta-hydroxycholesterol levels. After termination of rifampicin treatment plasma levels of 4beta-hydroxycholesterol decreased slowly with an apparent half-life of 17 days. The intraindividual variation in plasma levels of 4beta-hydroxycholesterol in untreated subjects was low, with coefficients of variation of between 4.8 and 13.2% over a period of 3 months. CONCLUSIONS: After termination of induction of CYP3A4/5, plasma 4beta-hydroxycholesterol levels decreased slowly during 8 weeks. The half-life of elimination (17 days) resembled that of cholesterol rather than other oxysterols. The long half-life results in stable plasma concentrations with time.

The TM6SF2 E167K genetic variant induces lipid biosynthesis and reduces apolipoprotein B secretion in human hepatic 3D spheroids.

There is a high unmet need for developing treatments for nonalcoholic fatty liver disease (NAFLD), for which there are no approved drugs today. Here, we used a human in vitro disease model to understand mechanisms linked to genetic risk variants associated with NAFLD. The model is based on 3D spheroids from primary human hepatocytes from five different donors. Across these donors, we observed highly reproducible differences in the extent of steatosis induction, demonstrating that inter-donor variability is reflected in the in vitro model. Importantly, our data indicates that the genetic variant TM6SF2 E167K, previously associated with increased risk for NAFLD, induces increased hepatocyte fat content by reducing APOB particle secretion. Finally, differences in gene expression pathways involved in cholesterol, fatty acid and glucose metabolism between wild type and TM6SF2 E167K mutation carriers (N = 125) were confirmed in the in vitro model. Our data suggest that the 3D in vitro spheroids can be used to investigate the mechanisms underlying the association of human genetic variants associated with NAFLD. This model may also be suitable to discover new treatments against NAFLD.

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.

HepaRG cells as an in vitro model for evaluation of cytochrome P450 induction in humans.

HepaRG is a highly differentiated cell line that displays several hepatocyte-like functions, including drug-metabolizing enzymes. In this study, the HepaRG cells were characterized and evaluated as an in vitro model to predict cytochrome P450 (P450) enzyme induction of drugs in humans. Exposure of HepaRG cells to prototypical inducers resulted in induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA, as well as phenacetin O-dealkylase, bupropion hydroxylase, diclofenac 4'-hydroxylase, and midazolam 1'-hydroxylase activities. The observed induction is consistent with the previously reported expression of the nuclear receptors pregnane X receptor, constitutive androstane receptor, and aryl hydrocarbon receptor, which are necessary for a P450 induction response. To avoid problems with toxicity and solubility, the induction potency of test compounds was evaluated by calculating the concentrations leading to a 2-fold increase of baseline mRNA or enzyme activity levels (F(2) values) instead of EC(50) values from full dose-response curves. For CYP3A4 mRNA, the obtained F(2) values were related to the in vivo exposure [area under the plasma concentration versus time curve (AUC)] of the inducer (AUC/F(2)). This score was then correlated with the decrease in AUC for a CYP3A probe drug, administered before and after treatment with the inducing agent. By using this method an excellent correlation (R(2) = 0.863) was obtained, which implies that the degree of CYP3A induction in vivo can be predicted from CYP3A4 mRNA induction in HepaRG cells. The present study shows that HepaRG cells are a valuable model to be used for prediction of induction of drug-metabolizing P450 enzymes in vivo in humans.

Human hepatocytes and cytochrome P450-selective inhibitors predict variability in human drug exposure more accurately than human recombinant P450s.

BACKGROUND AND PURPOSE: Drugs metabolically eliminated by several enzymes are less vulnerable to variable compound exposure in patients due to drug-drug interactions (DDI) or if a polymorphic enzyme is involved in their elimination. Therefore, it is vital in drug discovery to accurately and efficiently estimate and optimize the metabolic elimination profile. EXPERIMENTAL APPROACH: CYP3A and/or CYP2D6 substrates with well described variability in vivo in humans due to CYP3A DDI and CYP2D6 polymorphism were selected for assessment of fraction metabolized by each enzyme (fmCYP ) in two in vitro systems: (i) human recombinant P450s (hrP450s) and (ii) human hepatocytes combined with selective P450 inhibitors. Increases in compound exposure in poor versus extensive CYP2D6 metabolizers and by the strong CYP3A inhibitor ketoconazole were mathematically modelled and predicted changes in exposure were compared with in vivo data. KEY RESULTS: Predicted changes in exposure were within twofold of reported in vivo values using fmCYP estimated in human hepatocytes and there was a strong linear correlation between predicted and observed changes in exposure (r2  = 0.83 for CYP3A, r2  = 0.82 for CYP2D6). Predictions using fmCYP in hrP450s were not as accurate (r2  = 0.55 for CYP3A, r2  = 0.20 for CYP2D6). CONCLUSIONS AND IMPLICATIONS: The results suggest that variability in human drug exposure due to DDI and enzyme polymorphism can be accurately predicted using fmCYP from human hepatocytes and CYP-selective inhibitors. This approach can be efficiently applied in drug discovery to aid optimization of candidate drugs with a favourable metabolic elimination profile and limited variability in patients.

Critical differences in drug metabolic properties of human hepatic cellular models, including primary human hepatocytes, stem cell derived hepatocytes, and hepatoma cell lines.

Primary human hepatocytes (PHH), HepaRG™, HepG2, and two sources of induced pluripotent stem cell (iPSC) derived hepatocytes were characterized regarding gene expression and function of key hepatic proteins, important for the metabolic fate of drugs. The gene expression PCA analysis showed a distance between the two iPSC derived hepatocytes as well as the HepG2 and HepaRG™ cells to the three PHH donors and PHH pool, which were clustered more closely together. Correlation-based hierarchical analysis clustered HepG2 close to the stem cell derived hepatocytes both when the expression of 91 genes related to liver function or only cytochrome P450 (P450) genes were analyzed indicating the non-liver feature and a similar low P450 profile in these cell models. The specific P450 activities and the metabolic pattern of well-characterized drug substances in the cell models demonstrated that iPSC derived hepatocytes had modest levels of CYP3A and CYP2C9, while CYP1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 were barely detectable. High expression of several extrahepatic P450s such as CYP1A1 and 1B1 detected in the stem cell derived hepatocytes may have significant effects on metabolite profiles. However, one of the iPSC derived hepatocytes demonstrated significant combined P450 and conjugating enzyme activity of certain drugs. HepaRG™ cells showed many metabolic properties similar to PHHs and will in many respects be a good model in studies of metabolic pathways and induction of drug metabolism whereas there is still ground to cover before iPSC derived hepatocytes will be seen as a substitute to PHH in drug metabolism studies.

Publisher Correction: Functional coupling of human pancreatic islets and liver spheroids on-a-chip: Towards a novel human ex vivo type 2 diabetes model.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Functional coupling of human pancreatic islets and liver spheroids on-a-chip: Towards a novel human ex vivo type 2 diabetes model.

Human in vitro physiological models studying disease and drug treatment effects are urgently needed as more relevant tools to identify new drug targets and therapies. We have developed a human microfluidic two-organ-chip model to study pancreatic islet-liver cross-talk based on insulin and glucose regulation. We have established a robust co-culture of human pancreatic islet microtissues and liver spheroids maintaining functional responses up to 15 days in an insulin-free medium. Functional coupling, demonstrated by insulin released from the islet microtissues in response to a glucose load applied in glucose tolerance tests on different days, promoted glucose uptake by the liver spheroids. Co-cultures maintained postprandial glucose concentrations in the circulation whereas glucose levels remained elevated in both single cultures. Thus, insulin secreted into the circulation stimulated glucose uptake by the liver spheroids, while the latter, in the absence of insulin, did not consume glucose as efficiently. As the glucose concentration fell, insulin secretion subsided, demonstrating a functional feedback loop between the liver and the insulin-secreting islet microtissues. Finally, inter-laboratory validation verified robustness and reproducibility. Further development of this model using tools inducing impaired glucose regulation should provide a unique in vitro system emulating human type 2 diabetes mellitus.

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.