Kajsa P. Kanebratt

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 (F2 values) instead of EC50 values from full dose-response curves. For CYP3A4 mRNA, the obtained F2 values were related to the in vivo exposure [area under the plasma concentration versus time curve (AUC)] of the inducer (AUC/F2). 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 (R2 = 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.

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.

Cytochrome P450 Induction by Rifampicin in Healthy Subjects: Determination Using the Karolinska Cocktail and the Endogenous CYP3A4 Marker 4β‐Hydroxycholesterol

The Karolinska cocktail, comprising caffeine, losartan, omeprazole, and quinine, was given before and after administration of rifampicin (20, 100, or 500 mg daily) to measure induction of cytochrome P450 (P450) enzymes. Rifampicin was given for 14 days to eight healthy subjects (all of whom possessed at least one wild‐type CYP2C9 and one wild‐type CYP2C19 gene) in each dose group. 4β‐hydroxycholesterol was assessed as an endogenous marker of CYP3A4 induction. A fourfold induction of CYP3A4 was seen at the highest dose by both quinine:3′‐hydroxyquinine and 4β‐hydroxycholesterol measurements (P < 0.001). CYP3A4 was also induced at the two lower doses of rifampicin when measured by these two markers (P < 0.01 or P < 0.001). CYP1A2, CYP2C9, and CYP2C19 were induced after 500 mg rifampicin daily (1.2‐fold, P < 0.05; 1.4‐fold, P < 0.05; and 4.2‐fold, P < 0.01, respectively). In conclusion, we have shown that the Karolinska cocktail and 4β‐hydroxycholesterol can be used for an initial screening of the induction properties of a drug candidate.

4β‐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.

A comparison of 4β‐hydroxycholesterol : cholesterol and 6β‐hydroxycortisol : cortisol as markers of CYP3A4 induction

AIM To compare plasma 4β-hydroxycholesterol : cholesterol with urinary 6β-hydroxycortisol : cortisol as markers of cytochrome P4503A4 activity before and after treatment with rifampicin for 2 weeks. METHOD 6β-hydroxycortisol and cortisol were determined by liquid chromatography tandem mass spectrometry and 4β-hydroxycholesterol was determined by gas chromatography-mass spectrometry in three groups of healthy volunteers. RESULTS Induction ratios for 6β-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 4β-hydroxycholesterol : cholesterol were 1.5, 2.4 and 3.8. CONCLUSIONS Plasma 4β-hydroxycholesterol : cholesterol gave similar induction ratios to urinary 6β-hydroxycortisol : cortisol.

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.

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 4β-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.

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 AstraZenecas compound library, was used in an experimental evaluation of an optimal design approach (ODA) for the estimation of enzyme kinetic parameters (CLint, Vmax, and Km). 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. CLint 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 Vmax and Km estimates between the two methods as >80% of the estimates were within or almost within a 2-fold difference. The variability in Vmax and Km estimates were generally higher than for CLint estimates. In addition, decreased substrate turnover considerably increased the variability in Vmax and Km estimates, whereas only a modest increase was observed for CLint 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 CLint and also for Vmax and Km 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.

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

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