This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.

In contrast to microsomes, hepatocytes give a disproportionate prediction with increasing clearance suggesting limitations either at the active site, such as cofactor exhaustion, or with intracellular concentration equilibrium, such as rate-limiting cell permeability. A simple log linear empirical relationship can be used to correct hepatocyte predictions.

Although prediction of clearance using hepatocytes and liver microsomes has long played a decisive role in drug discovery, it is widely acknowledged that reliably accurate prediction is not yet achievable despite the predominance of hepatically cleared drugs. Physiologically mechanistic methodology tends to underpredict clearance by several fold, and empirical correction of this bias is confounded by imprecision across drugs. Understanding the causes of prediction uncertainty has been slow, possibly reflecting poor resolution of variables associated with donor source and experimental methods, particularly for the human situation. It has been reported that among published human hepatocyte predictions there was a tendency for underprediction to increase with increasing in vivo intrinsic clearance, suggesting an inherent limitation using this particular system. This implied an artifactual rate limitation in vitro, although preparative effects on cell stability and performance were not yet resolved from assay design limitations. Here, to resolve these issues further, we present an up-to-date and comprehensive examination of predictions from published rat as well as human studies (where = 128 and 101 hepatocytes and = 71 and 83 microsomes, respectively) to assess system performance more independently. We report a clear trend of increasing underprediction with increasing in vivo intrinsic clearance, which is similar both between species and between in vitro systems. Hence, prior concerns arising specifically from human in vitro systems may be unfounded and the focus of investigation in the future should be to minimize the potential in vitro assay limitations common to whole cells and subcellular fractions.

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The complexity of in vitro kinetic phenomena observed for CYP3A4 substrates (homo-or heterotropic cooperativity) confounds the prediction of drug-drug interactions, and an evaluation of alternative and/or pragmatic approaches and substrates is needed. The current study focused on the utility of the three most commonly used CYP3A4 in vitro probes for the prediction of 26 reported in vivo interactions with azole inhibitors (increase in area under the curve ranged from 1.2 to 24, 50% in the range of potent inhibition). In addition to midazolam, testosterone, and nifedipine, quinidine was explored as a more "pragmatic" substrate due to its kinetic properties and specificity toward CYP3A4 in comparison with CYP3A5. K i estimates obtained in human liver microsomes under standardized in vitro conditions for each of the four probes were used to determine the validity of substrate substitution in CYP3A4 drug-drug interaction prediction. Detailed inhibitor-related (microsomal binding, depletion over incubation time) and substrate-related factors (cooperativity, contribution of other metabolic pathways, or renal excretion) were incorporated in the assessment of the interaction potential. All four CYP3A4 probes predicted 69 to 81% of the interactions with azoles within 2-fold of the mean in vivo value. Comparison of simple and multisite mechanistic models and interaction prediction accuracy for each of the in vitro probes indicated that midazolam and quinidine in vitro data provided the best assessment of a potential interaction, with the lowest bias and the highest precision of the prediction. Further investigations with a wider range of inhibitors are required to substantiate these findings.CYP3A4 is the most abundant human P450 enzyme, metabolizing a wide range of structurally diverse therapeutic agents; hence, it is the target for many drug-drug interactions (DDI). A recent Food and Drug Administration report (Yuan et al., 2002) has shown testosterone to be the most commonly used in vitro CYP3A4 probe (50% of reported studies) in contrast to midazolam (15-20% of in vitro estimates of CYP3A4 activity) whereas nifedipine, felodipine, and erythromycin were used in less than 10% of studies. The existence of different substrate subgroups for CYP3A4 was based on correlation and cluster analysis of CYP3A4 inhibition data for a range of modifiers (Kenworthy et al., 1999). This was substantiated by others (Stresser et al., 2000) and further investigated in a number of detailed mechanistic kinetic studies that indicated the existence of distinct and preferential binding domains for each substrate subgroup, namely, midazolam, testosterone, and nifedipine. Due to the substrate-differential response observed for CYP3A4, the recommended approach to CYP3A4 DDI analysis is the use of multiprobes (Tucker et al., 2001;Bjornsson et al., 2003) where the lowest inhibition constant (K i ) obtained indicates the "worst case scenario" for a potential interaction. However, an inappropriate selection of a probe substrate may lead to fal...

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ABSTRACT:The hepatic uptake of quinine, fluvoxamine, and fluoxetine (0.1-10 M) was investigated with freshly isolated rat hepatocytes. The cell-to-medium concentration ratios (K p ) were concentration-dependent: the mean maximum K p values (at 0.1 M) were 150 (quinine), 500 (fluvoxamine), and 2000 (fluoxetine). There was also a large capacity site that was not saturable over the concentration range used (possibly partition into the phospholipid component of membranes); representing this site, the mean minimum K p values (at 10 M) were 30 (quinine), 200 (fluvoxamine), and 500 (fluoxetine). To eliminate concomitant metabolism, cells were pretreated with the irreversible P450 inhibitor, aminobenzotriazole. The saturable uptake was substantially eliminated after exposure to carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (ATP inhibitor). The difference between the maximum and minimum K p for these three amine drugs, as well as for dextromethorphan, propranolol, and imipramine, was within a limited range of 3-fold, indicating a common magnitude of saturable uptake. Basic, permeable drugs are expected to be sequestered into lysosomes, which actively maintain their low internal pH (ϳ5) using ATP, and this process is predictable from the combined effects of pH-driven ion accumulation and unsaturable binding representing partition into membranes. The resultant predicted maximum K p correlated strongly with the observed maximum K p . Thus, at low substrate concentrations, the fraction of drug unbound in the hepatocyte incubation (critical for assessing drug clearance and drug-drug interaction potential) may be dependent upon saturable as well as unsaturable binding, and for lipophilic, basic drugs, this can be readily estimated assuming a common degree of uptake into lysosomes.