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.

AimsIn theory, the magnitude of an in vivo drug-drug interaction arising from the inhibition of metabolic clearance can be predicted using the ratio of inhibitor concentration ([I]) to inhibition constant ( K i ). The aim of this study was to construct a database for the prediction of drug-drug interactions from in vitro data and to evaluate the use of the various estimates for the inhibitor concentrations in the term [I]/ K i . MethodsOne hundred and ninety-three in vivo drug-drug interaction studies involving inhibition of CYP3A4, CYP2D6 or CYP2C9 were collated from the literature together with in vitro K i values and pharmacokinetic parameters for inhibitors, to allow calculation of average/maximum systemic plasma concentration during the dosing interval and maximum hepatic input plasma concentration (both total and unbound concentration). The observed increase in AUC (decreased clearance) was plotted against the estimated [I]/ K i ratio for qualitative zoning of the predictions. ResultsThe incidence of false negative predictions (AUC ratio > 2, [I]/ K i < 1) was largest using the average unbound plasma concentration and smallest using the hepatic input total plasma concentration of inhibitor for each of the CYP enzymes. Excluding mechanism-based inhibition, the use of total hepatic input concentration resulted in essentially no false negative predictions, though several false positive predictions (AUC ratio < 2, [I]/ K i > 1) were found. The incidence of true positive predictions (AUC ratio > 2, [I]/ K i > 1) was also highest using the total hepatic input concentration. ConclusionsThe use of the total hepatic input concentration of inhibitor together with in vitro K i values was the most successful method for the categorization of putative CYP inhibitors and for identifying negative drug-drug interactions. However this approach should be considered as an initial discriminating screen, as it is empirical and requires subsequent mechanistic studies to provide a comprehensive evaluation of a positive result.

ABSTRACT:Human liver microsomes have typically resulted in marked underprediction of in vivo human intrinsic clearance (CL int ); therefore, the utility of cryopreserved hepatocytes as an alternative in vitro system has become an important issue. In this study, 10 compounds (tolbutamide, diclofenac, S-warfarin, S-mephenytoin, dextromethorphan, bufuralol, quinidine, nifedipine, testosterone, and terfenadine) were selected as substrate probes for CYP2C9, 2C19, 2D6, and 3A4, and the kinetics of metabolite formation (n ‫؍‬ 14 pathways) were investigated in three individual lots of cryopreserved hepatocytes and in a pool of human liver microsomes. For the majority of the compounds, lower unbound K M or S 50 values were observed in hepatocytes compared with microsomes, on average by 50% over a 200-fold range (0.5-140 M). Expressed on an equivalent liver weight basis, a good correlation between microsomal and hepatocyte V max values was observed for most pathways greater than 5 orders of magnitude (0.16-216 nmol/min/g liver). Unbound hepatocyte CL int (CL int,u ) values, when scaled to the whole liver (range 0.38-4000 ml/min/kg), were on average 2.5-fold higher than microsomal CL int,u values, with the exception of tolbutamide and diclofenac, for which lower hepatocellular CL int,u values were observed. Hepatocyte predicted CL int values were compared with human in vivo CL int values, and to supplement our data, in vitro data from cryopreserved hepatocytes were collated from four other published sources. These data show that for 37 drugs, there is, on average, a 4.5-fold under-prediction of the in vivo CL int using cryopreserved hepatocytes, representing a significant reduction in prediction bias compared with human microsomes.Human liver microsomes have traditionally been the most commonly used in vitro system for the prediction of metabolic clearance, in particular, for new chemical entities within drug discovery programs in the pharmaceutical industry. However, the use of this system has typically resulted in an underestimation of clearance, as illustrated by a data set of 55 compounds in which a 9-fold under-prediction of the in vivo intrinsic clearance (CL int ) was observed . As a result of this, attention in recent years has been placed on the use of alternative in vitro systems for clearance prediction. Fresh human hepatocytes are envisaged as a potentially more accurate system because of the full complement of both phase I and phase II metabolizing enzymes, along with the presence of transporter proteins, which should result in drug concentrations within the hepatocyte that are equivalent to in vivo concentrations within the liver. However, the limited availability of fresh human tissue and the cost implications involved in the preparation of freshly isolated human hepatocytes have resulted in cryopreserved hepatocytes emerging as the favored alternative, which also have the added advantage of being readily available commercially and more convenient to use (Li et al., 1999).Two major issues concerning the use ...

AimsSuccess of the quantitative prediction of drug-drug interactions via inhibition of CYPmediated metabolism from the inhibitor concentration at the enzyme active site ([ I ]) and the in vitro inhibition constant ( K i ) is variable. The aim of this study was to examine the impact of the fraction of victim drug metabolized by a par ticular CYP ( f m CYP ) and the inhibitor absorption rate constant ( k a ) on prediction accuracy. MethodsDrug-drug interaction studies involving inhibition of CYP2C9, CYP2D6 and CYP3A4 ( n = 115) were investigated. Data on f m CYP for the probe substrates of each enzyme and k a values for the inhibitors were incorporated into in vivo predictions, alone or in combination, using either the maximum hepatic input or the average systemic plasma concentration as a surrogate for [ I ]. The success of prediction (AUC ratio predicted within twofold of in vivo value) was compared using nominal values of f m CY P = 1 and k a = 0.1 min -1 . ResultsThe incorporation of f m CYP values into in vivo predictions using the hepatic input plasma concentration resulted in 84% of studies within twofold of in vivo value. The effect of k a values alone significantly reduced the number of over-predictions for CYP2D6 and CYP3A4; however, less precision was observed compared with the f m CYP . The incorporation of both f m CYP and k a values resulted in 81% of studies within twofold of in vivo value. ConclusionsThe incorporation of substrate and inhibitor-related information, namely f m CYP and k a , markedly improved prediction of 115 interaction studies with CYP2C9, CYP2D6 and CYP3A4 in comparison with [ I ]/ K i ratio alone.

G protein coupled receptor 119 (GPR119) is viewed as an attractive target for the treatment of type 2 diabetes and other elements of the metabolic syndrome. During a program toward discovering agonists of GPR119, we herein describe optimization of an initial lead compound, 2, into a development candidate, 42. A key challenge in this program of work was the insolubility of the lead compound. Small-molecule crystallography was utilized to understand the intermolecular interactions in the solid state and resulted in a switch from an aryl sulphone to a 3-cyanopyridyl motif. The compound was shown to be effective in wild-type but not knockout animals, confirming that the biological effects were due to GPR119 agonism.