Cytochrome P450 1A2 Messenger RNA is a More Reliable Marker than Cytochrome P450 1A2 Activity, Phenacetin O-Deethylation, for Assessment of Induction Potential of Drug-Metabolizing Enzymes Using HepaRG Cells.

BACKGROUND: The HepaRG cells have key drug metabolism functionalities comparable to those of primary human hepatocytes. Many studies have reported that this cell line can be used as a reliable in vitro model for human drug metabolism studies, including the assessment of cytochrome P450 (CYP) induction. OBJECTIVES: The objective of this study is to determine whether CYP mRNA level measurement is superior to the CYP enzyme activity measurement as a convenient high-throughput method for evaluating CYP induction potential using HepaRG cells. METHODS: QuantiGene Plex 2.0 Assay and LC/MS/MS. mRNA expression levels and enzyme activities of CYP1A2, CYP2B6, and CYP3A in HepaRG cells treated with prototypical inducers of each CYP isoform [omeprazole (OME) for CYP1A2, phenobarbital (PB) for CYP2B6, and rifampicin (RIF) for CYP3A] were evaluated. RESULTS: Although the activities of CYP2B6 and CYP3A were induced by treatment with PB and RIF, we found that the activity of phenacetin O-deethylase (PHOD), which is known as a marker of the activity of CYP1A2, was also enhanced by treatment with these non-CYP1A2 inducers in HepaRG cells. Based on previously published reports, we hypothesized that the expression ratio of CYP3A to CYP1A2 is much higher in HepaRG cells than in human hepatocytes; this may result in a nonnegligible contribution of CYP3A to the PHOD reaction in HepaRG cells. Studies using CYP3A inhibitor and pregnane X receptor-knockout HepaRG cells supported this hypothesis. CONCLUSION: The measurement of mRNA serves as a higher reliable indicator for the evaluation of CYP induction potential when using HepaRG cells.

New Screening Criteria Setting on Evaluation of Cytochrome P450 Induction Using HepaRG Cells with Multiplex Branched DNA Technologies in Early Drug Discovery.

BACKGROUND: Cytochrome P450 (CYP) enzymes are induced by some therapeutic drugs, leading to interactions reducing drug plasma concentrations. Recently, an assessment of CYP induction using messenger RNA (mRNA) levels has shown advantages over measurement of enzymatic activity; it has a larger dynamic range of induction and enables us to measure the intrinsic induction potential of time-dependent CYP inhibitors. In order to minimize the late-stage attrition of new chemical entities (NCE), it is important to evaluate CYP induction potency at mRNA levels in the early stage of drug development. OBJECTIVES: The aim of this study is to establish a new screening method to evaluate induction potency of CYP1A2, CYP2B6, and CYP3A4 at mRNA levels. METHODS: QuantiGene Plex 2.0 Assay using HepaRG cells. RESULTS: The results from our new CYP induction assay system corresponded well to the already reported results obtained by using human hepatocytes. The induction potency was evaluated by calculating the concentration of test compounds that gives 10% of positive control response (R10), which is measurable even when full dose-response curves cannot be obtained. Compared with the evaluation of CYP induction in human hepatocytes, the response at R10 in HepaRG cells suggested the possibility of exhibiting induction potency for corresponding CYPs. Interestingly, the results with our in-house 109 compounds showed that several compounds induced CYP1A2 or CYP2B6 expression without upregulation of CYP3A4. CONCLUSION: Our developed assay system, as well as the R10 value, is useful for evaluating the CYP induction potency of NCE in early drug discovery.

CYP3A4*16 and CYP3A4*18 alleles found in East Asians exhibit differential catalytic activities for seven CYP3A4 substrate drugs.

CYP3A4, the major form of cytochrome P450 (P450) expressed in the adult human liver, is involved in the metabolism of approximately 50% of commonly prescribed drugs. Several genetic polymorphisms in CYP3A4 are known to affect its catalytic activity and to contribute in part to interindividual differences in the pharmacokinetics and pharmacodynamics of CYP3A4 substrate drugs. In this study, catalytic activities of the two alleles found in East Asians, CYP3A4*16 (T185S) and CYP3A4*18 (L293P), were assessed using the following seven substrates: midazolam, carbamazepine, atorvastatin, paclitaxel, docetaxel, irinotecan, and terfenadine. The holoprotein levels of CYP3A4.16 and CYP3A4.18 were significantly higher and lower, respectively, than that of CYP3A4.1 when expressed in Sf21 insect cell microsomes together with human NADPH-P450 reductase. CYP3A4.16 exhibited intrinsic clearances (V(max)/K(m)) that were lowered considerably (by 84-60%) for metabolism of midazolam, carbamazepine, atorvastatin, paclitaxel, and irinotecan compared with CYP3A4.1 due to increased K(m) with or without decreased V(max) values, whereas no apparent decrease in intrinsic clearance was observed for docetaxel. On the other hand, K(m) values for CYP3A4.18 were comparable to those for CYP3A4.1 for all substrates except terfenadine; but V(max) values were lower for midazolam, paclitaxel, docetaxel, and irinotecan, resulting in partially reduced intrinsic clearance values (by 34-52%). These results demonstrated that the impacts of both alleles on CYP3A4 catalytic activities depend on the substrates used. Thus, to evaluate the influences of both alleles on the pharmacokinetics of CYP3A4-metabolized drugs and their drug-drug interactions, substrate drug-dependent characteristics should be considered for each drug.