ABSTRACT
Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CL(int)). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kinetics of metabolite formation from phenacetin, tolbutamide, and alprazolam and midazolam, selected as substrates probes for CYP1A2, CYP2C9, and CYP3A4, respectively, were characterized in this in vitro system. The magnitude of the K(m) or S(50) values observed in Ad-P450 cells was similar to those found in the literature for other human liver-derived systems. For each substrate, CL(int) (or CL(max)), values from Ad-P450 systems were scaled to human hepatocytes in primary culture using the relative activity factor (RAF) approach. Scaled Ad-P450 CL(int) values were approximately 3- to 6-fold higher (for phenacetin O-deethylation, tolbutamide 4-hydroxylation, and alprazolam 4-hydroxyaltion) or lower (midazolam 1'-hydroxylation) than those reported for human cryopreserved hepatocytes in suspension. Comparison with the in vivo data reveals that Ad-P450 cells provide a favorable prediction of CL(int) for the substrates studied (in a range of 20-200% in vivo observed CL(int)). This is an improvement compared with the consistent underpredictions (<10-50% in in vivo observed CL(int)) found in cryopreserved hepatocyte studies with the same substrates. These results suggest that the Ad-P450 cell is a promising in vitro system for clearance predictions of P450-metabolized drugs.
Subject(s)
Adenoviridae/genetics , Alprazolam/pharmacokinetics , Cytochrome P-450 Enzyme System/genetics , Hepatocytes/metabolism , Midazolam/administration & dosage , Phenacetin/pharmacokinetics , Tolbutamide/pharmacokinetics , Base Sequence , Cell Line , DNA Primers , HumansABSTRACT
HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.
Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/metabolism , Drug Evaluation, Preclinical/methods , Enzyme Induction/drug effects , Gene Expression Regulation/drug effects , Xenobiotics/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Line, Transformed , Cells, Cultured , Cytochrome P-450 Enzyme System/genetics , Glucuronosyltransferase/biosynthesis , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Glutathione Transferase/biosynthesis , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Isoenzymes/biosynthesis , Isoenzymes/genetics , Isoenzymes/metabolism , Membrane Transport Proteins/biosynthesis , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Omeprazole/pharmacology , Phenobarbital/pharmacology , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/biosynthesis , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Reproducibility of Results , Rifampin/pharmacology , Time FactorsABSTRACT
A fast and sensitive method was developed for the assessment of CYP induction in human hepatocytes cultured in 96-well plates. The effects on CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 were examined by means of mass spectrometry and the well-known cocktail strategy. The performance of the method was tested by using prototypic inducers such as methylcolanthrene, phenobarbital and rifampicin. The method was shown to be robust and predictable for the in vitro induction potential studies of drugs.
