Received for publication November 15, 2005.
Revised March 1, 2006.
Accepted for publication March 1, 2006.

CYTOCHROME P450 GENE INDUCTION IN RATS EX VIVO ASSESSED BY QUANTITATIVE REAL-TIME REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION (TAQMAN^®)

Abstract

Drug-induced changes in expression of cytochrome P450 (CYP) genes are a significant issue in the pre-clinical development of pharmaceuticals. For example, pre-clinically CYP induction can impact safety studies by reducing the systemic exposure of a compound undergoing toxicological evaluation, thus limiting the exposure that can be safely investigated in patients. Consequently, the induction potential of candidate drugs has been studied as part of the drug development process, typically utilising protein and/or catalytic end points. However, measuring changes in the levels of mRNA using TaqMan^® technology offers the opportunity to investigate this issue with the advantages of better dynamic range and specific enzyme identification. Here, we describe the TaqMan^® application to study ex vivo the P450 gene induction in the rat. Initially, livers from rats dosed with the prototypic CYP inducers -napthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) and clofibric acid (CLO) were analysed for mRNA levels of CYP1A1, 1A2, 2B1, 2B2, 2E1, 3A2, 3A23 and 4A1 and compared to control animals. The maximum fold induction of mRNA varied; 2500-fold for CYP1A1 with BNF, 680-fold for CYP2B1 with PB, 59-fold for CYP3A23 with DEX and 16-fold for CYP4A1 with CLO. This method was then applied to estimate the inductive potential of putative drug candidates undergoing rodent toxicological evaluation. We present a summary of these data which demonstrates the sensitivity and specificity of the TaqMan^® assay to distinguish between inducers and non-inducers and which offers a highly specific alternative to the quantification of drug effects on CYP expression using immunodetection and substrate metabolism.

Key words: CYP expression, CYP induction, cytochrome P450, drug development, drug-drug interactions, enzyme induction, induction

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