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Research ArticleArticle

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

Sandra J. Baldwin, Jo L. Bramhall, Charlotte A. Ashby, Lin Yue, Paul R. Murdock, Steven R. Hood, Andrew D. Ayrton and Stephen E. Clarke
Drug Metabolism and Disposition June 2006, 34 (6) 1063-1069; DOI: https://doi.org/10.1124/dmd.105.008185
Sandra J. Baldwin
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Jo L. Bramhall
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Charlotte A. Ashby
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Lin Yue
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Paul R. Murdock
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Steven R. Hood
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Andrew D. Ayrton
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Stephen E. Clarke
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Abstract

Drug-induced changes in expression of cytochrome P450 (P450) genes are a significant issue in the preclinical development of pharmaceuticals. For example, preclinically, P450 induction can affect safety studies by reducing the systemic exposure of a compound undergoing toxicological evaluation, thus limiting the exposure that can be safely investigated in patients. Therefore, the induction potential of candidate drugs has been studied as part of the drug development process, typically using 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 P450 inducers β-napthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX), and clofibric acid (CLO) were analyzed for mRNA levels of CYP1A1, 1A2, 2B1, 2B2, 2E1, 3A2, 3A23, and 4A1 and compared with 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 that demonstrates the sensitivity and specificity of the TaqMan assay to distinguish between inducers and noninducers and that offers a highly specific alternative to the quantification of drug effects on P450 expression using immunodetection and substrate metabolism.

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  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

  • doi:10.1124/dmd.105.008185.

  • ABBREVIATIONS: P450, cytochrome P450; DDI, drug-drug interaction; NCE, new chemical entity; qRT-PCR, quantitative real-time reverse transcriptase-polymerase chain reaction; DEPC, diethyl pyrocarbonate; RT, reverse transcriptase; BNF, β-napthoflavone; PB, phenobarbital; DEX, dexamethasone; CLO, clofibric acid; Tm, melting temperature; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PXR, pregnane X receptor; CAR, constitutively activated/androstane receptor; EROD, 7-ethoxyresorufin O-dealkylation; test 6β-oh, testosterone 6β-hydroxylation; TDI, time-dependent inhibitor.

    • Received November 15, 2005.
    • Accepted March 1, 2006.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 34 (6)
Drug Metabolism and Disposition
Vol. 34, Issue 6
1 Jun 2006
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Research ArticleArticle

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

Sandra J. Baldwin, Jo L. Bramhall, Charlotte A. Ashby, Lin Yue, Paul R. Murdock, Steven R. Hood, Andrew D. Ayrton and Stephen E. Clarke
Drug Metabolism and Disposition June 1, 2006, 34 (6) 1063-1069; DOI: https://doi.org/10.1124/dmd.105.008185

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Research ArticleArticle

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

Sandra J. Baldwin, Jo L. Bramhall, Charlotte A. Ashby, Lin Yue, Paul R. Murdock, Steven R. Hood, Andrew D. Ayrton and Stephen E. Clarke
Drug Metabolism and Disposition June 1, 2006, 34 (6) 1063-1069; DOI: https://doi.org/10.1124/dmd.105.008185
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