Cytochrome P450 gene induction in rats ex vivo assessed by quantitative real-time reverse transcriptase-polymerase chain reaction (TaqMan).
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 beta-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.
GlaxoSmithKline, Drug Metabolism and Pharmacokinetics, Ware, UK. sandra.j.baldwin@gsk.com
Sandra J
SJ
Baldwin
Jo L
JL
Bramhall
Charlotte A
CA
Ashby
Lin
L
Yue
Paul R
PR
Murdock
Steven R
SR
Hood
Andrew D
AD
Ayrton
Stephen E
SE
Clarke
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000235:genetics; D002384:Catalysis; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis / Q000235:genetics; D019362:Cytochrome P-450 CYP2B1 / Q000096:biosynthesis / Q000235:genetics; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D004790:Enzyme Induction; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D010634:Phenobarbital / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D019324:beta-Naphthoflavone / Q000494:pharmacology
D012333:RNA, Messenger; D019324:beta-Naphthoflavone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; D019914:Taq Polymerase; D010634:Phenobarbital