Quantitative PCR assay for cytochromes P450 2B and 3A induction in rat precision-cut liver slices: correlation study with induction in vivo.
BACKGROUND
In drug development, new chemical entities that cause cytochrome P450 induction are considered to be undesirable since P450 induction is linked to tumor formation and may compromise the evaluation of drug safety when autoinduction results in poor drug exposure.


METHODS
We evaluated the use of the precision-cut liver slice as a model for measuring induction of cytochrome P450 in rats. Quantitative real-time reverse-transcription polymerase chain reaction was used to analyze the induction of selected forms of cytochrome P450 at the mRNA level. Firstly, the system was validated against known inducers of CYP2B and 3A. Subsequently, 26 proprietary compounds were tested in rat liver slices and rats in vivo for CYP2B and 3A induction.


RESULTS
Exposure of liver slices to the known CYP2B inducers phenobarbital, benzoyl-pyridine, cabarmazepine, metyrapone, RU486 and dexamethasone caused elevation of CYP2B1/2 expression 10- to 40-fold compared to the control values. The CYP3A inducers PCN, dexamethasone, nicardipine, nifedipine, clotrimazole and RU486 induced a 4- to 50-fold expression of CYP3A14. For 26 proprietary compounds, a correlation with an R(2) value of 0.74 was established between the induction of CYP2B expression in liver slices and that in rats in vivo. When liver slice results were used to predict the induction of CYP2B in rats in vivo, the success rate was 91%. The induction of CYP3A in rats in vivo was analyzed by Western blot, then quantified by densitometry. There was a good correlation between CYP3A induction in liver slices and induction in vivo as assessed by Western blot, with an 86% positive prediction rate.


CONCLUSIONS
The use of liver slices in combination with TaqMan technology provides a good model for predicting CYP induction in the rat. This method is useful for identifying compounds with CYP2B and 3A induction liability in the early phase of drug discovery.
Cui
Xiaoming
X
Department of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, K15-D209, 2015 Galloping Hill Rd., Kenilworth, NJ 07033, USA. xiaoming.cui@spcorp.com
Thomas
Ann
A
Han
Yi
Y
Palamanda
Jairam
J
Montgomery
Diana
D
White
Ronald E
RE
Morrison
Richard A
RA
Cheng
K-C
KC
D000465:Algorithms; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D002220:Carbamazepine / Q000494:pharmacology; D019362:Cytochrome P-450 CYP2B1 / Q000235:genetics / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D017931:DNA Primers / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008797:Metyrapone / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D015735:Mifepristone / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D012333:RNA, Messenger; D015735:Mifepristone; D002220:Carbamazepine; D003907:Dexamethasone; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A; C025619:steroid 16-beta-hydroxylase; D008797:Metyrapone