A reporter gene cellular model for evaluating induction of CYP3A4 by new chemical entities

Abstract

AbstractA cell‐based reporter gene assay to study CYP3A4 induction was developed in the present study. The pregnane X receptor (PXR) gene or variant (PXR2) was cloned into the pSG5 vector and cotransfected into HepG2 cells, with a construct, pGL3‐3A4‐Luc, containing the promoter/enhancer region of the CYP3A4 gene. The two systems were validated using rifampicin (RIF). The variant, PXR2, did not mediate induction of CYP3A4 by rifampicin (10 µM) whereas PXR showed dose‐dependent induction of CYP3A4, with a fold change of 40–60, compared to the vehicle control, 0.1% dimethylsulfoxide (DMSO). Further validation of the PXR/3A4 system was performed using other inducers of CYP3A4, after which CYP3A4 inducibility by a Wyeth compound, Tanaproget (TNPR), and the synthetic steroid, 3‐ketodesogestrel (3‐KDG), were assessed. At the highest concentrations tested, troleandomycin (TROL) and phenobarbital (PB) induced CYP3A4 by 15–25‐fold, while carbamazepine (CMZ), dexamethasone (DEX), and erythromycin (ERY) produced fold induction of <3. Pregnenolone 16‐α carbonitrile (PCN), a rat‐specific CYP3A inducer, did not induce CYP3A4. TNPR did not induce CYP3A4 at concentrations up to 10 µM while 3‐KDG produced a concentration‐dependent induction, with a fold change of 14 at 20 µM. These data show that the variant PXR2 is not activated by rifampicin. The PXR/3A4 system described can be used to study CYP3A4 induction and provides a robust, specific, and reproducible in vitro system that can be used to assess CYP3A4 inducibility by compounds in the drug development process. Drug Dev. Res. 67:470–475, 2006. © 2006 Wiley‐Liss, Inc.