Human Pregnane X Receptor (hPXR) Activation Assay in Stable Cell Lines

Analysis of pregnane X receptor (PXR, NR1I2) activation to determine induction of drug metabolizing enzymes and transporters and predict drug-drug interactions (DDIs) is a wildly used technique among in vitro assays. Direct assessment of PXR activation is a cell-based assay that requires two major components, the PXR and a reporter gene linked to the promoter and enhancer regions of the CYP3A gene. Because of species differences in the ligand binding region of PXR, the receptor from the species of interest should be used when assessing activation. At present, PXR activation determined in stable cell lines can be assessed in medium (96-well) to high throughput (384 to 1,536-well) systems. Assays involving stable cell lines allow for simultaneous detection of PXR activation, CYP3A metabolism and cytotoxicity in a single well of a multi-well plate. In this manner, compounds that are toxic and are both inducers and inhibitors of CYP3A are readily identified. Here, we provide comprehensive step-by-step instructions for the application of screening for human PXR activation using commercially available stable cell lines harboring the PXR and a luciferase reporter gene linked to the promoters of the human CYP3A gene. These instructions provide detailed information on how to thaw, culture, passage and seed the cells in 96 well plates to use for screening of new drug entities to determine their ability to activate PXR. Instructions will also be provided for assessing not only nuclear receptor activation but also cytotoxicity and CYP3A4 metabolism simultaneously in the stable transformants. Finally, methods are provided for interpreting the results generated in the cell lines and a mechanistic model described for predicting clinical drug-drug interactions. The basic protocol provided here for identifying new drugs with the ability to activate human PXR and subsequently cause P450 enzyme induction can be miniaturized for higher throughput and extended to PXR from other species and additional nuclear receptors.