rifampin and mevastatin

The DPX-2 cell line, a derivative of HepG2 cells, harbors human PXR and a luciferase-linked CYP3A4 promoter. These cells were used in a panel of cell-based assays for a parallel assessment of CYP3A4 induction, metabolism, and inhibition at the cellular level. CYP3A4 induction in the DPX-2 cell line by various agents was monitored in 96-well plates by a luciferase-based transcriptional activation assay. Of the prototypical CYP3A4 inducers examined, all exhibited elevated luciferase activity in DPX-2 cells. CYP3A4 enzyme activity in noninduced and rifampicin-induced DPX-2 cells was also assessed using Vivid fluorogenic substrates. Significantly elevated CYP3A4 activity levels (2.8-fold +/- 0.2-fold above DMSO-treated cells) were found in DPX-2 cells after 48 hours of exposure to rifampicin, but were undetectable in parental HepG2 cells. Rifampicin-induced activity levels were found to be suitable for assessing the inhibitory potential of new chemical entities in downstream CYP3A4 inhibition assays. The elevated CYP3A4 activity was inhibited 85% by 10 microM ketoconazole. In addition, a cytotoxicity assay to correct for possible toxic effects of compounds at the cellular level was applied. The comparative data obtained with a combination of the above assays suggests that the application of several independent in vitro technologies used in DPX-2 cells is the best possible strategy for the assessment of the complex phenomena of CYP3A4 induction and inhibition.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Chromans; Clotrimazole; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dexamethasone; Dimethyl Sulfoxide; Enhancer Elements, Genetic; Enzyme Induction; Genes, Reporter; Genes, Synthetic; Humans; Ketoconazole; Liver Neoplasms; Lovastatin; Luciferases; Mifepristone; Neoplasm Proteins; Nifedipine; Omeprazole; Paclitaxel; Phenytoin; Pregnane X Receptor; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Recombinant Fusion Proteins; Rifampin; Thiazolidinediones; Transcription, Genetic; Troglitazone; Troleandomycin

Assessing the inducibility of CYP3A4 by various xenobiotics can predict potential drug interactions. In the present investigation, human hepatoma cells were stably integrated with either the CYP3A4 enhancer region and a luciferase reporter gene or the CYP3A4-luciferase construct and the human pregnane X receptor (PXR). Several colonies containing one to three copies of luciferase per cell were identified by Southern blot analysis. Those transformants producing high luciferase activity in response to rifampicin were used to standardize a 96-well plate screening system with minimal inter- and intraplate variability. Standardization also consisted of assessing viability of cells cultured in medium containing various serum concentrations. In cells maintained for 48 h in medium with less than 5% serum, a significant (p < 0.01) decline was observed in viability accompanied by altered induction. A defined serum-free medium also produced less viable cells but did not alter the inductive response. Treatment of transformants with various concentrations of rifampicin produced a dose-response curve with maximal induction at 10 microM (5.6 +/- 0.18- and 2.1 +/- 0.3-fold above dimethyl sulfoxide (DMSO)-treated cells in transformants with and without PXR, respectively). Of additional agents examined for their ability to induce CYP3A4, omeprazole (200 microM) was the most potent inducer (12.8 +/- 1.9- and 2.4 +/- 0.2-fold above DMSO-treated cells in transformants with and without PXR, respectively). Mifepristone and mevastatin produced modest induction (approximately 3-fold) in the cell line containing exogenous PXR, but produced less than 1.2-fold increases in cells lacking PXR. Thus, only potent inducers can be identified in the cell line without PXR. In contrast, cells containing the receptor can be used to rank CYP3A4 induction. Because a high volume of chemicals can be readily and accurately screened for their ability to induce CYP3A4 with this format, such a system could be valuable in the initial stages of preclinical drug development.

Topics: Base Sequence; Blotting, Northern; Cell Line, Transformed; Cell Survival; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; DNA Primers; Enzyme Induction; Genes, Reporter; Hepatocytes; Humans; Liver; Lovastatin; Luciferases; Mifepristone; Mixed Function Oxygenases; Omeprazole; Polychlorinated Dibenzodioxins; Pregnane X Receptor; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Rifampin; RNA, Messenger; Transcription, Genetic; Transfection