Received for publication July 28, 2004.
Revised September 28, 2004.
Accepted for publication September 30, 2004.

HIGH VOLUME BIOASSAYS TO ASSESS CYP3A4-MEDIATED DRUG INTERACTIONS: INDUCTION AND INHIBITION IN A SINGLE CELL LINE

Abstract

Exposure to certain xenochemicals can alter the catalytic activity of the major drug-metabolizing enzyme, CYP3A4, either by enhancing expression of this P450 or inhibiting its activity. Such alterations can result in adverse consequences stemming from drug-drug interactions. A simplified and reliable tool for detecting the ability of candidate drugs to alter CYP3A4 levels or inhibit catalytic activity was developed by stable integration of human pregnane X receptor (hPXR) and a luciferase vector harboring the CYP3A4 enhancers. Treatment of stable transformants, namely DPX-2, with various concentrations of inducers including rifampicin, mifepristone, troglitazone, methoxychlor, and kava produced dose-dependent increases in luciferase expression (between 2 to 40 fold above DMSO treated cells). Northern blot analyses of CYP3A4 mRNA in DPX-2 cells exhibited a good correlation to results generated with the reporter gene assay (r²=0.5, p<0.01). Induction of CYP3A4 protein was examined by measuring catalytic activity with the CYP3A4 substrate, luciferin 6' benzyl ether (luciferin BE). Metabolism of luciferin BE by DPX2 cells was enhanced 5.2 fold above DMSO treated cells by treatment with rifampicin. Constitutive androstane receptor (CAR) -mediated regulation of CYP3A4 protein was addressed by measuring catalytic activity in a separate cell line over-expressing this receptor. Phenobarbital and dexamethasone produced 1.5 and 2.0-fold increases, respectively, above control in luciferin BE metabolism. To determine the utility of DPX2 cells for identifying inhibitors of CYP3A4 catabolism, luciferin BE activity was measured in the presence of various concentrations of ketoconazole, erythromycin, or kava. These agents exhibited dose dependent decreases in CYP3A4 activity with IC50 values of 0.3 µM for ketoconazole, 108 µM for erythromycin, and 15.5 µg/ml for kava. Collectively, DPX2 cells were used to identify xenobiotics that induce or inhibit CYP3A4 in a high throughput manner, demonstrating their applicability to early stage drug development.

Key words: CYP3A, drug interactions, enzyme induction, enzyme inhibitors, high throughput screening, nuclear receptors, PXR

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