HIGH VOLUME BIOASSAYS TO ASSESS CYP3A4-MEDIATED DRUG INTERACTIONS: INDUCTION AND INHIBITION IN A SINGLE CELL LINE
| Autor: | Judy L. Raucy, Mei-Fei Yueh, Marleen Kawahara |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Pharmaceutical Science
Pharmacology Cell Line Xenobiotics Cytochrome P-450 Enzyme System Cell Line Tumor Cytochrome P-450 CYP3A Cytochrome P-450 Enzyme Inhibitors Humans Drug Interactions Luciferase Enzyme Inhibitors Cells Cultured Pregnane X receptor Reporter gene Dose-Response Relationship Drug biology CYP3A4 Cytochrome P450 Luciferin Biochemistry Cell culture Enzyme Induction biology.protein Biological Assay |
| Zdroj: | Drug Metabolism and Disposition. 33:38-48 |
| ISSN: | 1521-009X 0090-9556 |
| DOI: | 10.1124/dmd.104.001594 |
| Popis: | Exposure to certain xenochemicals can alter the catalytic activity of the major drug-metabolizing enzyme, CYP3A4, either by enhancing expression of this cytochrome 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 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- and 40-fold above dimethyl sulfoxide-treated cells). Northern blot analyses of CYP3A4 mRNA in DPX-2 cells exhibited a good correlation to results generated with the reporter gene assay (r(2) = 0.5, p0.01). 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 DPX-2 cells was enhanced 5.2-fold above dimethyl sulfoxide-treated cells by treatment with rifampicin. Constitutive androstane receptor-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 DPX-2 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 IC(50) values of 0.3 microM for ketoconazole, 108 microM for erythromycin, and 15.5 microg/ml for kava. Collectively, DPX-2 cells were used to identify xenobiotics that induce or inhibit CYP3A4 in a high throughput manner, demonstrating their applicability to early-stage drug development. |
| Databáze: | OpenAIRE |
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