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Research ArticleArticle

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

Mei-Fei Yueh, Marleen Kawahara and Judy Raucy
Drug Metabolism and Disposition January 2005, 33 (1) 38-48; DOI: https://doi.org/10.1124/dmd.104.001594
Mei-Fei Yueh
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Marleen Kawahara
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Judy Raucy
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Abstract

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 (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 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 IC50 values of 0.3 μM for ketoconazole, 108 μM for erythromycin, and 15.5 μg/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.

Footnotes

  • This work was supported by National Institutes of Health Grants GM58287 (M.-F.Y.), GM49511 (J.R.), and AA08990 (J.R.) and by the Liver Transplant, Procurement, and Distribution System (N01-DK-9-2310).

  • doi:10.1124/dmd.104.001594.

  • ABBREVIATIONS: P450, cytochrome P450; PXR, pregnane X receptor; hPXR, human PXR; CAR, constitutive androstane receptor; hCAR, human CAR; luciferin BE, luciferin-6′ benzyl ether; ANF, α-naphthoflavone; PCN, pregnenolone 16α-carbonitrile; XREM, xenobiotic response element module; rRNA, recombinant RNA; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CITCO, 6-(4-chlorophenyl) imidazo [2,1-b][1,3] thiazole-5-carbaldehyde O-(3,4,dichlorobenzyl) oxime; HIV, human immunodeficiency virus; bp, base pair(s); RLU, relative light unit; AU, arbitrary unit(s); Ah, aryl hydrocarbon; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    • Received July 26, 2004.
    • Accepted September 30, 2004.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 33 (1)
Drug Metabolism and Disposition
Vol. 33, Issue 1
1 Jan 2005
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Research ArticleArticle

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

Mei-Fei Yueh, Marleen Kawahara and Judy Raucy
Drug Metabolism and Disposition January 1, 2005, 33 (1) 38-48; DOI: https://doi.org/10.1124/dmd.104.001594

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Research ArticleArticle

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

Mei-Fei Yueh, Marleen Kawahara and Judy Raucy
Drug Metabolism and Disposition January 1, 2005, 33 (1) 38-48; DOI: https://doi.org/10.1124/dmd.104.001594
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