High volume bioassays to assess CYP3A4-mediated drug interactions: Induction and inhibition in a single cell line
High volume bioassays to assess CYP3A4-mediated drug interactions: Induction and inhibition in a single cell line
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DOI：
10.1124/dmd.104.001594
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发表时间：
2005-01-01
影响因子：
3.9
通讯作者：
Raucy, J
中科院分区：
文献类型：
--
作者：
Yueh, MF;Kawahara, M;Raucy, J
暴露于某些异种化学品可以改变主要药物代谢酶CYP 3A 4的催化活性，通过增强这种细胞色素P450的表达或抑制其活性。这种改变可能导致药物相互作用引起的不良后果。通过稳定整合人胆甾烷X受体和携带CYP 3A 4增强子的荧光素酶载体，开发了一种用于检测候选药物改变CYP 3A 4水平或抑制催化活性的能力的简化且可靠的工具。稳定的转化体，即DPX-2，与各种浓度的诱导剂，包括利福平，米非司酮，曲格列酮，甲氧滴滴涕，和卡瓦产生剂量依赖性增加荧光素酶的表达（2- 40倍以上二甲基亚砜处理的细胞）。DPX-2细胞中CYP 3A 4 mRNA的北方印迹分析显示与报告基因分析产生的结果具有良好的相关性（r（2）= 0.5，p < 0.01）。通过测定CYP 3A 4底物6'苄基醚（Bisphenin BE）的催化活性，检查CYP 3A 4蛋白的诱导作用。通过用利福平处理，DPX-2细胞对荧光素BE的代谢比二甲基亚砜处理的细胞增强5.2倍。通过测量过表达该受体的单独细胞系中的催化活性，解决了组成型雄烷受体介导的CYP 3A 4蛋白调节。苯巴比妥和地塞米松分别使BE代谢增加1.5倍和2.0倍。为了确定DPX-2细胞用于鉴定CYP 3A 4催化剂抑制剂的效用，在存在不同浓度的酮康唑、红霉素或卡瓦的情况下测量了阿托伐他汀BE活性。这些药物表现出CYP 3A 4活性的剂量依赖性降低，酮康唑的IC 50值为0.3 μ M，红霉素为108 μ M，卡瓦为15.5 μ g/ml。总的来说，DPX-2细胞用于以高通量方式鉴定诱导或抑制CYP 3A 4的外源性物质，证明其适用于早期药物开发。
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, 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 mu M for ketoconazole, 108 mu M for erythromycin, and 15.5 mu g/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.