Miller V P, Stresser D M, Blanchard A P, Turner S, Crespi C L
GENTEST Corporation, Woburn, Massachusetts 01801, USA.
Ann N Y Acad Sci. 2000;919:26-32. doi: 10.1111/j.1749-6632.2000.tb06864.x.
Rapid screening for cytochrome P450 inhibitors is part of the current paradigm for avoiding development of drugs likely to give clinical pharmacokinetic drug-drug interactions and associated toxicities. We have developed microtiter plate-based, direct, fluorometric assays for the activities of the principal human drug-metabolizing enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, as well as for CYP2A6, which is an important enzyme in environmental toxicology. These assays are rapid and compatible with existing high-throughput assay instrumentation. For CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6, the potency of enzyme inhibition (IC50) is consistent regardless of the probe substrate or assay method employed. In contrast, CYP3A4 inhibition for an individual inhibitor shows significant differences in potency (>300-fold) depending on the probe substrate being used. We have investigated these differences through the use of several structurally distinct fluorescent substrates for CYP3A4 and several classical substrate probes (e.g., testosterone, nifedipine, and midazolam), with a panel of known, clinically significant, CYP3A4 inhibitors. The use of multiple probe substrates appears to be needed to characterize the inhibition potential of xenobiotics for CYP3A4.
细胞色素P450抑制剂的快速筛选是当前避免开发可能导致临床药代动力学药物-药物相互作用及相关毒性的药物范式的一部分。我们已经开发了基于微量滴定板的直接荧光测定法，用于检测主要的人类药物代谢酶CYP1A2、CYP2C8、CYP2C9、CYP2C19、CYP2D6和CYP3A4的活性，以及用于检测环境毒理学中一种重要酶CYP2A6的活性。这些测定方法快速且与现有的高通量检测仪器兼容。对于CYP1A2、CYP2C8、CYP2C9、CYP2C19和CYP2D6，无论使用何种探针底物或测定方法，酶抑制效力（IC50）都是一致的。相比之下，对于单个抑制剂，CYP3A4的抑制效力根据所使用的探针底物显示出显著差异（>300倍）。我们通过使用几种结构不同的CYP3A4荧光底物和几种经典底物探针（如睾酮、硝苯地平和咪达唑仑）以及一组已知的、具有临床意义的CYP3A4抑制剂来研究这些差异。似乎需要使用多种探针底物来表征外源性物质对CYP3A4的抑制潜力。
