Testino Samuel A, Patonay Gabor
Bioanalytical Department, Solvay Pharmaceuticals, Inc., 901 Sawyer Road, Marietta, GA 30062, USA.
J Pharm Biomed Anal. 2003 Jan 1;30(5):1459-67. doi: 10.1016/s0731-7085(02)00480-6.
A method has been developed for the high-throughput inhibition screening of the major human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) using an in vitro substrate cocktail and liquid chromatography-tandem mass spectrometry (LC-MS-MS). A cocktail consisting of the selective substrates phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), bufuralol (CYP2D6), and midazolam (CYP3A4) was incubated with human liver microsomes. The metabolic reactions were terminated with methanol containing dextrorphan as an internal standard. Following centrifugation, the supernatant was analyzed by LC-MS-MS employing a fast gradient. The concentrations of the substrate metabolites-paracetamol, 4-hydroxytolbutamide, 5-hydroxyomeprazole, 1'-hydroxybufuralol, and 1'-hydroxymidazolam-in each sample were determined by LC-MS-MS in a single assay. The method was validated by incubating known CYP inhibitors (furafylline, CYP1A2; sulfaphenazole, CYP2C9; s-mephenytoin, CYP2C19; quinidine, CYP2D6; and troleandomycin, CYP3A4) with the individual substrates they were known to inhibit and with the substrate cocktail. IC50s (microM) determined using the substrate cocktail were in good agreement with those obtained with individual substrates (furafylline, 2.9 vs. 2.0; sulfaphenazole, 0.75 vs. 0.72; s-mephenytoin, 170 vs. 180; quinidine, 0.17 vs. 0.24; troleandomycin, 2.6 vs. 3.2) and with previously reported values in the literature.
已开发出一种使用体外底物混合物和液相色谱 - 串联质谱法（LC-MS-MS）对主要人类细胞色素P450（CYP）酶（CYP1A2、CYP2C9、CYP2C19、CYP2D6和CYP3A4）进行高通量抑制筛选的方法。将由选择性底物非那西丁（CYP1A2）、甲苯磺丁脲（CYP2C9）、奥美拉唑（CYP2C19）、布福洛尔（CYP2D6）和咪达唑仑（CYP3A4）组成的混合物与人肝微粒体一起孵育。代谢反应以含有右啡烷作为内标的甲醇终止。离心后，采用快速梯度通过LC-MS-MS分析上清液。通过LC-MS-MS在一次测定中确定每个样品中底物代谢物——对乙酰氨基酚、4 - 羟基甲苯磺丁脲、5 - 羟基奥美拉唑、1'-羟基布福洛尔和1'-羟基咪达唑仑的浓度。通过将已知的CYP抑制剂（呋拉茶碱，CYP1A2；磺胺苯吡唑，CYP2C9；s - 美芬妥英，CYP2C19；奎尼丁，CYP2D6；和醋竹桃霉素，CYP3A4）与它们已知抑制的单个底物以及底物混合物一起孵育来验证该方法。使用底物混合物测定的IC50（微摩尔）与用单个底物获得的IC50（呋拉茶碱，2.9对2.0；磺胺苯吡唑，0.75对0.72；s - 美芬妥英，170对180；奎尼丁，0.17对0.24；醋竹桃霉素，2.6对3.2）以及文献中先前报道的值高度一致。
