A comprehensive assay for nine major cytochrome P450 enzymes activities with 16 probe reactions on human liver microsomes by a single LC/MS/MS run to support reliable in vitro inhibitory drug-drug interaction evaluation
A comprehensive assay for nine major cytochrome P450 enzymes activities with 16 probe reactions on human liver microsomes by a single LC/MS/MS run to support reliable in vitro inhibitory drug-drug interaction evaluation
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DOI：
10.3109/00498254.2015.1036954
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发表时间：
2015-01-01
期刊：
影响因子：
1.8
通讯作者：
Sun, Jianguo
中科院分区：
文献类型：
--
作者：
Peng, Ying;Wu, Hui;Sun, Jianguo
1.利用16种CYP催化的14种底物与3套鸡尾酒孵育装置的反应和一次LC/MS/MS分析，建立了一种同时表征外源化合物对人肝微粒体中九种主要CYP酶的抑制作用的综合方法。为了最大限度地减少有机溶剂、多不饱和脂肪酸和相互底物相互作用的影响，开发了三个鸡尾酒亚组：第一组由甲苯丁胺(CYP2C9)、S-美芬妥因(CYP2C19)、睾酮(CYP3A4)、右美沙芬(CYP2D6)组成；第二组由硝苯地平(CYP3A4)、咪达唑仑(CYP3A4)、香豆素(CYP2A6)、安非他酮(CYP2B6)、双氯芬酸(CYP2C9)组成；第三组由非那西汀(CYP1A2)、氯唑沙宗(CYP2E1)、奥美拉唑(CYP2C19和CYP3A4)、紫杉醇(CYC8)、(+)-呋喃西林(CYP2C6)组成。对于CYP2C9、CYP2C19、CYP2D6和CYP3A4，由于多个底物结合口袋和底物依赖抑制的现象，使用了多个探针底物。在一次LC/MS/MS运行中，采用极性切换模式同时分析所有探针代谢物。用12种具有良好特性的CYP抑制剂和两个新实体(GT0918，MDV3100)对该方法进行了单探针底物分析验证。鸡尾酒中各抑制剂的IC50值与单个探针药物的IC50值以及以往文献报道的值符合得很好。
1. A comprehensive method for the simultaneous characterization of xenobiotic compound inhibition of nine major CYP enzymes in human liver microsomes was established by using 16 CYP-catalyzed reactions of 14 probe substrates with three cocktail incubation sets and a single LC/MS/MS analysis.2. The three cocktail subgroups were developed to minimize the effects of organic solvents, polyunsaturated fatty acids and mutual substrate interactions: Group I was composed of tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), testosterone (CYP3A4), dextromethorphan (CYP2D6); Group II was composed of nifedipine (CYP3A4), midazolam (CYP3A4), coumarin (CYP2A6), bupropion (CYP2B6), diclofenac (CYP2C9); Group III was composed of phenacetin (CYP1A2), chlorzoxazone (CYP2E1), omeprazole (CYP2C19 and CYP3A4), paclitaxel (CYP2C8), (+)-bufuralol (CYP2D6). In the case of CYP2C9, CYP2C19, CYP2D6 and CYP3A4, multiple probe substrates were used due to the phenomenon of multiple substrate-binding pockets and substrate-dependent inhibition. All probe metabolites were simultaneously analyzed with a polarity switching mode in a single LC/MS/MS run.3. This method was validated against the single probe substrate assay using 12 wellcharacterized CYP inhibitors and two new entities (GT0918, MDV3100). The IC50 values of each inhibitor in the cocktail agreed well with that of the individual probe drug as well as with values reported in previous literatures.