An in-vitro cocktail assay for assessing compound-mediated inhibition of six major cytochrome P450 enzymes  

作　　者：Jing-Jing Wang Jian-Jun Guo Jenny Zhan Hai-Zhi Bu Jiunn H.Lin 

机构地区：[1]First Affiliated Hospital of Kunming Medical University [2]3D Biooptima Co.,Ltd

出　　处：《Journal of Pharmaceutical Analysis》2014年第4期270-278,共9页药物分析学报（英文版）

摘　　要：An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 （CYP） enzymes. This method employed a cocktail of six probe substrates （i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYPIA2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively） as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites （i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S- mephenytoin, dextrorphan and 1-OH-midazolam） in the incubates were quantified using LC-MS/MS methods either by an internal standard （IS） calibration curve or a simpfified analyte-to-IS peak area ratio approach. The results showed that the IC5o values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition.An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 （CYP） enzymes. This method employed a cocktail of six probe substrates （i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYPIA2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively） as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites （i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S- mephenytoin, dextrorphan and 1-OH-midazolam） in the incubates were quantified using LC-MS/MS methods either by an internal standard （IS） calibration curve or a simpfified analyte-to-IS peak area ratio approach. The results showed that the IC5o values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition.

关 键 词：LC-MS/MS Cytochrome P450 Cocktail-probe Inhibition assessment Drug screenning 

分 类 号：TQ460.1[化学工程—制药化工] Q55[生物学—生物化学]