混合探针底物法同时预测细胞色素P450酶5种亚型的抑制作用

Simultaneous determination of the inhibitory potency of compounds on the activity of five cytochrome P-450 enzymes using a cocktail probe substrates method

本研究建立了混合探针底物法同时预测细胞色素P450(cytochrome P-450,CYP450)酶5种亚型的抑制作用。将CYP450酶5种亚型的特异性探针底物非那西丁(CYP1A2)、右美沙芬(CYP2D6)、甲苯磺丁脲(CYP2C9)、奥美拉唑(CYP2C19)及咪达唑仑(CYP3A4)同时与人肝微粒体在体外进行孵化反应,采用液相色谱-串联质谱分析方法同时测定5个特异性底物及其生成的对应的5种代谢产物(对乙酰氨基酚、右啡烷、4-羟基甲苯磺丁脲、5-羟基奥美拉唑和1′-羟基咪达唑仑)。并选择5种细胞色素P450酶的特异性抑制剂α-萘黄酮(CYP1A2)、奎尼丁(CYP2D6)、磺胺苯吡唑(CYP2C9)、氟康唑(CYP2C19)和酮康唑(CYP3A4)加入到其所对应酶的单个探针底物及混合探针底物的反应体系中,测定生成的代谢物,计算相应IC50值,对方法进行验证。5种特异性的抑制剂与混合探针底物反应后所得的IC50值和与单个探针底物反应后所得的IC50值具有很好的一致性且与文献报道的基本一致。本研究建立的混合探针底物法可以用于快速高通量地同时预测化合物对CYP450酶5种亚型活性的抑制作用。

This study developed a method for simultaneously assessing the inhibitory potency of compounds on five major cytochrome P-450 （CYP450） enzymes using a cocktail of probe substrates. A cocktail selective substrates consisting of the phenacetin （PN, CYP1A2 ）, dextromethorphan （DM, CYP2D6）, tolbutamide （TB, CYP2C9 ）, omeprazole （OPZ, CYP2CI9 ） and midazolam （MPZ, CYP3A4） was incubated with human liver microsomes. The concentrations of the substrate metabolites -- paracetamol, dextrorphan, 4-hydroxytolbutamide, 5-hydroxyomeprazole and 1＇-hydroxymidazolam were determined by LC/MS/MS in a single assay sample. The method was validated by incubating known CYP inhibitors- α-naphthoflavone （ANF, CYP1A2）, quinidine （QND, CYP2D6）, sulfaphenazole （SUL, CYP2C9）, fluconazole （ FLU, CYP2C19） and ketoconazole （ KET, CYP3A4） with the individual substrates and with the substrate cocktail. The IC50 values were then determined. The IC50s （ μmol·L^-1 ） were in good agreement with those obtained with individual substrates （α-naphthoflavone, 0. 18 vs O. 26; quinidine, 0. 058 5 vs0. 058 4 ; sulfaphenazole, 0.48 vs O. 45 ; fluconazol, 17.5 vs 11.4 ; ketoconazole, 0.22 vs O. 24） and with previously reported values in the literature. This cocktail probe substrate method can be utilized for the rapid simultaneous determination of the inhibition potential of compounds on the five CYP450 enzymes.