Probing CYP2C19 and CYP3A4 activities in Chinese liver microsomes by quantification of 5-hydroxyomeprazole and omeprazole sulphone^1  

定量5-羟奥美拉唑和奥美拉唑砜以探测中国人肝微粒体中CYP2C19和CYP3A4的活性(英文)

作　　者：舒焱[1] 王连生[1] 肖卫民[2] 王伟[1] 黄松林[1] 周宏灏[1] 

机构地区：[1]湖南医科大学遗传药理学研究室,长沙中国410078 [2]湖南医科大学病理生理学教研室,长沙中国410078

出　　处：《Acta Pharmacologica Sinica》2000年第8期84-89,共6页中国药理学报（英文版）

基　　金：Project supported by the Nationd Natural Science Foundation of China, № F3930230, and by China Medial Board of America, № 92-568 and 99-697.

摘　　要：To develop an analytical method for simultaneous quantification of 5-hydroxyomeprazole (5-OH-OP) and omeprazole sulfone (OPS), and explore whether omepra-zole (OP) is an appropriate phenotypic probe for CYP2C19 and CYP3A4 in Chinese liver microsomes. METHODS: OP metabolism in vitro was conducted in Chinese liver microsomes, and the major metabolites 5-OH-OP and OPS were determined using high pressure liquid chromatography (HPLC) . Monoclonal antibodies anti-CYP2C8/9/19 and anti-CYP3A4 were employed to conduct inhibition experiments. The protein contents of CYP2C19 and CYP3A4 were quantified using Western blot analysis and densitometric scanning. RESULTS: 5-OH-OP and OPS gave a baseline resolution in the HPLC analysis. The detection limits for both compounds were 0.01 nmol and the recovery (98 % - 102 %) had good precision with relative standard deviation of <9.5%. Both anti-CYP2C8/9/19 and anti-CYP3A4 had a significant inhibitory effect (P< 0.05) on the 5-OH-OP formation in a substrate concentration-dependent manner, and anti-CYP3A4 alone could almost abolish the formation of OPS ( > 87 %). At a substrate concentration of 2μmol/L OP, good correlations were found between OP 5-hydroxylation and S-mephenytoin 4'-hydrox-ylation activities (r = 0.72, P < 0.01), OP 5-hydroxylation activities and CYP2C19 contents ( r = 0.82, P < 0.01), and OP sulfoxidation activities and CYP3A4 contents (r = 0.78, P < 0.01) in Chinese liver microsomes. CONCLUSION: OP metabolism is mediated mainly by CYP2C19 and CYP3A4, and OP can be used to probe CYP2C19 and CYP3A4 activities in Chinese liver microsomes at appropriate substrate concentrations with the HPLC method presently developed.目的:建立同时定量5-羟奥美拉唑(5-OH-OP)和奥美拉唑砜(OPS)的分析方法,探讨奥美拉唑(OP)在中国人肝微粒体中能否作为细胞色素 P450 2C19(CYP2C19)及3A4(CYP3A4)的活性表型探针药.方法:在中国人肝微粒体中进行OP的体外代谢,以高压液相色谱法(HPLC)检测主要代谢产物5-OH-OP及OPS;抗CYP2C8/9/19及抗CYR3A4的单克隆抗体被用于抑制实验;以Westem斑点法和光密度扫描定量肝微粒体中CYP2C19及CYP3A4的含量.结果:在HPLC分析中5-OH-OP及OPS有很好的洗脱,二者检测限均为0.01nmol,回收率为98％—102％并且变异系数小于9.5％;抗CYP2C8/9/19及抗CYP3A4均对5-OH-OP的生成有显著的抑制作用(P<0.05),二者的抑制作用呈底物浓度依赖,并且抗CYP3A4几乎可单独去除OPS的生成(>87％);在底物浓度为2μmol/L OP时,中国人肝微粒体中OP的5-羟化与美芬妥英的4′-羟化活性之间(r=0.72,P<0.01)、OP的5-羟化活性与CYP2C19含量之间(r=0.82,P<0.01)以及OP的硫代氧化活性与CYP3A4含量之间(r=O.78,P<O.01)均有很好的相关性.结论:中国人肝微粒体中OP的代谢主要由CYP2C19和CYP3A4介导;采用本研究建立的HPLC方法,在适当的底物浓度下,OP能用于体外探测中国人肝微粒体中CYP2C19及CYP3A4的活性.

关 键 词：liver microsomes cytochrome P-450 CYP2C19 cycochrome P-450 CYP3A4 high pressure liquid chromatography OMEPRAZOLE MEPHENYTOIN metabolism 

分 类 号：R96[医药卫生—药理学]