LC-MS/MS同时测定5种探针底物代谢产物和快速评价细胞色素P450同工酶的活性

中国药物警戒 ›› 2013, Vol. 10 ›› Issue (5): 263-268.

LC-MS/MS同时测定5种探针底物代谢产物和快速评价细胞色素P450同工酶的活性

叶林虎,孔令提,肖冰心,王倩,何晓曦,曹方瑞,常琪

中国医学科学:北京协和医学:药用植物研究所,北京 100193

收稿日期:2013-03-28 修回日期:2016-03-09 出版日期:2013-05-08 发布日期:2016-03-09
通讯作者: 常琪,女,博士生导师,药物代谢研究。E-mail:qchang@implad.ac.cn
作者简介:叶林虎,男,在读博士,药物代谢研究。
基金资助:
科技部对欧盟科技合作专项（1108）和重大新药创制“科技重大专项”（2012ZX09301002-001）。

Simultaneous Determination of the Activities of Five Major Cytochrome P450 Enzymes by an in Vitro Cocktail Method Coupled with LC-MS/MS Assay

YE Lin -Hu ,KONG Ling –Ti, XIAO Bing -Xin ,WANG Qian, HE Xiao –Xi, CAO Fang -Rui ,CHANG Qi^*

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China

Received:2013-03-28 Revised:2016-03-09 Online:2013-05-08 Published:2016-03-09

摘要/Abstract

摘要： 目的建立同时测定奥美拉唑（CYP2C19）、右美沙芬（CYP2D6）、睾酮（CYP3A4）、氯唑沙宗（CYP2E1）和甲苯磺丁脲（CYP2C9）5 种探针底物代谢产物的Cocktail 体外方法,快速评价药物对人肝微粒体细胞色素P450同工酶活性的影响。方法通过人肝微粒体与5种探针底物在还原系统NADPH的作用下,于37℃水浴条件下共同孵育,采用液质联用（LC-MS/MS）分析方法,测定探针底物的代谢产物生成量来评价各P450同工酶的活性。结果建立了同时测定5 种P450 同工酶活性的酶反应条件和LC-MS/MS 分析方法,数据显示该方法具有良好的线性和分析灵敏度,以及具有良好的准确度、精密度、重现性,可用于药物间相互作用的体外快速评价。结论可用于体外快速评价药物对相应的CYP450 亚型酶活性的影响,以预测潜在的药物相互作用。

关键词: 细胞色素 P450, Cocktail, LC-MS/MS, 药物相互作用, 体外

Abstract: Objective To develop an in vitro Cocktail method coupled with liquid chromatography/tandem mass spectrometry ( LC -MS/ MS ) assay for simultaneous determination of the activities of five major cytochrome P450 (CYP450) isoenzymes by monitoring the metabolites of five probe substances including omeprazole(CYP2C19), dex-tromethorphan (CYP2D6), testosterone (CYP3A4), chlorzoxazone (CYP2E1), tolbutamide (CYP2C9). Methods The five probes were incubated with human microsome CYP450 isoenzymes at 37 ℃ in the presence of NATPH. The concentrations of produced metabolites in the reaction solution were determined by LC-MS/MS method, and compared for evaluating the activities of isoenzymes. Results The various conditions for enzyme reactions and LC -MS/MS method have been established for monitoring the activities of five CYPP450 isoenzymes. The data showed that the method possesseda good linearity and sensitivity, as well as the excellent accuracy and repeatability, and can be used for the rapid evaluation of drug -drug interactions. Conclusion A sensitive and validated Cocktail Method coupled with LC -MS/MS assay has been developed and can be applied to rapidly evaluate the CYP450 enzyme activities for predicting the potential drug-drug interactions.

Key words: P450, Cocktail, LC-MS/MS, drug-drug interactions, in vitro

中图分类号:

R969

叶林虎,孔令提,肖冰心,王倩,何晓曦,曹方瑞,常琪 . LC-MS/MS同时测定5种探针底物代谢产物和快速评价细胞色素P450同工酶的活性[J]. 中国药物警戒, 2013, 10(5): 263-268.

YE Lin -Hu ,KONG Ling –Ti, XIAO Bing -Xin ,WANG Qian, HE Xiao –Xi, CAO Fang -Rui ,CHANG Qi. Simultaneous Determination of the Activities of Five Major Cytochrome P450 Enzymes by an in Vitro Cocktail Method Coupled with LC-MS/MS Assay [J]. Chinese Journal of Pharmacovigilance, 2013, 10(5): 263-268.

参考文献

[1] CHANG G W, KAM P C. The physiological and pharmacologicalnroles of cytochrome P450 isoenzymes[J]. Anaesthesia,1999,54(1):42-50.
[2] LEUCUTA S E, VLASE L. Pharmacokinetics and metabolic drug interactions[J].Current clinical pharmacology, 2006,1(1):5-20.
[3] 郭晓昕,杜晓曦,薛松林.从美国药品撤市看药品安全风险管理[J].中国药物警戒, 2011, 2(04): 212-216.
[4] 张石革, 宋菲. 回顾若干处方药的淘汰史,重视药物的临床再评价[J].中国医院用药评价与分析, 2002,8(04): 195-198.
[5] ALFARO C L. Emerging role of drug interaction studies in drug development:the good, the bad, and the unknown[J]. Psychopharmacology bulletin, 2001, 35(4): 80-93.
[6] FDA. Guidance for Industry Drug Interaction Studies-Study Design, Data Analysis, and Implications for Dosing and Labelinge.Draft Guidance[EB/OL].(2006-09-11).[2010-11-01].http://wwwfdagov/cder/guidance/indexhtm, 2006.
[7] ZHANG L, REYNOLDS K S, ZHAO P, et al. Drug interactions evaluation: an integrated part of risk assessment of therapeutics[J]. Toxicol Appl Pharmacol, 2010, 243(2): 134-145.
[8] BREIMER D D, SCHELLENS J H. A 'cocktail' strategy to assess in vivo oxidative drug metabolism in humans[J].Trends Pharmacol Sci, 1990, 11(6): 223-225.
[9] CHAURET N, GAUTHIER A,NICOLL-GRIFFITH D A. Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes[J]. Drug Metab Dispos, 1998, 26(1): 1-4.
[10] BUSBYWF, JR., ACKERMANN J M, et al. Effect of methanol,ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450[J]. Drug Metab Dispos, 1999, 27(2): 246-249.
[11] HE F, BI H C, XIE Z Y, et al. Rapid determination of six metabolites from multiple cytochrome P450 probe substrates in human liver microsome by liquid chromatography/mass spectrometry:
application to high-throughput inhibition screening of terpenoids[J]. Rapid communications in mass spectrometry: RCM,2007, 21(5): 635-643.
[12] TURPEINEN M, UUSITALO J, JALONEN J, et al. Multiple P450 substrates in a single run: rapid and comprehensive in vitro interaction assay[J]. Eur J Pharm Sci, 2005, 24(1): 123-132.
[13] WUJ, HUGHESC S, PICARDP, et al. High-throughput cytochrome P450 inhibition assays using laser diode thermal desorption-atmospheric pressure chemical ionization-tandemmass spectrometry[J].Anal Chem, 2007, 79(12): 4657-4665.
[14] SHADERRI,GRANDABW,VONMOLTKE LL, et al. Inhibition of human cytochrome P450 isoforms in vitro by zafirlukast [J].Biopharm Drug Dispos,1999, 20(8): 385-388.
[15] TESTINO S A, JR., PATONAY G. High-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry[J]. J Pharm Biomed Anal, 2003, 30(5): 1459-1467.
[16] 王丹,刘萍霞,张振清,等.LC-MS/MS 同时测定6 种CYP450 探针药物及快速评价小鼠CYP450 同工酶活性[J].军事医学科学院院刊, 2008,32(6):545-549.