A novel testosterone 6β-hydroxylase activity assay for the study of CYP3A-mediated metabolism, inhibition, and induction in vitro
A novel testosterone 6β-hydroxylase activity assay for the study of CYP3A-mediated metabolism, inhibition, and induction in vitro
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DOI：
10.1016/s1056-8719(02)00168-5
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发表时间：
2001-09-01
影响因子：
1.9
通讯作者：
Ruterbories, KJ
中科院分区：
文献类型：
--
作者：
Fayer, JL;Petullo, DM;Ruterbories, KJ
前言：为了研究细胞色素P3A在体外的代谢，建立了一种独特的分析方法来检测6β-羟睾酮(6β-OHT)的形成。该方法已被确定用于体外抑制和诱导相关药物-药物相互作用的研究，并且包括简单的孵育和样品处理程序。方法：建立一套主要分析条件和三套备用分析条件，以检测受试化合物与6β-羟色胺或内标之间的干扰。结果：初步的条件集通过三批验证得到验证，其余的条件集通过一批验证得到验证，所有这些条件都是在人的肝微粒体中进行的。该初步试验还在原代人肝细胞中进行了单批次的CYP3A诱导研究。6β-羟色胺形成的酶动力学参数(K-m，V-max)在人肝微粒体中是重复性的。讨论：试验化合物酮康唑和利福平分别证实了该方法在抑制研究和诱导研究中的有效性。此外，还在三个方面证明了该方法优于现有方法：样品制备容易、运行时间短和检测下限低。(C)2002 Elsevier Science Inc.保留所有权利。
Introduction: In order to examine CYP3A-mediated metabolism in vitro, a unique analytical assay was developed to detect the formation of 6beta-hydroxytestosterone (6beta-OHT). This assay has been determined to be useful for the study of both inhibition- and induction-related drug-drug interactions in vitro and involves simple incubation and sample handling procedures. Methods: A primary and three backup sets of analytical conditions were developed to detect interference between a test compound and either 6beta-OHT or the internal standard. Results: The primary set of conditions was validated with a three-batch validation, and the remaining sets of conditions were validated with one-batch validations, all in human liver microsomes. The primary assay was also validated with a single batch for CYP3A induction studies in primary human hepatocytes. Enzyme kinetic parameters of 6beta-OHT formation (K-m, V-max) were determined to be reproducible in human liver microsomes. Discussion: Utility of the assay in inhibition studies and induction studies, respectively, was confirmed with the test compounds ketoconazole and rifampicin. In addition, superiority to existing methods was demonstrated in three areas: ease of sample preparation, short run times, and low detection limits. (C) 2002 Elsevier Science Inc. All rights reserved.