J L Fayer1, D M Petullo, B J Ring, S A Wrighton, K J Ruterbories. 1. Lilly Research Laboratories, Department of Drug Disposition, Eli Lilly and Company, Lilly Corporate Center, DC0730, Indianapolis, IN 46285, USA. fayer_jessica@lilly.com
Abstract
INTRODUCTION: In order to examine CYP3A-mediated metabolism in vitro, a unique analytical assay was developed to detect the formation of 63-hydroxytestosterone (63-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 (Km, Vmax) 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.
INTRODUCTION: In order to examine CYP3A-mediated metabolism in vitro, a unique analytical assay was developed to detect the formation of 63-hydroxytestosterone (63-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 (Km, Vmax) 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.
Authors: Siamak Cyrus Khojasteh; Saileta Prabhu; Jane R Kenny; Jason S Halladay; Anthony Y H Lu Journal: Eur J Drug Metab Pharmacokinet Date: 2011-02-19 Impact factor: 2.441
Authors: Guolin Shen; Cheng Wang; Lili Zhou; Lei Li; Huiming Chen; Wenlian Yu; Haishan Li Journal: Int J Environ Res Public Health Date: 2015-09-02 Impact factor: 3.390