期刊
DRUG METABOLISM AND DISPOSITION
卷 44, 期 10, 页码 1653-1661出版社
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/dmd.116.072017
关键词
-
资金
- Intramural Research Program of the National Center for Advancing Translational Sciences/National Institutes of Health
Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t(1/2)) method] was chosen to determine CLint. The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t(1/2) compounds, carbamazepine and antipyrine (t(1/2) > 30 minutes); one moderate t(1/2) compound, ketoconazole (10 < t(1/2) < 30 minutes); and two short t(1/2) compounds, loperamide and buspirone (t(1/2) < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (similar to 12%) for the linear range observed. Using this assay, CYP3A4 CLint and t(1/2) values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes.
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