Abstract

Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CL_int) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CL_int 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 CL_int. 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_½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CL_int 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.