An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software.

Paradigms and Technologies
Methods Development


Advancement of in silico tools would be enabled by availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure dataset by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (CYP) isozyme. In order to achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle large volume of compound sets. The substrate depletion method (in vitro half-life (t1/2) method) was chosen to determine CLint The assay (384-well format) consisted of three parts: a robotic system for incubation and sample clean up; two different, integrated, ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and an automated data analysis system. The CYP3A4 assay was evaluated using two long-t1/2 compounds, carbamazepine and antipyrine (t1/2>30 min), one moderate-t1/2 compound, ketoconazole (10<t1/2<30 min), and two short-t1/2 compounds, loperamide and buspirone (t1/2<10 min). Inter-day and intra-day precision and accuracy of the assay was within acceptable range (~12%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 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 CYP isozymes.


Shah, Pranav; Obach, R Scott; Nguyen, Trung; Kerns, Edward; Wang, Amy; Zakharov, Alexey; Simeonov, Anton; Hop, Cornelis E C A; McKew, John; Xu, Xin;

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