title: An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software.
abstract: 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 (t1/2) 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 t1/2 compounds, carbamazepine and antipyrine (t1/2 > 30 minutes); one moderate t1/2 compound, ketoconazole (10 < t1/2 < 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t½ < 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 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 P450 isozymes.
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
authors.forename: Pranav
authors.initials: P
authors.lastname: Shah
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
authors.forename: Edward
authors.initials: E
authors.lastname: Kerns
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
authors.forename: Dac-Trung
authors.initials: DT
authors.lastname: Nguyen
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
authors.forename: R Scott
authors.initials: RS
authors.lastname: Obach
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
authors.forename: Amy Q
authors.initials: AQ
authors.lastname: Wang
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
authors.forename: Alexey
authors.initials: A
authors.lastname: Zakharov
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
authors.forename: John
authors.initials: J
authors.lastname: McKew
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H).
authors.forename: Cornelis E C A
authors.initials: CE
authors.lastname: Hop
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville Maryland (P.S, E.K, D-T.N, A Q.W, A.Z, J.M, A.S, X.X.); Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer. Groton, Connecticut (R.S.O.); and Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (C.E.C.A.H) xin.xu3@nih.gov.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
mesh_terms: D001331:Automation; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D013058:Mass Spectrometry; D012984:Software
chemical_list: D003577:Cytochrome P-450 Enzyme System