title: In vitro assay of six UDP-glucuronosyltransferase isoforms in human liver microsomes, using cocktails of probe substrates and liquid chromatography-tandem mass spectrometry.
abstract: UDP-glucuronosyltransferase (UGT)-mediated drug-drug interactions are commonly evaluated during drug development. We present a validated method for the simultaneous evaluation of drug-mediated inhibition of six major UGT isoforms, developed in human liver microsomes through the use of pooled specific UGT probe substrates (cocktail assay) and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The six probe substrates used in this assay were estradiol (UGT1A1), chenodeoxycholic acid (UGT1A3), trifluoperazine (UGT1A4), 4-hydroxyindole (UGT1A6), propofol (UGT1A9), and naloxone (UGT2B7). In a cocktail incubation, UGT1A1, UGT1A9, and UGT2B7 activities were substantially inhibited by other substrates. This interference could be eliminated by dividing substrates into two incubations: one containing estradiol, trifluoperazine, and 4-hydroxyindole, and the other containing chenodeoxycholic acid, propofol, and naloxone. Incubation mixtures were pooled for the simultaneous analysis of glucuronyl conjugates in a single LC-MS/MS run. The optimized cocktail method was further validated against single-probe substrate assays using compounds known to inhibit UGTs. The degree of inhibition of UGT isoform activities by such known inhibitors in this cocktail assay was not substantially different from that in single-probe assays. This six-isoform cocktail assay may be very useful in assessing the UGT-based drug-interaction potential of candidates in a drug-discovery setting.
authors.affiliation: Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (K.-A.S., H.-J.K., E.S.J., N.A., D.-H.K., J.-G.S.); and Department of General Surgery, Inje University Busan Paik Hospital, Busan, Korea (C.-S.C.).
authors.forename: Kyung-Ah
authors.initials: KA
authors.lastname: Seo
authors.affiliation: Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (K.-A.S., H.-J.K., E.S.J., N.A., D.-H.K., J.-G.S.); and Department of General Surgery, Inje University Busan Paik Hospital, Busan, Korea (C.-S.C.).
authors.forename: Hyo-Ji
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (K.-A.S., H.-J.K., E.S.J., N.A., D.-H.K., J.-G.S.); and Department of General Surgery, Inje University Busan Paik Hospital, Busan, Korea (C.-S.C.).
authors.forename: Eun Sook
authors.initials: ES
authors.lastname: Jeong
authors.affiliation: Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (K.-A.S., H.-J.K., E.S.J., N.A., D.-H.K., J.-G.S.); and Department of General Surgery, Inje University Busan Paik Hospital, Busan, Korea (C.-S.C.).
authors.forename: Nagi
authors.initials: N
authors.lastname: Abdalla
authors.affiliation: Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (K.-A.S., H.-J.K., E.S.J., N.A., D.-H.K., J.-G.S.); and Department of General Surgery, Inje University Busan Paik Hospital, Busan, Korea (C.-S.C.).
authors.forename: Chang-Soo
authors.initials: CS
authors.lastname: Choi
authors.affiliation: Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (K.-A.S., H.-J.K., E.S.J., N.A., D.-H.K., J.-G.S.); and Department of General Surgery, Inje University Busan Paik Hospital, Busan, Korea (C.-S.C.) phshinjg@inje.ac.kr dhkim@inje.ac.kr.
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Department of Pharmacology and PharmacoGenomics Research Center, Inje University College of Medicine, Busan, Korea (K.-A.S., H.-J.K., E.S.J., N.A., D.-H.K., J.-G.S.); and Department of General Surgery, Inje University Busan Paik Hospital, Busan, Korea (C.-S.C.) phshinjg@inje.ac.kr dhkim@inje.ac.kr.
authors.forename: Jae-Gook
authors.initials: JG
authors.lastname: Shin
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D014453:Glucuronosyltransferase