RT Journal Article
SR Electronic
T1 In Vitro Assay of Six UDP-Glucuronosyltransferase Isoforms in Human Liver Microsomes, Using Cocktails of Probe Substrates and Liquid Chromatography–Tandem Mass Spectrometry
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1803
OP 1810
DO 10.1124/dmd.114.058818
VO 42
IS 11
A1 Kyung-Ah Seo
A1 Hyo-Ji Kim
A1 Eun Sook Jeong
A1 Nagi Abdalla
A1 Chang-Soo Choi
A1 Dong-Hyun Kim
A1 Jae-Gook Shin
YR 2014
UL http://dmd.aspetjournals.org/content/42/11/1803.abstract
AB 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.