Development of an Assay Using 4-Trifluoromethylumbelliferyl as a Marker Substrate for Assessment of Drug–Drug Interactions to Multiple Isoforms of UDP-Glucuronosyltransferases

Abstract

Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs) and is one of the most important pathways for elimination of xenobiotics. The aim of the present study was to develop an in vitro assay for assessment of drug–drug interactions related to UGTs applicable to early drug discovery. 4-Trifluoromethylumbelliferyl was tested as a marker substrate for six human recombinant expressed UGT isoforms: 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7. It was shown that 4- trifluoromethylumbelliferyl was glucuronidated by all UGTs tested, except UGT1A4. By using a short HPLC gradient (7 min) and fluorescence detection, the enzyme kinetic parameters for these reactions were obtained. All reactions were found to follow classical Michaelis–Menten kinetics, with K_m values between 29 μM (UGT1A9) and 80 μM (UGT1A3). The method was validated by using several known competitive inhibitors of UGTs. The most potent inhibition was observed for the reaction between 17α-ethynylestradiol and UGT1A1 (K_i = 10.5 μM), and the weakest interaction was detected for acetaminophen and UGT1A9 (IC₅₀ > 1 mM). Taken together, we report the development of an assay using 4-trifluoromethylumbelliferyl as a marker substrate for five different human UGT isoforms suitable for the assessment of drug–drug interactions related to UGTs during early drug discovery.