Characterization of niflumic acid as a selective inhibitor of human liver microsomal UDP-glucuronosyltransferase 1A9: Application to the reaction phenotyping of acetaminophen glucuronidation. Drug Metab. Dispos

Enzyme selective inhibitors represent the most valuable experi-mental tool for reaction phenotyping. However, only a limited num-ber of UDP-glucuronosyltransferase (UGT) enzyme-selective inhib-itors have been identified to date. This study characterized the UGT enzyme selectivity of niflumic acid (NFA). It was demonstrated that 2.5 M NFA is a highly selective inhibitor of recombinant and human liver microsomal UGT1A9 activity. Higher NFA concentra-tions (50–100 M) inhibited UGT1A1 and UGT2B15 but had little effect on the activities of UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B17. NFA inhibited 4-methylumbelliferone and propofol (PRO) glucuronidation by recombinant UGT1A9 and PRO glucuronidation by human liver microsomes (HLM) according to a mixed (competitive-noncompetitive) mechanism, with Ki values ranging from 0.10 to 0.40 M. Likewise, NFA was a mixed or noncompetitive inhibitor of recombinant and human liver micro-somal UGT1A1 (Ki range 14–18 M), whereas competitive inhibition (Ki 62 M) was observed with UGT2B15. NFA was subsequently applied to the reaction phenotyping of human liver microsomal acetaminophen (APAP) glucuronidation. Consistent with previous reports, APAP was glucuronidated by recombinant UGT1A1, UGT1A6, UGT1A9, and UGT2B15. NFA concentrations in the range of 2.5 to 100 M inhibited APAP glucuronidation by UGT1A1, UGT1A9, and UGT2B15 but not by UGT1A6. The mean Vmax for APAP glucuronidation by HLM was reduced by 20, 35, and 40%, respectively, in the presence of 2.5, 50, and 100 M NFA. Mean Km values decreased in parallel with Vmax, although the magni-tude of the decrease was smaller. Taken together, the NFA inhibition data suggest that UGT1A6 is the major enzyme in-volved in APAP glucuronidation