Enzyme selective inhibitors represent the most valuable experimental tool for reaction phenotyping. However, only a limited number of UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors have been identified to date. This study characterized the UGT enzyme selectivity of niflumic acid (NFA). It was demonstrated that 2.5 μM NFA is a highly selective inhibitor of recombinant and human liver microsomal UGT1A9 activity. Higher NFA concentrations (50–100 μM) 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 μM. Likewise, NFA was a mixed or noncompetitive inhibitor of recombinant and human liver microsomal UGT1A1 (Ki range 14–18 μM), whereas competitive inhibition (Ki 62 μM) 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 μM 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 μM NFA. Mean Km values decreased in parallel with Vmax, although the magnitude of the decrease was smaller. Taken together, the NFA inhibition data suggest that UGT1A6 is the major enzyme involved in APAP glucuronidation.
Footnotes
This work was supported by the National Health and Medical Research Council of Australia.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.037036.
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ABBREVIATIONS:
- UGT
- UDP-glucuronosyltransferase
- HLM
- human liver microsomes
- NFA
- niflumic acid
- APAP
- acetaminophen (N-acetyl-p-aminophenol)
- APAPG
- acetaminophen β-d-glucuronide
- COD
- codeine
- 4MU
- 4-methylumbelliferone
- β-E2
- β-estradiol
- PRO
- propofol
- TST
- testosterone
- TFP
- trifluoperazine
- UDPGA
- UDP-glucuronic acid
- AZT
- zidovudine
- DEFG
- deferiprone 3-O-β-d-glucuronide
- LTG
- lamotrigine
- DEF
- deferiprone
- HPLC
- high-performance liquid chromatography
- EI
- enzyme-inhibitor
- ESI
- enzyme-inhibitor substrate
- HEK
- human embryonic.
- Received November 3, 2010.
- Accepted January 18, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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