Aims To estimate the relative contribution of liver, kidney and jejunum to MPA elimination via glucuronidation from in vitro kinetic data. Methods The kinetics of MPA glucuronidation by human liver, kidney and jejunum microsomes were characterized. Mycophenolic acid glucuronide (MPAG) concentrations in microsomal incubations were determined using a speci®c h.p.l.c. procedure. Non-speci®c microsomal binding of MPA was excluded using an equilibrium dialysis approach. Results Microsomes from all three tissues catalysed the conversion of MPA to MPAG. Mean microsomal intrinsic clearances for MPAG formation by liver, kidney and jejunum microsomes were 46.6, 73.5 and 24.5 ml (min mg) x1 , respectively. When extrapolated to the whole organ, however, hepatic intrinsic clearance was 21-and 38-fold higher than the respective intrinsic clearances for kidney and small intestine. Conclusions The data suggest that the liver is the organ primarily responsible for the systemic clearance of MPA, with little contribution from the kidney, and that the small intestine would be expected to contribute to ®rst-pass extraction to a minor extent only.

ABSTRACT: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 M NFA is a highly selective inhibitor of recombinant and human liver microsomal UGT1A9 activity. Higher NFA concentrations (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 K i values ranging from 0.10 to 0.40 M. Likewise, NFA was a mixed or noncompetitive inhibitor of recombinant and human liver microsomal UGT1A1 (K i range 14-18 M), whereas competitive inhibition (K i 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 V max 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 K m values decreased in parallel with V max , 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.

AimsTo characterize the kinetics of S-naproxen ('naproxen') acyl glucuronidation and desmethylnaproxen acyl and phenolic glucuronidation by human liver microsomes and identify the human UGT isoform(s) catalysing these reactions. MethodsNaproxen and desmethylnaproxen glucuronidation were investigated using microsomes from six and five livers, respectively. Human recombinant UGTs were screened for activity towards naproxen and desmethylnaproxen. Where significant activity was observed, kinetic parameters were determined. Naproxen and desmethylnaproxen glucuronides were measured by separate high-per formance liquid chromatography methods. ResultsNaproxen acyl glucuronidation by human liver microsomes followed biphasic kinetics. Mean apparent K m values ( ± SD, with 95% confidence interval in parentheses) for the high-and low-affinity components were 29 ± 13 m M (16, 43) and 473 ± 108 m M (359, 587), respectively. UGT 1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10 and 2B7 glucuronidated naproxen. UGT2B7 exhibited an apparent K m (72 m M ) of the same order as the high-affinity human liver microsomal activity, which was inhibited by the UGT2B7 selective 'probe' fluconazole. Although data for desmethylnaproxen phenolic glucuronidation by human liver microsomes were generally adequately fitted to either the single-or two-enzyme Michaelis-Menten equation, model fitting was inconclusive for desmethylnaproxen acyl glucuronidation. UGT 1A1, 1A7, 1A9 and 1A10 catalysed both the phenolic and acyl glucuronidation of desmethylnaproxen, while UGT 1A3, 1A6 and 2B7 formed only the acyl glucuronide. Atypical glucuronidation kinetics were variably observed for naproxen and desmethylnaproxen glucuronidation by the recombinant UGTs. ConclusionUGT2B7 is responsible for human hepatic naproxen acyl glucuronidation, which is the primary elimination pathway for this drug.K. Bowalgaha et al. 42460 :4 Br J Clin Pharmacol