ABSTRACT:Relatively few selective substrate and inhibitor probes have been identified for human UDP-glucuronosyltransferases (UGTs). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone, as inhibitors, for human UGTs. Selectivity was assessed using UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, and 2B15 expressed in HEK293 cells. TFP was confirmed as a highly selective substrate for UGT1A4. However, TFP bound extensively to both HEK293 lysate and human liver microsomes in a concentration-dependent manner (fu inc 0.20-0.59). When corrected for nonspecific binding, K m values for TFP glucuronidation were similar for both UGT1A4 (4.1 M) and human liver microsomes (6.1 ؎ 1.2 M) as the enzyme sources. Of the compounds screened as inhibitors, hecogenin, alone, was selective; significant inhibition was observed only for UGT1A4 (IC 50 1.5 M). Using phenylbutazone and quinine as "models," inhibition kinetics were variously described by competitive and noncompetitive mechanisms. Inhibition of UGT2B7 by quinidine was also investigated further, because the effects of this compound on morphine pharmacokinetics (a known UGT2B7 substrate) have been ascribed to inhibition of P-glycoprotein. Quinidine inhibited human liver microsomal and recombinant UGT2B7, with respective K i values of 335 ؎ 128 M and 186 M. In conclusion, TFP and hecogenin represent selective substrate and inhibitor probes for UGT1A4, although the extensive nonselective binding of the former should be taken into account in kinetic studies. Amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone are nonselective UGT inhibitors.

Studies were performed to elucidate the mechanism responsible for the reduction in K m values of UDP-glucuronosyltransferase 2B7 (UGT2B7) substrates observed for incubations conducted in the presence of albumin. Addition of bovine serum albumin (BSA) and fatty acid-free human serum albumin (HSA-FAF), but not "crude" HSA, resulted in an approximate 90% reduction in the K m values for the glucuronidation of zidovudine (AZT) by human liver microsomes (HLM) and UGT2B7 and a 50 to 75% reduction in the S 50 for 4-methylumbelliferone (4MU) glucuronidation by UGT2B7, without affecting V max . Oleic, linoleic, and arachidonic acids were shown to be the most abundant unsaturated long-chain fatty acids present in crude HSA and in the membranes of HLM and human embryonic kidney (HEK)293 cells, and it was demonstrated that these and other unsaturated long-chain fatty acids were UGT2B7 substrates.Glucuronides with R f (retention factor) values corresponding to the glucuronides of linoleic and arachidonic acid were detected when HLM and HEK293 cell lysates were incubated with radiolabeled cofactor, and the intensity of the bands was modulated by the presence of crude HSA (increased) and BSA or HSA-FAF (decreased). Oleic, linoleic, and arachidonic acid inhibited AZT and 4MU glucuronidation by HLM and/or UGT2B7, due to an increase in K m /S 50 without a change in V max . Addition of BSA and HSA-FAF reversed the inhibition. Likewise, coexpression of UGT2B7 and HSA in HEK293 cells reduced the K m /S 50 values of these substrates. It is postulated that BSA and HSA-FAF sequester inhibitory fatty acids released during incubations, and the apparent high K m values observed for UGT2B7 substrates arise from the presence of these endogenous inhibitors.

ABSTRACT:Studies were performed to investigate the UDP-glucuronosyltransferase enzyme (

AimsUsing the fluconazole-zidovudine (AZT) interaction as a model, to determine whether inhibition of UDP-glucuronosyltransferase (UGT) catalysed drug metabolism in vivo could be predicted quantitatively from in vitro kinetic data generated in the presence and absence bovine serum albumin (BSA). MethodsKinetic constants for AZT glucuronidation were generated using human liver microsomes (HLM) and recombinant UGT2B7, the principal enzyme responsible for AZT glucuronidation, as the enzyme sources with and without fluconazole. K i values were used to estimate the decrease in AZT clearance in vivo . ResultsAddition of BSA (2%) to incubations decreased the K m values for AZT glucuronidation by 85-90% for the HLM (923 ± 357 to 91 ± 9 µ M ) and UGT2B7 (478-70 µ M ) catalysed reactions, with little effect on V max . Fluconazole, which was shown to be a selective inhibitor of UGT2B7, competitively inhibited AZT glucuronidation by HLM and UGT2B7. Like the K m , BSA caused an 87% reduction in the K i for fluconazole inhibition of AZT glucuronidation by HLM (1133 ± 403 to 145 ± 36 µ M ) and UGT2B7 (529 to 73 µ M ). K i values determined for fluconazole using HLM and UGT2B7 in the presence (but not absence) of BSA predicted an interaction in vivo . The predicted magnitude of the interaction ranged from 41% to 217% of the reported AUC increase in patients, depending on the value of the in vivo fluconazole concentration employed in calculations. ConclusionsK i values determined under certain experimental conditions may quantitatively predict inhibition of UGT catalysed drug glucuronidation in vivo .