Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation

Abstract

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 .

Human UDP-Glucuronosyltransferase (UGT) 2B10: Validation of Cotinine as a Selective Probe Substrate, Inhibition by UGT Enzyme-Selective Inhibitors and Antidepressant and Antipsychotic Drugs, and Structural Determinants of Enzyme Inhibition

“…The potent inhibition of UGT2B10 by several drugs observed in this study is consistent with previous reports from this laboratory, demonstrating that inhibition of other UGT enzymes (e.g., UGT 1A1, 1A9, 2B4, and 2B7) may potentially precipitate DDIs and drugendobiotic interactions (Boyd et al, 2006;Uchaipichat et al, 2006b;Miners et al, 2010b;Raungrut et al, 2010;Pattanawongsa et al, 2015). These observations reinforce the recent recommendations of Regulatory Agencies that new drugs should be evaluated for their potential to inhibit UGT enzymes.…”

“…The IC 50 for niflumic acid inhibition of UGT2B10 observed in this study (168 mM) confirms the UGT1A9 inhibition selectivity of niflumic acid at a low concentration, but indicates that this compound will significantly inhibit UGT2B10 as well as UGT1A1 and UGT2B15 at a concentration of 100 mM. As noted above, hecogenin does not inhibit UGT2B10, consistent with the reported inhibition selectivity for UGT1A4 (Uchaipichat et al, 2006b). By contrast, fluconazole (2.5 mM), which is considered a selective inhibitor of UGT2B4 and UGT2B7, inhibited UGT2B10 to a similar extent to that reported for UGT2B4/7 (Uchaipichat et al, 2006a;Raungrut et al, 2010).…”

“…Positive control inhibitors were used in all inhibition screening experiments, as described in Pattanawongsa et al (2015). The magnitude of inhibition of each positive control inhibitor (data not shown) was as expected from previous studies in this laboratory (Uchaipichat et al, 2004(Uchaipichat et al, , 2006a(Uchaipichat et al, , 2006bRaungrut et al, 2010;Miners et al, 2011).…”

“…Niflumic acid, which inhibits UGT1A9 with a K i of 0.10 mM and UGT1A1 and UGT2B15 with respective K i values of 18 and 62 mM (Miners et al, 2011), inhibited UGT2B10 with an IC 50 of 168 6 0.14 mM (Table 2). Fluconazole, employed as a selective inhibitor of UGT2B4 and UGT2B7 (Uchaipichat et al, 2006b;Raungrut et al, 2010), inhibited UGT2B10 with an IC 50 of 1136 6 88.4 mM ( Table 2), whereas phenylbutazone, which has been reported to be a relatively selective inhibitor of UGT1A subfamily enzyme activities (Uchaipichat et al, 2006a), inhibited UGT2B10 with an IC 50 of 220 6 35.4 mM. The effect of fluconazole on UGT2B10 prompted us to investigate the effects of two other azole antifungal agents, itraconazole and ketoconazole.…”

Phase Ii Drug Metabolizing Enzymes

“…Hecogenin (steroidal saponin) is responsible for inhibition of UGT1A4, and flucoconazole inhibits UGT2B7 activity 27,28 . Bilirubin, the specific substrate of UGT1A1, has been shown to inhibit the enzymatic activity of UGT1A4 29 .…”