TY - JOUR T1 - Glucuronidation of Dihydrotestosterone and Trans-androsterone by Recombinant UGT1A4: Evidence for Multiple UGT1A4 Aglycone Binding Sites JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.109.028712 SP - dmd.109.028712 AU - Jin Zhou AU - Timothy S Tracy AU - Rory P Remmel Y1 - 2009/01/01 UR - http://dmd.aspetjournals.org/content/early/2009/12/09/dmd.109.028712.abstract N2 - UGT1A4-catalyzed glucuronidation is an important drug elimination pathway. Though atypical kinetic profiles (non-hyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucuronidation have been occasionally reported, systematic kinetic studies to explore the existence of multiple aglycone binding sites in UGT1A4 have not been conducted. To this end, two positional isomers dihydrotestosterone (DHT) and trans-androsterone (t-AND) were used as probe substrates and their glucuronidation kinetics with HEK293-expressed UGT1A4 were evaluated both alone and in the presence of a UGT1A4 substrate (tamoxifen (TAM) or lamotrigine (LTG)). Interestingly, co-incubation with TAM, a high affinity UGT1A4 substrate, resulted in concentration-dependent activation/ inhibition effect on DHT and t-AND glucuronidation, whereas LTG, a low affinity UGT1A4 substrate, non-competitively inhibited both processes. The glucuronidation kinetics of TAM were then evaluated both alone and in the presence of different concentrations of DHT or t-AND. TAM displayed substrate inhibition kinetics, suggesting that TAM may have two binding sites in UGT1A4. However, the substrate inhibition kinetic profile of TAM became more hyperbolic, as DHT or t-AND concentration was increased. Various two-site kinetic models adequately explained the interactions between TAM and DHT or TAM and t-AND. Also, the effect of TAM on LTG glucuronidation was evaluated. In contrast to the mixed effect of TAM on DHT and t-AND glucuronidation, TAM inhibited LTG glucuronidation. Our results suggest that multiple aglycone binding sites exist within UGT1A4, which may result in atypical kinetics (both homotropic and heterotropic) in a substrate-dependent fashion.The American Society for Pharmacology and Experimental Therapeutics ER -