Abstract

Inhibition of UGT1A1-catalyzed bilirubin glucuronidation by drug compounds may potentially be of clinical concern. However, in drug discovery and development settings, bilirubin is less than an ideal in vitro probe for assessing a new chemical entity's potential to inhibit bilirubin glucuronidation. In part, this is due to propensity of bilirubin to photodegrade and to the instability of its metabolites. To this end, the utility of estradiol-3-glucuronidation as a surrogate in-vitro, predictor for interactions with bilirubin was evaluated. The glucuronidation kinetics of bilirubin and estradiol were carefully characterized with recombinant UGT1A1 expressed in HEK293 cells. Consistent with previous reports, estradiol-3-glucuronidation displayed sigmoidal kinetics, whereas bilirubin glucuronidation exhibited typical hyperbolic kinetics. The two compounds also mutually inhibited the metabolism of the other. Sixteen UGT1A1 substrates/inhibitors were evaluated as effectors of each reaction. Fourteen compounds inhibited both bilirubin and estradiol glucurdoniation. However, two compounds (ethinylestradiol and daidzein) exhibited mixed effects (concentration-dependent activation and inhibition) on estradiol-3-glucuronidation whereas bilirubin glucuronidation was inhibited by both compounds. In addition, SN-38, a substrate of UGT1A1 (reported K_m =24 μM) appeared to be a weak inhibitor of bilirubin glucuronidation (IC₅₀=356.4 μM), but a partial inhibitor of estradiol-3-glucuronidation. The IC₅₀ values of the inhibitors against estradiol-3-glucuronidation were strongly correlated with IC₅₀ values against bilirubin glucuronidation, resulting in a R² value of 0.9604 (activator excluded) or 0.8287 (activator included). Thus, estradiol-3-glucuronidation can serve as a good surrogate for predicting inhibition of bilirubin glucuronidation with the caveat that occasionally compounds may demonstrate activation of estradiol-3-glucuronidation.