Atypical (non-Michaelis-Menten) kinetics confound straightforward in vitro-in vivo extrapolations on clearance and inhibition potentials of new chemical entities. However, unlike cytochrome P450s, studies on atypical kinetics of uridine 5'-diphospho-glucuronosyltransferases (UGTs) are much less prevalent. With the use of model substrates, the atypical kinetics of two important glucuronidation enzymes, UGT1A1 and UGT1A4, were explored.
In Chapter 2 (Part I), two positional isomers dihydrotestosterone (DHT) and trans-androsterone (t-AND) were used as probe substrates and their glucuronidation kinetics with recombinant UGT1A4 were evaluated 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 a concentration-dependent activation/ inhibition effect on DHT and t-AND glucuronidation. The glucuronidation kinetics of TAM and the interactions of DHT or t-AND on TAM glucuronudation were then evaluated. TAM displayed substrate inhibition kinetics, however, the substrate inhibition kinetic profile of TAM became more hyperbolic, as DHT or t-AND concentration was increased. Kinetic analysis with two-site kinetic models demonstrated that these atypical interactions can be explained by the existence of multiple aglycone substrate binding sites in UGT1A4. In this chapter, the glucuronidation kinetics of DHT, t-AND and TAM with two UGT1A4 variants (UGT1A4 P24T and UGT1A4 L48V) (Part II) and the interactions of ethinylestradiol and estradiol-3-sulfate on UGT1A4-catalyzed LTG glucuronidation (Part III) were also described.
In Chapter 3, a robust bilirubin glucuronidation assay with recombinant UGT1A1 was established (Part I). With this assay, the correlation between UGT1A1-catalyzed bilirubin glucuronidation and estradiol-3-glucuronidation was studied in the presence model UGT1A1 substrates or inhibitors. Through this evaluation, we found estradiol-3-glucuronidation is a reasonable surrogate in vitro predictor for interactions with bilirubin even though they displayed different kinetic profiles. However, atypical interactions by some effectors were observed with estradiol-3-glucuronidation but not with bilirubin glucuronidation. The atypical interactions of daidzein and SN-38 on ethinylestradiol-3-glucuronidation were also analyzed with multi-site kinetic models.
In conclusion, through kinetic studies with prototype substrates, we found evidence to support the existence of multiple aglycone substrate binding sites in UGT1A1 and UGT1A4. Thus, multiple probe substrates may be needed to evaluate drug-drug interactions involving UGT1A1/UGT1A4-catalyzed metabolism.