Differential modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to human liver microsomes.

Previous results demonstrating homotropic activation of human UDP-glucuronosyltransferase (UGT) 1A1-catalyzed estradiol-3-glucuronidation led us to investigate the effects of 16 compounds on estradiol glucuronidation by human liver microsomes (HLM). In confirmation of previous work using alamethicin-treated HLM pooled from four livers, UGT1A1-catalyzed estradiol-3-glucuronidation demonstrated homotropic activation kinetics (S(50) = 22 microM, Hill coefficient, n = 1.9) whereas estradiol-17-glucuronidation (catalyzed by other UGT enzymes) followed Michaelis-Menten kinetics (K(m) = 7 microM). Modulatory effects of the following compounds were investigated: bilirubin, eight flavonoids, 17alpha-ethynylestradiol (17alpha-EE), estriol, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), anthraflavic acid, retinoic acid, morphine, and ibuprofen. Although the classic UGT1A1 substrate bilirubin was a weak competitive inhibitor of estradiol-3-glucuronidation, the estrogens and anthraflavic acid activated or inhibited estradiol-3-glucuronidation dependent on substrate and effector concentrations. For example, at substrate concentrations of 5 and 10 microM, estradiol-3-glucuronidation activity was stimulated by as much as 80% by low concentrations of 17alpha-EE but was unaltered by flavanone. However, at higher substrate concentrations (25-100 microM) estradiol-3-glucuronidation was inhibited by about 55% by both compounds. Anthraflavic acid and PhIP were also stimulators of estradiol 3-glucuronidation at low substrate concentrations. The most potent inhibitor of estradiol 3-glucuronidation was the flavonoid tangeretin. The UGT2B7 substrates morphine and ibuprofen had no effect on estradiol 3-glucuronidation, whereas retinoic acid was slightly inhibitory. Estradiol-17-glucuronidation was inhibited by 17alpha-EE, estriol, and naringenin but was not activated by any compound. This study demonstrates that the interactions of substrates and inhibitors at the active site of UGT1A1 are complex, yielding both activation and competitive inhibition kinetics.

Comparative metabolic capabilities of CYP3A4, CYP3A5, and CYP3A7.

The human cytochromes P450 (P450) CYP3A contribute to the biotransformation of 50% of oxidatively metabolized drugs. The predominant hepatic form is CYP3A4, but recent evidence indicates that CYP3A5 contributes more significantly to the total liver CYP3A than was originally thought. CYP3A7 is the major fetal form and is rarely expressed in adults. To compare the metabolic capabilities of CYP3A forms for 10 substrates, incubations were performed using a consistent molar ratio (1:7:9) of recombinant CYP3A, P450 reductase, and cytochrome b5. A wide range of substrate concentrations was examined to determine the best fit to kinetic models for metabolite formation. In general, K(m) or S(50) values for the substrates were 3 to 4 times lower for CYP3A4 than for CYP3A5 or CYP3A7. For a more direct comparison of these P450 forms, clearance to the metabolites was determined as a linear relationship of rate of metabolite formation for the lowest substrate concentrations examined. The clearance for 1'-hydroxy midazolam formation at low substrate concentrations was similar for CYP3A4 and CYP3A5. For CYP3A5 versus CYP3A4, clearance values at low substrate concentrations were 2 to 20 times lower for the other biotransformations. The clearance values for CYP3A7-catalyzed metabolite formation at low substrate concentrations were substantially lower than for CYP3A4 or CYP3A5, except for clarithromycin, 4-OH triazolam, and N-desmethyl diltiazem (CYP3A5 - CYP3A7). The CYP3A forms demonstrated regioselective differences in some of the biotransformations. These results demonstrate an equal or reduced metabolic capability for CYP3A5 compared with CYP3A4 and a significantly lower capability for CYP3A7.