Abstract

In human liver microsomes, triazolam is principally metabolized by CYP3A4 to form two metabolites, 1′-hydroxytriazolam (1′OHTz) and 4-hydroxytriazolam (4OHTz). The velocity of 1′OHTz formation was found to decrease at higher triazolam concentrations (>200 μM), indicative of “substrate inhibition”. Coincubation of [¹⁴C]triazolam with authentic metabolite standards of either 1′OHTz or 4OHTz up to 30 μM did not significantly inhibit the rate of [¹⁴C]1′OHTz formation. The effects of secondary compounds on triazolam oxidation were shown to be product-specific, producing either activation or inhibition depending on the triazolam metabolite monitored. When human liver microsomes were supplemented with exogenous human cytochrome b₅, it was observed that substrate inhibition was attenuated and the resulting increase in 1′OHTz formation, relative to control (nonsupplemented) incubations, corresponded to a decrease in the ratio of 4OHTz to 1′OHTz. In contrast, when cofactor (e.g., 100 μM NADPH) was rate limiting, the metabolite ratio (4OHTz/1′OHTz) was markedly increased over the entire substrate concentration range (0.5–1000 μM). To explain these kinetic observations, a two-site binding model is proposed in which triazolam is hypothesized to bind within the CYP3A4 active site in spatially distinct orientations, which may lead to the formation of either the 1′-hydroxytriazolam or 4-hydroxytriazolam. Differential inhibition/activation is consistent with this two-site model and substrate inhibition is hypothesized to result from competition between the two sites for reactive oxygen.