Abstract

During the course of our research efforts to understand the kinetics of human AOX as a xenobiotic clearing enzyme, we investigated the effect of 8 different inhibitors on the oxidation of the probe substrate phthalazine. Saturation kinetic parameters for phthalazine oxidation in human liver cytosol were found to be the following: K_m = 8.0 ± 0.4 µM and V_max = 4.3 ± 0.1 nmol/min/mg protein. Inhibitory potency of the inhibitors tested ranged from 0.1 µM to 5 µM. Of the 8 different inhibitor compounds tested, 7 were observed to inhibit through a mixed mode and 1 through a strictly competitive mode. A ratio of the K_ii and K_is values was used to assess the relative competitiveness of each inhibitor. For the mixed inhibitors, the mode of inhibition varied from mostly uncompetitive to predominantly competitive (K_ii / K_is values ranging from 0.1 to 15). The implications for potential drug-drug interactions and inhibition mechanism are discussed. We found two inhibitors, clozapine and chlorpromazine, that have a moderate predicted risk of drug-drug interactions based on the Ki value relative to inhibitor concentration in human plasma, having a calculated [I]/K_i value of 0.4 and 0.8 respectively.