Inhibition of human liver aldehyde oxidase: implications for potential drug-drug interactions

Abstract

During the course of our research efforts to understand the kinetics of human aldehyde oxidase as a xenobiotic-clearing enzyme, we investigated the effect of eight 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(-1) · mg protein(-1). Inhibitory potency of the inhibitors tested ranged from 0.1 to 5 μM. Of the eight different inhibitor compounds tested, seven were observed to inhibit through a mixed mode and one 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 K(i) value relative to the inhibitor concentration in human plasma, having a calculated [I]/K(i) value of 0.4 and 0.8, respectively.

Fig. 1.

Structures of human liver aldehyde oxidase inhibitors.

Fig. 2.

Oxidation reaction of phthalazine to 1-phthalazinone. The structure of the internal standard (IS) used for mass spectrometry assay is provided on the right.

Fig. 3.

Saturation kinetics plot for oxidation of phthalazine. Each point represents an average ± S.E. for a minimum of eight different experiments.

Fig. 4.

Lineweaver-Burk plots and slope/intercept replots of phthalazine oxidation inhibition by eight different inhibitors: DCPIP (A), estradiol (B), menadione (C), ethinyl estradiol (D), domperidone (E), chlorpromazine (F), clozapine (G), and 6-chloroquinazolinone (6-CQ) (H). Each point reflects the average of triplicate determinations.