In vitro identification of the human cytochrome P-450 enzymes involved in the N-demethylation of azelastine

Azelastine hydrochloride [4-[(4-chlorophenyl)methyl]-2-(hexahydro-1-methyl-1H-azepin-4yl)-1-(2H)-phthalazinone monohydrochloride], is a long-acting antiallergic and antiasthmatic drug. The human cytochrome P-450 (CYP) isoform responsible for azelastine N-demethylation, the major metabolic pathway for azelastine, has been examined. Eadie-Hofstee plots of azelastine N-demethylation in human liver microsomes were biphasic. In microsomes from baculovirus-infected insect cells, recombinant CYP3A4, 2D6, 1A2, and 2C19 exhibited high azelastine N-demethylase activity. The K-m values of the recombinant CYP2D6 (3.75 mu M) and CYP3A4 (43.7 mu M) were relatively close to that of high-affinity (14.1 mu M) and low-affinity (54.7 mu M) components in human liver microsomes, respectively. Azelastine N-demethylase activity was inhibited only by the anti-CYP3A antibody, in contrast to antibodies for CYP1A, 2D6, and 2C. In addition, desmethylazelastine formation was significantly inhibited by ketoconazole and troleandomycin but only weakly by omeprazole, sulfaphenazole, and furafylline. These observations suggested that the N-demethylation of azelastine is most extensively catalyzed by the CYP2D6 and 3A4 isoforms in humans.