Azelastine N-demethylation by cytochrome P-450 (CYP) 3A4, CYP2D6, and CYP1A2 in human liver microsomes: Evaluation of approach to predict the contribution of multiple CYPS

Azelastine, an antiallergy and antiasthmatic drug, has been reported to be mainly N-demethylated to desmethylazelastine in humans. In the present stud y, Eadie-Hofstee plots of azelastine N-demethylation in human liver microso mes were biphasic. In microsomes from human B-lymphoblast cells, recombinan t cytochrome P-450 (CYP)2D6 and CYP1A1 exhibited higher azelastine N-demeth ylase activity than did other CYP enzymes. On the other hand, recombinant C YP3A4 and CYP1A2 as well as CYP1A1 and CYP2D6 in microsomes from baculoviru s-infected insect cells were active in azelastine N-demethylation. The K-M value of the recombinant CYP2D6 (2.1 mu M) from baculovirus-infected insect cells was similar to the K-M value of the high-affinity (2.4 +/- 1.3 mu M) component in human liver microsomes. On the other hand, the K-M values of the recombinant CYP3A4 (51.1 mu M) and CYP1A2 (125.4 mu M) from baculovirus -infected insect cells were similar to the K-M value of the low-affinity (7 9.7 +/- 12.8 mu M) component in human liver microsomes. Bufuralol inhibited the high-affinity component, making the Eadie-Hofstee plot in human liver microsomes monophasic. Azelastine N-demethylase activity in human liver mic rosomes (5 mu M azelastine) was inhibited by ketoconazole, erythromycin, an d fluvoxamine (IC50 = 0.08, 18.2, and 17.2 mu M, respectively). Azelastine N-demethylase activity in microsomes from twelve human livers was significa ntly correlated with testosterone 6 beta-hydroxylase activity (r = 0.849, p < .0005). The percent contributions of CYP1A2, CYP2D6, and CYP3A4 in human livers were predicted using several approaches based on the concept of cor rection with CYP contents or relative activity factors (RAFs). Our data sug gested that the approach using RAF(CL) (RAF as the ratio of clearance) is m ost predictive of the N-demethylation clearance of azelastine because it be st reflects the observed N-demethylation clearance in human liver microsome s. Summarizing the results, azelastine N-demethylation in humans liver micr osomes is catalyzed mainly by CYP3A4 and CYP2D6, and CYP1A2 to a small exte nt (in average, 76.6, 21.8, and 3.9%, respectively), although the percent c ontribution of each isoform varied among individuals.