人肝微粒体中细胞色素P-450（CYP）3A4、CYP2D6和CYP1A2对氮卓斯汀的N-去甲基化作用：预测多种细胞色素P-450贡献方法的评估

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.

作者信息

Nakajima M, Nakamura S, Tokudome S, Shimada N, Yamazaki H, Yokoi T

机构信息

Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.

出版信息

Drug Metab Dispos. 1999 Dec;27(12):1381-91.

PMID:10570018

Abstract

Azelastine, an antiallergy and antiasthmatic drug, has been reported to be mainly N-demethylated to desmethylazelastine in humans. In the present study, Eadie-Hofstee plots of azelastine N-demethylation in human liver microsomes were biphasic. In microsomes from human B-lymphoblast cells, recombinant cytochrome P-450 (CYP)2D6 and CYP1A1 exhibited higher azelastine N-demethylase activity than did other CYP enzymes. On the other hand, recombinant CYP3A4 and CYP1A2 as well as CYP1A1 and CYP2D6 in microsomes from baculovirus-infected insect cells were active in azelastine N-demethylation. The K(M) value of the recombinant CYP2D6 (2.1 microM) from baculovirus-infected insect cells was similar to the K(M) value of the high-affinity (2.4+/-1.3 microM) component in human liver microsomes. On the other hand, the K(M) values of the recombinant CYP3A4 (51.1 microM) and CYP1A2 (125.4 microM) from baculovirus-infected insect cells were similar to the K(M) value of the low-affinity (79.7+/-12.8 microM) 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 microsomes (5 microM azelastine) was inhibited by ketoconazole, erythromycin, and fluvoxamine (IC(50) = 0.08, 18.2, and 17.2 microM, respectively). Azelastine N-demethylase activity in microsomes from twelve human livers was significantly correlated with testosterone 6beta-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 correction with CYP contents or relative activity factors (RAFs). Our data suggested that the approach using RAF(CL) (RAF as the ratio of clearance) is most predictive of the N-demethylation clearance of azelastine because it best reflects the observed N-demethylation clearance in human liver microsomes. Summarizing the results, azelastine N-demethylation in humans liver microsomes is catalyzed mainly by CYP3A4 and CYP2D6, and CYP1A2 to a small extent (in average, 76.6, 21.8, and 3.9%, respectively), although the percent contribution of each isoform varied among individuals.

摘要

氮卓斯汀是一种抗过敏和抗哮喘药物，据报道在人体内主要通过N-去甲基化生成去甲基氮卓斯汀。在本研究中，人肝微粒体中氮卓斯汀N-去甲基化的伊迪-霍夫斯蒂图呈双相。在人B淋巴细胞微体中，重组细胞色素P-450（CYP）2D6和CYP1A1表现出比其他CYP酶更高的氮卓斯汀N-去甲基酶活性。另一方面，杆状病毒感染昆虫细胞微体中的重组CYP3A4、CYP1A2以及CYP1A1和CYP2D6在氮卓斯汀N-去甲基化中具有活性。杆状病毒感染昆虫细胞中的重组CYP2D6的K(M)值（2.1微摩尔）与人肝微粒体中高亲和力（2.4±1.3微摩尔）成分的K(M)值相似。另一方面，杆状病毒感染昆虫细胞中的重组CYP3A4（51.1微摩尔）和CYP1A2（125.4微摩尔）的K(M)值与人肝微粒体中低亲和力（79.7±12.8微摩尔）成分的K(M)值相似。布非洛尔抑制高亲和力成分，使人肝微粒体中的伊迪-霍夫斯蒂图变为单相。酮康唑、红霉素和氟伏沙明抑制人肝微粒体中氮卓斯汀N-去甲基酶活性（IC(50)分别为0.08、18.2和17.2微摩尔）。来自12个人肝脏的微体中氮卓斯汀N-去甲基酶活性与睾酮6β-羟化酶活性显著相关（r = 0.849，p<0.0005）。基于用CYP含量或相对活性因子（RAF）校正的概念，采用几种方法预测了人肝脏中CYP1A2、CYP2D6和CYP3A4的贡献百分比。我们的数据表明，使用RAF(CL)（RAF作为清除率的比值）的方法对氮卓斯汀N-去甲基化清除率的预测性最强，因为它最能反映人肝微粒体中观察到的N-去甲基化清除率。总结结果，人肝微粒体中氮卓斯汀N-去甲基化主要由CYP3A4和CYP2D6催化，CYP1A2的催化作用较小（平均分别为76.6%、21.8%和3.9%），尽管每种同工型的贡献百分比在个体之间有所不同。