Venkatakrishnan K, von Moltke L L, Duan S X, Fleishaker J C, Shader R I, Greenblatt D J
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA 02111, USA.
J Pharm Pharmacol. 1998 Mar;50(3):265-74. doi: 10.1111/j.2042-7158.1998.tb06859.x.
The kinetics of the N-demethylation of adinazolam to N-desmethyladinazolam (NDMAD), and of NDMAD to didesmethyladinazolam (DDMAD), were studied with human liver microsomes using substrate concentrations in the range 10-1000 microM. The specific cytochrome P450 (CYP) isoforms mediating the biotransformations were identified using microsomes containing specific recombinant CYP isozymes expressed in human lymphoblastoid cells, and by the use of CYP isoform-selective chemical inhibitors. Adinazolam was demethylated by human liver microsomes to NDMAD, and NDMAD was demethylated to DDMAD; the substrate concentrations, Km, at which the reaction velocities were 50% of the maximum were 92 and 259 microM, respectively. Another metabolite of yet undetermined identity (U) was also formed from NDMAD (Km 498 microM). Adinazolam was demethylated by cDNA-expressed CYP 2C19 (Km 39 microM) and CYP 3A4 (Km 83 microM); no detectable activity was observed for CYPs 1A2, 2C9, 2D6 and 2E1. Ketoconazole, a relatively specific CYP 3A4 inhibitor, inhibited the reaction; the concentration resulting in 50% of maximum inhibition, IC50, was 0.15 microM and the inhibition constant, Ki, was < 0.04 microM in five of six livers tested. Troleandomycin, a specific inhibitor of CYP 3A4, inhibited adinazolam N-demethylation with an IC50 of 1.96 microM. The CYP 2C19-inhibitor omeprazole resulted in only partial inhibition (IC50 21 microM) and sulphaphenazole, alpha-naphthoflavone, quinidine and diethyldithiocarbamate did not inhibit the reaction. NDMAD was demethylated by cDNA-expressed CYP 3A4 (Km 220 microM, Hill number A 1.21), CYP 2C19 (Km 187 microM, Hill number A 1.29) and CYP 2C9 (Km 1068 microM). Formation of U was catalysed by CYP 3A4 alone. Ketoconazole strongly inhibited NDMAD demethylation (IC50 0.14 microM) and formation of U (IC50 < 0.1 microM) whereas omeprazole and sulphaphenazole had no effect on reaction rates. These results show that CYP 3A4 is the primary hepatic CYP isoform mediating the N-demethylation of adinazolam and NDMAD. Co-administration of adinazolam with CYP 3A4 inhibitors such as ketoconazole or erythromycin might lead to reduced efficacy, since adinazolam by itself has relatively weak benzodiazepine agonist activity, with much of the pharmacological activity of adinazolam being attributable to its active metabolite NDMAD.
采用人肝微粒体，在10 - 1000微摩尔范围内的底物浓度下，研究了阿地唑仑N - 去甲基化生成N - 去甲基阿地唑仑（NDMAD）以及NDMAD生成二去甲基阿地唑仑（DDMAD）的动力学过程。使用在人淋巴母细胞中表达的特定重组CYP同工酶的微粒体，并通过使用CYP同工酶选择性化学抑制剂，来鉴定介导生物转化的特定细胞色素P450（CYP）同工酶。阿地唑仑被人肝微粒体去甲基化为NDMAD，NDMAD又被去甲基化为DDMAD；反应速度达到最大值50%时的底物浓度Km分别为92和259微摩尔。NDMAD还生成了另一种身份尚未确定的代谢物（U）（Km为498微摩尔）。阿地唑仑被cDNA表达的CYP 2C19（Km为39微摩尔）和CYP 3A4（Km为83微摩尔）去甲基化；未观察到CYPs 1A2、2C9、2D6和2E1有可检测到的活性。酮康唑是一种相对特异的CYP 3A4抑制剂，可抑制该反应；在六个测试肝脏中的五个中，导致最大抑制50%的浓度IC50为0.15微摩尔，抑制常数Ki < 0.04微摩尔。三乙酰竹桃霉素是CYP 3A4的特异性抑制剂，抑制阿地唑仑N - 去甲基化的IC50为1.96微摩尔。CYP 2C19抑制剂奥美拉唑仅导致部分抑制（IC50为21微摩尔），而磺胺苯吡唑、α - 萘黄酮、奎尼丁和二乙基二硫代氨基甲酸盐不抑制该反应。NDMAD被cDNA表达的CYP 3A4（Km为220微摩尔，希尔系数A为1.21）、CYP 2C19（Km为187微摩尔，希尔系数A为1.29）和CYP 2C9（Km为1068微摩尔）去甲基化。U的形成仅由CYP 3A4催化。酮康唑强烈抑制NDMAD去甲基化（IC50为0.14微摩尔）和U的形成（IC50 < 0.1微摩尔），而奥美拉唑和磺胺苯吡唑对反应速率无影响。这些结果表明，CYP 3A4是介导阿地唑仑和NDMAD N - 去甲基化的主要肝CYP同工酶。阿地唑仑与CYP 3A4抑制剂如酮康唑或红霉素合用时，可能会导致疗效降低，因为阿地唑仑本身的苯二氮䓬激动剂活性相对较弱，其大部分药理活性归因于其活性代谢物NDMAD。
