Jones D R, Gorski J C, Hamman M A, Mayhew B S, Rider S, Hall S D
Indiana University School of Medicine, Department of Medicine, Division of Clinical Pharmacology, Wishard Memorial Hospital, Indianapolis, Indiana, USA.
J Pharmacol Exp Ther. 1999 Sep;290(3):1116-25.
Diltiazem (DTZ) N-demethylation occurs by cytochrome P-450 (CYP) 3A based on the following observations: 1) a single enzyme Michaelis-Menten model of metabolite formation, 2) high correlations of DTZ N-demethylation activity to other CYP3A activities, 3) inhibition of DTZ N-demethylation activity by triacetyloleandomycin, and 4) DTZ N-demethylation activity by expressed CYP3A enzymes only. The mean K(m)s for DTZ N-demethylation in human liver microsomes and expressed CYP3A4(+b(5)) were 53 and 16 microM, respectively. A 30-min preincubation of DTZ in expressed CYPs inhibited CYP3A4(+b(5)) by 100%, of which 55% was due to formation of a metabolite intermediate complex (MIC), which is an inactive form of CYP. MIC was observed in human liver microsomes and cDNA-expressed CYP3A only. In experiments to assess simultaneous MIC formation and loss of CYP3A activity, DTZ caused greater than 80% inhibition of midazolam hydroxylation after a 60-min preincubation in human liver microsomes. The rate constants for MIC formation and loss of midazolam hydroxylation activity were equivalent for the line of best fit for both data sets, which illustrates that MIC formation causes the inhibition of CYP3A activity. The mechanistic inhibition was characterized in expressed CYP3A4(+b(5)), which exhibited a concentration-dependent formation of MIC by DTZ (1-100 microM) with an estimated k(inact) of 0.17 min(-1) and K(I) of 2.2 microM. The partition ratio for expressed CYP3A4(+b(5)) was substrate concentration dependent and varied from 13 to 86. This study showed that DTZ inhibition of CYP3A substrate metabolism occurs primarily by MIC formation.
基于以下观察结果，地尔硫䓬（DTZ）的N - 去甲基化由细胞色素P - 450（CYP）3A介导：1）代谢产物形成的单酶米氏动力学模型；2）DTZ N - 去甲基化活性与其他CYP3A活性高度相关；3）三乙酰夹竹桃霉素对DTZ N - 去甲基化活性的抑制作用；4）仅由表达的CYP3A酶介导的DTZ N - 去甲基化活性。人肝微粒体和表达的CYP3A4（+b5）中DTZ N - 去甲基化的平均米氏常数（K(m)）分别为53和16 microM。DTZ在表达的细胞色素P450酶中预孵育30分钟可使CYP3A4（+b5）完全抑制，其中55%是由于代谢产物中间体复合物（MIC）的形成，这是一种无活性的细胞色素P450形式。仅在人肝微粒体和cDNA表达的CYP3A中观察到MIC。在评估同时形成MIC和CYP酶活性丧失的实验中，DTZ在人肝微粒体中预孵育60分钟后，对咪达唑仑羟化的抑制率超过80%。两个数据集的最佳拟合线显示，MIC形成和咪达唑仑羟化活性丧失的速率常数相等，这表明MIC的形成导致CYP3A酶活性的抑制。在表达的CYP3A4（+b5）中对这种机制性抑制进行了表征，DTZ（1 - 100 microM）使其呈现浓度依赖性MIC形成，估计失活常数（k(inact)）为0.17 min(-1)，抑制常数（K(I)）为2.2 microM。表达的CYP3A4（+b5）的分配比取决于底物浓度，范围为13至86。该研究表明，DTZ对CYP3A底物代谢的抑制主要通过MIC的形成发生。
