Venkatakrishnan K, Greenblatt D J, von Moltke L L, Schmider J, Harmatz J S, Shader R I
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA 02111, USA.
J Clin Pharmacol. 1998 Feb;38(2):112-21. doi: 10.1002/j.1552-4604.1998.tb04399.x.
The human cytochromes P450 (CYPs) mediating amitriptyline N-demethylation have been identified using a combination of enzyme kinetic and chemical inhibition studies. Amitriptyline was N-demethylated to nortriptyline by microsomes from cDNA transfected human lymphoblastoid cells expressing human CYPs 1A2, 2C9, 2C19, 2D6, and 3A4. CYP 2E1 showed no detectable activity. While CYP 2C19 and CYP 2D6 showed high affinity, CYP 3A4 showed low affinity; CYP 2C9 and 1A2 showed intermediate affinities. Based on these kinetic parameters and estimated relative abundance of the different CYPs in human liver, CYP 2C19 was identified as the major amitriptyline N-demethylase at low (therapeutically relevant) amitriptyline concentrations, whereas CYP 3A4 may be more important at higher amitriptyline concentrations. Chemical inhibition studies with ketoconazole and omeprazole indicate that CYP 3A4 is the major amitriptyline N-demethylase at 100 mumol/L amitriptyline, while CYP 2C19 is equally important at a substrate concentration of 5 mumol/L. The CYP 1A2 inhibitor alpha-naphthoflavone and the CYP 2C9 inhibitor sulfaphenazole produced much less inhibition of amitriptyline N-demethylation at both substrate concentrations. Quinidine produced no detectable inhibition. The kinetics of amitriptyline N-demethylation by human liver microsomes were consistent with a two enzyme model, with the high affinity component exhibiting Michaelis Menten kinetics and the low affinity component exhibiting Hill enzyme kinetics. No difference was apparent in the kinetics of amitriptyline N-demethylation in two liver samples with low levels of CYP 2C19 activity compared with two other samples with relatively normal 2C19 activity. This may reflect the importance of higher substrate concentration values in estimation of kinetic parameters in vitro.
通过酶动力学和化学抑制研究相结合的方法，已鉴定出介导阿米替林N - 去甲基化的人细胞色素P450（CYP）。阿米替林被表达人CYP 1A2、2C9、2C19、2D6和3A4的cDNA转染人淋巴母细胞样细胞的微粒体N - 去甲基化为去甲替林。CYP 2E1未显示出可检测到的活性。虽然CYP 2C19和CYP 2D6显示出高亲和力，但CYP 3A4显示出低亲和力；CYP 2C9和1A2显示出中等亲和力。基于这些动力学参数以及人肝脏中不同CYP的估计相对丰度，可以确定在低（治疗相关）阿米替林浓度下，CYP 2C19是主要的阿米替林N - 去甲基酶，而在较高阿米替林浓度下，CYP 3A4可能更重要。酮康唑和奥美拉唑的化学抑制研究表明，在100 μmol/L阿米替林浓度下，CYP 3A4是主要的阿米替林N - 去甲基酶，而在5 μmol/L底物浓度下，CYP 2C19同样重要。CYP 1A2抑制剂α - 萘黄酮和CYP 2C9抑制剂磺胺苯吡唑在两种底物浓度下对阿米替林N - 去甲基化的抑制作用都小得多。奎尼丁未产生可检测到的抑制作用。人肝脏微粒体对阿米替林N - 去甲基化的动力学符合双酶模型，高亲和力组分表现出米氏动力学，低亲和力组分表现出希尔酶动力学。与另外两个具有相对正常2C19活性的样品相比，在两个CYP 2C19活性水平较低的肝脏样品中，阿米替林N - 去甲基化的动力学没有明显差异。这可能反映了较高底物浓度值在体外动力学参数估计中的重要性。
