Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
Drug Metab Dispos. 2010 Mar;38(3):376-85. doi: 10.1124/dmd.109.030551. Epub 2009 Dec 10.
We identify here for the first time the low-affinity cytochrome P450 (P450) isoforms that metabolize paroxetine, using cDNA-expressed human P450s measuring substrate depletion and paroxetine-catechol (product) formation by liquid chromatography-tandem mass spectrometry. CYP1A2, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were identified as paroxetine-catechol-forming P450 isoforms, and CYP2C19 and CYP2D6 were identified as metabolizing P450 isoforms by substrate depletion. Michaelis-Menten constants K(m) and V(max) were determined by product formation and substrate depletion. Using selective inhibitory studies and a relative activity factor approach for pooled and single-donor human liver microsomes, we confirmed involvement of the identified P450 isoforms for paroxetine-catechol formation at 1 and 20 muM paroxetine. In addition, we used the population-based simulator Simcyp to estimate the importance of the identified paroxetine-metabolizing P450 isoforms for human metabolism, taking mechanism-based inhibition into account. The amount of active hepatic CYP2D6 and CYP3A4 (not inactivated by mechanism-based inhibition) was also estimated by Simcyp. For extensive and poor metabolizers of CYP2D6, Simcyp-estimated pharmacokinetic profiles were in good agreement with those reported in published in vivo studies. Considering the kinetic parameters, inhibition results, relative activity factor calculations, and Simcyp simulations, CYP2D6 (high affinity) and CYP3A4 (low affinity) are most likely to be the major contributors to paroxetine metabolism in humans. For some individuals CYP1A2 could be of importance for paroxetine metabolism, whereas the importance of CYP2C19 and CYP3A5 is probably limited.
我们首次鉴定了代谢帕罗西汀的低亲和力细胞色素 P450（CYP）同工酶，使用 cDNA 表达的人 CYP 进行液体色谱-串联质谱法测量底物耗竭和帕罗西汀儿茶酚（产物）形成。鉴定 CYP1A2、CYP2C19、CYP2D6、CYP3A4 和 CYP3A5 为帕罗西汀儿茶酚形成的 CYP 同工酶，CYP2C19 和 CYP2D6 为通过底物耗竭代谢的 CYP 同工酶。通过产物形成和底物耗竭确定米氏常数 K(m)和 V(max)。使用选择性抑制研究和混合和单个供体人肝微粒体的相对活性因子方法，我们确认了鉴定的 CYP 同工酶在 1 和 20 μM 帕罗西汀时对帕罗西汀儿茶酚形成的参与。此外，我们使用基于人群的模拟器 Simcyp 来估计所鉴定的帕罗西汀代谢 CYP 同工酶对人体代谢的重要性，同时考虑到基于机制的抑制作用。Simcyp 还估计了活性肝 CYP2D6 和 CYP3A4（不受基于机制的抑制作用失活）的数量。对于 CYP2D6 的广泛和不良代谢者，Simcyp 估计的药代动力学曲线与已发表的体内研究报告的曲线吻合良好。考虑到动力学参数、抑制结果、相对活性因子计算和 Simcyp 模拟，CYP2D6（高亲和力）和 CYP3A4（低亲和力）很可能是帕罗西汀在人体中代谢的主要贡献者。对于某些个体，CYP1A2 可能对帕罗西汀代谢很重要，而 CYP2C19 和 CYP3A5 的重要性可能有限。
