Kim Kyoung-Ah, Chung Jaegul, Jung Dong-Hae, Park Ji-Young
Department of Pharmacology, Gil Medical Center, Gachon Medical School, 1198 Kuwol-dong, 405-760, Incheon, Namdong-gu, Korea.
Eur J Clin Pharmacol. 2004 Oct;60(8):575-81. doi: 10.1007/s00228-004-0815-3. Epub 2004 Sep 8.
The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes.
Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values.
The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent Km and Vmax values were 21.1 microM and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 microM and 412.0 pmol/min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (rs=0.829; P<0.01), CYP2B6-catalzyed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (rs=0.691; P<0.05), and CYP2C8-catalyzed paclitaxel 6alpha-hydroxylation (rs=0.797; P<0.05).
CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.
本研究旨在阐明人肝微粒体中参与将洛哌丁胺（LOP）代谢为N-去甲基洛哌丁胺（DLOP）的细胞色素P450（P450）同工酶。
采用三种既定方法，利用人肝微粒体和cDNA表达的P450同工酶鉴定负责LOP N-去甲基化的P450同工酶：（1）一组人肝微粒体中LOP N-去甲基化活性与标记P450活性的相关性，（2）P450选择性抑制剂对酶活性的抑制作用，（3）cDNA表达的P450同工酶对DLOP形成的测定。通过应用相对活性因子（RAF）值估算人肝微粒体中参与LOP N-去甲基化的P450同工酶的相对贡献。
DLOP的形成速率呈现双相动力学，提示涉及多种P450同工酶。高亲和力组分的表观Km和Vmax值分别为21.1 microM和每毫克蛋白质122.3 pmol/min，低亲和力组分分别为83.9 microM和每毫克蛋白质412.0 pmol/min。在所测试的cDNA表达的P450中，CYP2B6、CYP2C8、CYP2D6和CYP3A4催化LOP N-去甲基化。与槲皮素（一种CYP2C8抑制剂）和酮康唑（一种CYP3A4抑制剂）共同孵育时，LOP N-去甲基化分别被显著抑制40%和90%，但所测试的其他化学抑制剂显示出较弱的抑制作用或无显著抑制作用。DLOP的形成与CYP3A4催化的咪达唑仑1-羟化反应（rs = 0.829；P < 0.01）、CYP2B6催化的7-乙氧基-4-三氟甲基香豆素O-去乙基化反应（rs = 0.691；P < 0.05）以及CYP2C8催化的紫杉醇6α-羟化反应（rs = 0.797；P < 0.05）高度相关。
CYP2B6、CYP2C8、CYP2D6和CYP3A4在人肝微粒体中催化LOP N-去甲基化，其中CYP2C8和CYP3A4在LOP治疗浓度下的代谢中可能起关键作用。这些P450抑制剂的共同给药可能导致与LOP的药物相互作用。然而，CYP2C8和CYP3A4抑制剂对LOP代谢潜在相互作用的临床意义应进一步研究。
