A Specific Probe Substrate for Evaluation of CYP4A11 Activity in Human Tissue Microsomes and a Highly Selective CYP4A11 Inhibitor: Luciferin-4A and Epalrestat
A Specific Probe Substrate for Evaluation of CYP4A11 Activity in Human Tissue Microsomes and a Highly Selective CYP4A11 Inhibitor: Luciferin-4A and Epalrestat
复制标题
DOI：
10.1124/jpet.118.249557
复制
发表时间：
2018-07
期刊：
影响因子：
--
通讯作者：
Satoshi Yamaori;Noriyuki Araki;Mio Shionoiri;Kurumi Ikehata;Shinobu Kamijo;S. Ohmori;Kazuhito Watanabe
中科院分区：
文献类型：
--
作者：
Satoshi Yamaori;Noriyuki Araki;Mio Shionoiri;Kurumi Ikehata;Shinobu Kamijo;S. Ohmori;Kazuhito Watanabe
研究了细胞色素P450 4A 11（CYP 4A 11）对人肝微粒体（HLM）和人肾微粒体（HRM）中的CYP 4A 11-O-去甲基化的特异性以及依帕司他对CYP 4A 11抑制的选择性。对HLM进行动力学分析，得到的Vmax和S50值分别为39.7 pmol/min/mg蛋白质和43.2 μM（希尔系数1.24），HRM为39.4 pmol/min/mg蛋白质和33.8 μM（希尔系数1.34）。在检测的选择性CYP 14抑制剂中，HET 0016（CYP 4抑制剂）仅抑制HLM和HRM引起的CYP 14 -4A O-去甲基化。此外，抗CYP 4A 11抗体几乎消除了两种组织微粒体的活性。HLM的荧光素-4A O-脱甲基酶活性与CYP 4A 11的标志物月桂酸ω-羟化酶活性呈显著正相关（r = 0.904，P < 0.0001）。接下来，检查依帕司他对CYP介导的药物氧化的影响。在检测的17种重组酶中，依帕司他对CYP 4A 11的抑制作用最强（IC 50 = 1.82 μM）。依帕司他对CYP 4A 11的抑制作用至少比对CYP 4F 2、CYP 4F 3B和CYP 4F 12的抑制作用强10倍。相反，对于已知的CYP 4抑制剂，HET 0016抑制CYP 4A 11和CYP 4F 2的活性（IC 50 = 0.0137-0.0182 μM）; 17-十八炔酸降低CYP 4A 11、CYP 4F 2、CYP 4F 3B和CYP 4F 12活性的程度相似（IC 50 = 5.70-17.7 μM）。依帕司他选择性有效抑制HLM（IC 50 = 0.913 μM）和HRM（IC 50 = 0.659 μM）的CYP 4A 11活性。这些结果表明，依帕司他-4A O-脱甲基酶活性是HLM和HRM的良好CYP 4A 11标志物，与已知CYP 4抑制剂相比，依帕司他是一种选择性更高的CYP 4A 11抑制剂。
The specificity of cytochrome P450 4A11 (CYP4A11) against luciferin-4A O-demethylation in human liver microsomes (HLMs) and human renal microsomes (HRMs) and selectivity of CYP4A11 inhibition by epalrestat were investigated. Kinetic analysis of luciferin-4A O-demethylation yielded Vmax and S50 values of 39.7 pmol/min per milligram protein and 43.2 μM for HLMs (Hill coefficient 1.24) and 39.4 pmol/min per milligram protein and 33.8 μM for HRMs (Hill coefficient 1.34), respectively. Among the selective CYP inhibitors tested, HET0016 (CYP4 inhibitor) exclusively inhibited luciferin-4A O-demethylation by HLMs and HRMs. Furthermore, anti-CYP4A11 antibody nearly abolished the activity of both tissue microsomes. Luciferin-4A O-demethylase activity of HLMs was significantly correlated with lauric acid ω-hydroxylase activity, a marker of CYP4A11 activity (r = 0.904, P < 0.0001). Next, effects of epalrestat on CYP-mediated drug oxidations were examined. Epalrestat showed the most potent inhibition against CYP4A11 (IC50 = 1.82 μM) among the 17 recombinant enzymes tested. The inhibitory effect of epalrestat on CYP4A11 was at least 10-fold stronger than those on CYP4F2, CYP4F3B, and CYP4F12. For known CYP4 inhibitors, in contrast, HET0016 inhibited the activities of CYP4A11 and CYP4F2 (IC50 = 0.0137–0.0182 μM); 17-octadecynoic acid reduced activities of CYP4A11, CYP4F2, CYP4F3B, and CYP4F12 to a similar extent (IC50 = 5.70–17.7 μM). Epalrestat selectively and effectively inhibited the CYP4A11 activity of HLMs (IC50 = 0.913 μM) and HRMs (IC50 = 0.659 μM). These results indicated that luciferin-4A O-demethylase activity is a good CYP4A11 marker of HLMs and HRMs, and that epalrestat is a more selective CYP4A11 inhibitor compared with known CYP4 inhibitors.