Kahma Helinä, Aurinsalo Laura, Neuvonen Mikko, Katajamäki Jani, Paludetto Marie-Noëlle, Viinamäki Jenni, Launiainen Terhi, Filppula Anne M, Tornio Aleksi, Niemi Mikko, Backman Janne T
Department of Clinical Pharmacology, University of Helsinki, Helsinki, Finland; Individualized Drug Therapy Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland; Department of Clinical Pharmacology, HUS Diagnostic Center, Helsinki University Hospital, Helsinki, Finland.
Department of Clinical Pharmacology, University of Helsinki, Helsinki, Finland; Individualized Drug Therapy Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
Eur J Pharm Sci. 2021 Jul 1;162:105810. doi: 10.1016/j.ejps.2021.105810. Epub 2021 Mar 19.
We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-β-glucuronide and clopidogrel acyl-β-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and K values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-β-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-β-glucuronide was a strong (>90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 μM, while the selectivity of clopidogrel acyl-β-D-glucuronide was limited at concentrations above its IC for CYP2C8. The time-dependent IC values of these glucuronides for CYP2C8 were 8.1 and 38 µM, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting time-dependent inhibition. Moreover, gemfibrozil 1-O-β-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.
我们开发了一种针对九种主要药物代谢CYP酶的体外高通量混合测定法，该方法针对时间依赖性抑制的筛选进行了优化。该方法用于确定时间依赖性CYP2C8抑制剂吉非贝齐1-O-β-葡萄糖醛酸苷和氯吡格雷酰基-β-D-葡萄糖醛酸苷的选择性。使用自动液体处理技术在96孔板中进行CYP选择性探针底物与混合人肝微粒体的体外孵育，并通过定量UHPLC-MS/MS分析测量代谢物浓度。在确定每个反应的底物间相互作用和K值后，将探针底物分为混合物I（他克林/CYP1A2、安非他酮/CYP2B6、阿莫地喹/CYP2C8、甲苯磺丁脲/CYP2C9和咪达唑仑/CYP3A4/5）和混合物II（香豆素/CYP2A6、S-美芬妥英/CYP2C19、右美沙芬/CYP2D6和阿司咪唑/CYP2J2）。时间依赖性抑制剂（呋拉茶碱/CYP1A2、司来吉兰/CYP2A6、氯吡格雷/CYP2B6、吉非贝齐1-O-β-葡萄糖醛酸苷/CYP2C8、替尼酸/CYP2C9、噻氯匹定/CYP2C19、帕罗西汀/CYP2D6和利托那韦/CYP3A）和直接抑制剂（特非那定/CYP2J2）在单底物法和混合法中表现出相似的抑制作用。已确定的时间依赖性抑制剂通过混合法导致IC倍数变化范围为2.2至30。在时间依赖性抑制条件下，吉非贝齐1-O-β-葡萄糖醛酸苷在60至300μM的浓度范围内是CYP2C8的强效（>90%抑制）和选择性（对其他CYPs的抑制<<20%）抑制剂，而氯吡格雷酰基-β-D-葡萄糖醛酸苷在高于其对CYP2C8的IC浓度时选择性有限。这些葡萄糖醛酸苷对CYP2C8的时间依赖性IC值分别为8.1和38μM。总之，开发、优化并验证了一种可靠的包含九种最重要药物代谢CYP酶的混合法，用于检测时间依赖性抑制。此外，吉非贝齐1-O-β-葡萄糖醛酸苷被确定为CYP2C8的选择性抑制剂，可在体外研究中用作诊断抑制剂。
