AIMMS-Division of Molecular Toxicology, Department of Chemistry & Pharmaceutical Sciences, Vrije Universiteit Amsterdam, O|2 building, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.
Kaly-Cell, 20A Rue du Général Leclerc, Plobsheim, France; UNISTRA, 4 Rue Blaise Pascal, Strasbourg, France.
Eur J Pharm Sci. 2017 Nov 15;109:96-110. doi: 10.1016/j.ejps.2017.07.032. Epub 2017 Aug 1.
UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.
UDP-葡糖醛酸基转移酶（UGTs）和细胞色素 P450s（CYPs）是参与药物在肝脏中代谢的主要酶。通常使用人肝微粒体（HLM）或原代人肝细胞（PHH）来研究肝药物代谢。我们描述了一种灵敏的测定法，用于通过分析选择性探针底物的葡糖醛酸化来表型鉴定 6 种主要肝 UGT 同工型（UGT1A1、UGT1A3、UGT1A4、UGT1A6、UGT1A9 和 UGT2B7）在完整 PHH 中的活性。为了进行比较，还包括非选择性的通用底物 7-羟基香豆素。对于每个肝供体制剂（5 个供体），比较了冷冻保存悬浮和种植 PHH 中的 UGT 活性与来自同一供体的 HLM。同时平行进行了 7 种主要同工型的 CYP 反应表型测定。对于所有供体，PHH 和 HLM 中 CYP 和 UGT 同工型的活性谱相似，表明用选择性探针底物在完整细胞中进行的反应表型主要反映了各自的 CYP 或 UGT 活性。仍然发现了对 UGT 和 CYP 同工型活性的系统依赖性影响。虽然 UGT1A1 在种植和悬浮 PHH 中的活性相当，但 UGT1A3、UGT1A6 和 UGT2B7 的活性在悬浮 PHH 中较高，UGT1A9 和 UGT1A4 的活性在种植 PHH 中较高。证实了众所周知的在种植 PHH 中与悬浮 PHH 相比大多数 CYP 同工型活性降低的现象。重要的是，我们发现 HLM 中 CYP2C19 和 CYP2B6 的活性显著降低，活性低于完整细胞。综上所述，这些发现表明，取决于代谢特定化合物的 UGT 或 CYP 同工型，代谢测定的结果强烈依赖于体外系统的选择。目前描述的 UGT 和 CYP 活性分析方法可作为完整细胞中的标准测定法，尤其有助于对可用细胞数量有限的体外系统进行反应表型鉴定。
