Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Br J Pharmacol. 2013 May;169(1):21-33. doi: 10.1111/bph.12126.
Hepatic uptake (e.g. by OATP1B1), phase I and II metabolism (e.g. by CYP3A4, UGT1A1) and subsequent biliary excretion (e.g. by MRP2) are key determinants for the pharmacokinetics of numerous drugs. However, stably transfected cell models for the simultaneous investigation of transport and phase I and II metabolism of drugs are lacking.
A newly established quadruple-transfected MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 cell line was used to investigate metabolism and transcellular transport of the endothelin receptor antagonist bosentan.
Intracellular accumulation of bosentan equivalents (i.e. parent compound and metabolites) was significantly lower in all cell lines expressing MRP2 compared to cell lines lacking this transporter (P < 0.001). Accordingly, considerably higher amounts of bosentan equivalents were detectable in the apical compartments of cell lines with MRP2 expression (P < 0.001). HPLC and LC-MS measurements revealed that mainly unchanged bosentan accumulated in intracellular and apical compartments. Furthermore, the phase I metabolites Ro 48-5033 and Ro 47-8634 were detected intracellularly in cell lines expressing CYP3A4. Additionally, a direct glucuronide of bosentan could be identified intracellularly in cell lines expressing UGT1A1 and in the apical compartments of cell lines expressing UGT1A1 and MRP2.
These in vitro data indicate that bosentan is a substrate of UGT1A1. Moreover, the efflux transporter MRP2 mediates export of bosentan and most likely also of bosentan glucuronide in the cell system. Taken together, cell lines simultaneously expressing transport proteins and metabolizing enzymes represent additional useful tools for the investigation of the interplay of transport and metabolism of drugs.
肝脏摄取（例如，通过 OATP1B1）、I 相和 II 相代谢（例如，通过 CYP3A4、UGT1A1）以及随后的胆汁排泄（例如，通过 MRP2）是许多药物药代动力学的关键决定因素。然而，缺乏用于同时研究药物转运和 I 相和 II 相代谢的稳定转染细胞模型。
使用新建立的四重转染 MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 细胞系来研究内皮素受体拮抗剂波生坦的代谢和细胞间转运。
与缺乏该转运体的细胞系相比，表达 MRP2 的所有细胞系中，波生坦当量（即母体化合物和代谢物）的细胞内积累显著降低（P < 0.001）。因此，在表达 MRP2 的细胞系的顶端隔室中可检测到明显更高量的波生坦当量（P < 0.001）。HPLC 和 LC-MS 测量显示，主要未改变的波生坦在细胞内和顶端隔室中积累。此外，在表达 CYP3A4 的细胞系中检测到 I 相代谢物 Ro 48-5033 和 Ro 47-8634 细胞内。此外，在表达 UGT1A1 的细胞系中以及在表达 UGT1A1 和 MRP2 的细胞系的顶端隔室中可鉴定到波生坦的直接葡萄糖醛酸苷。
这些体外数据表明，波生坦是 UGT1A1 的底物。此外，外排转运蛋白 MRP2 介导波生坦及其可能的波生坦葡萄糖醛酸苷在细胞系统中的外排。综上所述，同时表达转运蛋白和代谢酶的细胞系代表了用于研究药物转运和代谢相互作用的额外有用工具。
