Department of Metabolism and Pharmacokinetics, Pharmaceutical Candidate Optimization, Bristol-Meyers Squibb, Princeton, New Jersey 08543-4000, USA.
Drug Metab Dispos. 2010 Jul;38(7):1064-71. doi: 10.1124/dmd.109.031526. Epub 2010 Apr 1.
17alpha-Ethinylestradiol (EE2), a synthetic and potent estrogen receptor agonist, is extensively metabolized in both intestine and liver and is largely excreted in bile and urine as the 3-O-sulfate (EE2-Sul) and 3-O-glucuronide. In the present study, EE2-Sul was evaluated as a substrate of various transporters known to be expressed in the kidney. Uptake studies were performed with human epithelial cells [human embryonic kidney (HEK)-293] that contained individually expressed organic cation transporter 2 (OCT2), organic anion transporter (OAT) forms 3 and 4, and multidrug and toxin extrusion 1 (MATE1). The transporter phenotyping studies were extended to include insect cell (Sf9) membrane vesicles that expressed multidrug resistance-associated protein 4 (MRP4) and Madin-Darby canine kidney cells that expressed OAT1. Based on the results obtained, we concluded that EE2-Sul serves as a substrate of OAT3 and OAT4, but not OCT2, OAT1, MATE1, and MRP4. First, EE2-Sul uptake was highly increased in OAT3/HEK-293 cells (versus mock/HEK-293 cells) and was inhibited by OAT3 inhibitors such as bromosulfophthalein (BSP), cimetidine, and probenecid. OAT3-mediated uptake also conformed to single-K(m) (Michaelis constant) kinetics (K(m) = 21.1 microM). Second, EE2-Sul uptake was also significantly higher in OAT4/HEK-293 cells and was inhibited by BSP, methotrexate, and probenecid. In contrast to OAT3, OAT4-dependent uptake was characterized by a two-K(m) model (K(m1) = 1.6 microM; K(m2) = 195 microM). Based on the results of this study, we hypothesize that EE2-Sul is taken up into renal proximal tubule cells by OAT3, and OAT4 plays a role in its secretion into the renal brush border lumen.
17α-乙炔基雌二醇（EE2）是一种合成的、有效的雌激素受体激动剂，在肠道和肝脏中广泛代谢，并主要以 3-O-硫酸盐（EE2-Sul）和 3-O-葡糖苷酸的形式从胆汁和尿液中排泄。在本研究中，评估 EE2-Sul 作为已知在肾脏中表达的各种转运体的底物。使用分别表达有机阳离子转运体 2（OCT2）、有机阴离子转运体（OAT）形式 3 和 4 以及多药和毒素外排蛋白 1（MATE1）的人上皮细胞[人胚肾（HEK）-293]进行摄取研究。转运体表型研究扩展到包括表达多药耐药相关蛋白 4（MRP4）的昆虫细胞（Sf9）膜囊泡和表达 OAT1 的马迪逊-达比犬肾细胞。根据获得的结果，我们得出结论，EE2-Sul 是 OAT3 和 OAT4 的底物，但不是 OCT2、OAT1、MATE1 和 MRP4 的底物。首先，EE2-Sul 的摄取在 OAT3/HEK-293 细胞（与 mock/HEK-293 细胞相比）中高度增加，并且被 OAT3 抑制剂如溴磺酞（BSP）、西咪替丁和丙磺舒抑制。OAT3 介导的摄取也符合单-K(m)（米氏常数）动力学（K(m) = 21.1 μM）。其次，EE2-Sul 的摄取在 OAT4/HEK-293 细胞中也显著增加，并且被 BSP、甲氨蝶呤和丙磺舒抑制。与 OAT3 不同，OAT4 依赖性摄取的特征是双-K(m)模型（K(m1) = 1.6 μM；K(m2) = 195 μM）。根据本研究的结果，我们假设 EE2-Sul 被 OAT3 摄取到肾近端小管细胞中，而 OAT4 在其分泌到肾刷状缘腔中起作用。
