Chu Xiao-Yan, Huskey Su-E W, Braun Matthew P, Sarkadi Balazs, Evans David C, Evers Raymond
Department of Drug Metabolism, RY80M-112, Merck & Co. Inc., 126 East Lincoln Avenue, Rahway, NJ, USA.
J Pharmacol Exp Ther. 2004 Apr;309(1):156-64. doi: 10.1124/jpet.103.062091. Epub 2004 Jan 13.
Ethinylestradiol (EE) is one of the key constituents of oral contraceptives. Major metabolites of EE in humans are the glucuronide and sulfate conjugates, EE-3-O-glucuronide (EE-G) and EE-3-O-sulfate (EE-S). In the present study, transport of EE-G and EE-S by the human multidrug resistance proteins MRP1, MRP2, and MRP3 was investigated using inside-out membrane vesicles, isolated from Sf9 cells expressing human MRP1, MRP2, or MRP3. Vesicular uptake studies showed that EE-G was not a substrate for MRP1, whereas an ATP-dependent and saturable transport of [(3)H]EE-G was observed in MRP2 (K(m) of 35.1 +/- 3.5 microM) and MRP3 (K(m) of 9.2 +/- 2.3 microM) containing vesicles. EE-S was not transported by either MRP1, MRP2, or MRP3. However, low concentrations of EE-S stimulated MRP2-mediated uptake of ethacrynic acid glutathione. EE-S also stimulated MRP2 and MRP3-mediated uptake of 17beta-estradiol-17beta-D-glucuronide. Interestingly, EE-S stimulated strongly MRP2- and MRP3-mediated uptake of EE-G by increasing its apparent transport affinity, whereas no reciprocal stimulation of EE-S uptake by EE-G was observed. These data indicate that EE-S allosterically stimulates MRP2- and MRP3-mediated transport of EE-G and is not cotransported with EE-G. Our studies demonstrate specific active transport of a pharmacologically relevant drug conjugate by human MRP2 and MRP3, involving complex interactions with other organic anions. We also suggest that caution needs to be taken when using only competition studies as screening tools to identify substrates or inhibitors of MRP-mediated transport.
炔雌醇（EE）是口服避孕药的关键成分之一。EE在人体内的主要代谢产物是葡萄糖醛酸和硫酸酯结合物，即EE - 3 - O - 葡萄糖醛酸（EE - G）和EE - 3 - O - 硫酸酯（EE - S）。在本研究中，使用从表达人MRP1、MRP2或MRP3的Sf9细胞分离得到的内翻膜囊泡，研究了人多药耐药蛋白MRP1、MRP2和MRP3对EE - G和EE - S的转运。囊泡摄取研究表明，EE - G不是MRP1的底物，而在含有MRP2（米氏常数K(m)为35.1±3.5 microM）和MRP3（K(m)为9.2±2.3 microM）的囊泡中观察到[(3)H]EE - G的ATP依赖性和饱和性转运。EE - S既不被MRP1、MRP2也不被MRP3转运。然而，低浓度的EE - S刺激MRP2介导的依他尼酸谷胱甘肽摄取。EE - S还刺激MRP2和MRP3介导的17β - 雌二醇 - 17β - D - 葡萄糖醛酸摄取。有趣的是，EE - S通过增加其表观转运亲和力强烈刺激MRP2和MRP3介导的EE - G摄取，而未观察到EE - G对EE - S摄取的反向刺激。这些数据表明，EE - S变构刺激MRP2和MRP3介导的EE - G转运，且不与EE - G共转运。我们的研究证明了人MRP2和MRP3对一种具有药理学相关性的药物结合物的特异性主动转运，涉及与其他有机阴离子的复杂相互作用。我们还建议，仅将竞争研究用作筛选工具来鉴定MRP介导转运的底物或抑制剂时需谨慎。
