Sasaki Makoto, Suzuki Hiroshi, Aoki Jun, Ito Kousei, Meier Peter J, Sugiyama Yuichi
Department of Biopharmaceutics, School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Mol Pharmacol. 2004 Sep;66(3):450-9. doi: 10.1124/mol.66.3..
We have proposed previously that the evaluation of transcellular transport across the double-transfected Madin-Darby canine kidney II (MDCK II) monolayer that expresses both human organic anion transporting polypeptide 4 (OATP2/SLC21A6) and multidrug resistance associated protein 2 (MRP2/ABCC2) on the basal and apical membranes, respectively, may be useful in characterizing human biliary excretion (J Biol Chem 277: 6497-6503, 2002). However, to demonstrate that this in vitro system represents in vivo biliary excretion, it is essential to compare in vitro data with in vivo biliary excretion. The problem is that we cannot determine the human biliary excretion for many ligands. In the present study, we have established a double-transfected MDCK II monolayer that expresses both rat Oatp4/Slc21a10 and Mrp2/Abcc2 on the basal and apical membranes, respectively, for the purpose of quantitatively comparing the clearance for transcellular transport with that for in vivo biliary excretion. The basal-to-apical transport of 17beta-estradiol-17beta-d-glucuronide, pravastatin, leukotriene C(4), cyclo-[D-Asp-Pro-d-Val-Leu-d-Trp] (BQ123), temocaprilat, and taurolithocholate 3-sulfate was significantly higher than that in the opposite direction in the double transfectant. Kinetic analysis suggested that that the rate-determining step of these compounds is the uptake process. The extent of the transcellular transport across the rat double-transfectant correlated well with that across the double-transfectant for human OATP2/SLC21A6 and MRP2/ABCC2. Moreover, considering the scaling factor, the clearance values for in vitro transcellular transport correlated well with those for in vivo biliary clearance. The double-transfected MDCK II monolayer may be useful in analyzing the hepatic vectorial transport of organic anions and in predicting in vivo biliary clearance.
我们之前曾提出，对在基底膜和顶膜分别表达人有机阴离子转运多肽4（OATP2/SLC21A6）和多药耐药相关蛋白2（MRP2/ABCC2）的双转染马-达二氏犬肾II（MDCK II）单层细胞进行跨细胞转运评估，可能有助于表征人胆汁排泄（《生物化学杂志》277: 6497 - 6503, 2002）。然而，为了证明这个体外系统代表体内胆汁排泄，将体外数据与体内胆汁排泄进行比较至关重要。问题在于，对于许多配体，我们无法确定其人体胆汁排泄情况。在本研究中，我们建立了一种双转染MDCK II单层细胞，其在基底膜和顶膜分别表达大鼠Oatp4/Slc21a10和Mrp2/Abcc2，目的是定量比较跨细胞转运清除率与体内胆汁排泄清除率。在双转染细胞中，17β-雌二醇-17β-D-葡萄糖醛酸、普伐他汀、白三烯C4、环-[D-天冬氨酸-脯氨酸-D-缬氨酸-亮氨酸-D-色氨酸]（BQ123）、替莫卡普利拉和牛磺石胆酸3-硫酸盐的基底到顶膜转运显著高于相反方向。动力学分析表明，这些化合物的限速步骤是摄取过程。大鼠双转染细胞的跨细胞转运程度与表达人OATP2/SLC21A6和MRP2/ABCC2的双转染细胞的跨细胞转运程度密切相关。此外，并考虑到比例因子，体外跨细胞转运的清除率值与体内胆汁清除率值密切相关。双转染MDCK II单层细胞可能有助于分析有机阴离子的肝脏向性转运，并预测体内胆汁清除率。
