Siissalo Sanna, Hannukainen Jenni, Kolehmainen Johanna, Hirvonen Jouni, Kaukonen Ann Marie
Division of Pharmaceutical Technology, University of Helsinki, Helsinki, Finland.
Eur J Pharm Biopharm. 2009 Feb;71(2):332-8. doi: 10.1016/j.ejpb.2008.08.010. Epub 2008 Aug 19.
The aim of this work was to develop a screening method for MRP2 efflux substrates using the well-characterized, human-based intestinal Caco-2 cell model as a platform. MRP2 has a significant role in drug absorption and disposition and is known to co-operate with phase II metabolic enzymes. Caco-2 cells grown in a 96-well plate were loaded with non-fluorescent CDCFDA (diacetate ester of 5(6)-carboxy-2',7'-dichlorofluorescein), which is hydrolyzed to fluorescent CDCF by intracellular esterases. De-esterification in Caco-2 was comparable to that in porcine liver esterases. CDCFDA enters the cells passively, while CDCF is effluxed out of the cells by the apically localized MRP2 and/or basolateral MRPs. The method was optimized with regard to several factors. In the concluding protocol, Caco-2 cells are grown on clear 96-well plates for 8 days. The loading conditions were optimized to 10 min incubation with 5 microM CDCFDA. The highest responses were obtained for samples taken at t=30 min. The samples were analyzed in black 96-well plates with a fluorescence plate reader. The Caco-2 based method utilizing the probe pair CDCFDA/CDCF provides a fast screening tool for MRP2 substrates and/or inhibitors, along with compounds having metabolites formed in Caco-2 that interact with MRP2.
本研究的目的是开发一种筛选方法，以特征明确的人源肠道Caco-2细胞模型为平台，筛选多药耐药相关蛋白2（MRP2）外排底物。MRP2在药物吸收和处置中发挥重要作用，并且已知其与II相代谢酶协同作用。将生长在96孔板中的Caco-2细胞用非荧光的5(6)-羧基-2',7'-二氯荧光素二乙酸酯（CDCFDA）加载，其被细胞内酯酶水解为荧光性的CDCF。Caco-2细胞中的去酯化作用与猪肝酯酶中的相当。CDCFDA被动进入细胞，而CDCF则通过顶端定位的MRP2和/或基底外侧的多药耐药相关蛋白（MRP）排出细胞。该方法针对几个因素进行了优化。在最终方案中，将Caco-2细胞接种在透明的96孔板中培养8天。加载条件优化为用5 microM CDCFDA孵育10分钟。在t = 30分钟时采集的样品获得了最高响应。样品在黑色96孔板中用荧光酶标仪进行分析。基于Caco-2细胞、利用CDCFDA/CDCF探针组的方法为MRP2底物和/或抑制剂以及在Caco-2细胞中形成的与MRP2相互作用的代谢物的化合物提供了一种快速筛选工具。
