期刊
EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS
卷 71, 期 2, 页码 332-338出版社
ELSEVIER
DOI: 10.1016/j.ejpb.2008.08.010
关键词
Caco-2 cell line; Screening method; MRP2 efflux protein; Intracellular de-esterification; CDCF; 5(6)-Carboxy-2 ',7 '-dichlorofluorescein
资金
- TEKES (Finnish Funding Agency for Technology)
- Orion Pharma
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)-carboxy2',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 mu M 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. (C) 2008 Elsevier B.V. All rights reserved.
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