A Caco-2 cell based screening method for compounds interacting with MRP2 efflux protein.
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.
Siissalo
Sanna
S
Division of Pharmaceutical Technology, University of Helsinki, Helsinki, Finland. sanna.siissalo@helsinki.fi
Hannukainen
Jenni
J
Kolehmainen
Johanna
J
Hirvonen
Jouni
J
Kaukonen
Ann Marie
AM
D001692:Biological Transport; D018938:Caco-2 Cells; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000187:drug effects / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism
D005452:Fluoresceins; D005456:Fluorescent Dyes; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D004364:Pharmaceutical Preparations; C000715087:ABCC2 protein, human; C053897:5(6)-carboxy-2',7'-dichlorofluorescein