| Assay Detail |
Cell permeability was determined by using native wild-type MDCK cells. Madin-Darby canine kidney (MDCK) cells are an epithelial cell line of canine kidney origin. MDCK cells have low expression of transporter proteins and low metabolic activity. MDCK cells were used as acellular barrier model to assay the polarized transport of the compounds of interest through the epi- thelia. Under standard cell-culture conditions, MDCK cells develop tight junctions and form monolayers of polarized cells. The cells were seeded on a24-well plate (BD Falcon) and formed aconfluent monolayer within 4days prior to the experiment. On day 4, the test compound was added to the apical side of the membrane, and transport of the compound across the monolayer was monitored after a 2 h incubation period. For studies related to drug efflux, it was also necessary to examine the transport of the compound from the basolateral compartment to the apical compartment declared as the efflux ratio. The permeability in both direc- tions, A to Band B to A, was derived from the compound concentration (measured by LC–MS/MS) in apical and basal compartments after a 2 h incubation time.
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