| Assay Detail |
Cell permeability was determined using RRCK (MDCK-LE) cells (Pfizer, Inc. Groton, CT). RRCK cells were generated in house as a subclone of Madin-Darby CanineKidney wild-type (MDCK-WT) cells that displayed low expression of endogenous pglycoprotein (~ 1-2% of MDCK-WT cells, based on mRNA level). Cells were cultured in minimal essential medium α with supplements and passaged when 70-80% confluent. Cell monolayer flux studies were conducted five days after seeding in 24-well transwell inserts (RRCK in 1.0 μm pore size (Becton Dickinson, Cowley, UK) at 4.2 x 10^4 cells/cm^2. Donor and acceptor solutions were prepared from HBSS containing HEPES at 20 mM, pH 7.4. Stock solutions of test compounds, prepared at 10 mM in DMSO, were used to prepare donor solutions of 2 μM compound in 0.05% (v/v) DMSO. Apparent permeability (Papp) was determined in apical to basolateral (AB) direction in triplicate by incubating with compound for 2 h at 37 °C. Samples of the medium were analyzed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). Papp values were calculated according to the equation Papp = (Q/t) x 1/C0 x 1/A, where Q is the sampled concentration in the acceptor compartment, t is the incubation time, C0 isthe initial concentration in the donor compartment and A is the area of the filter of the transwell plate.
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