| Assay Detail |
Passive permeability was determined using a low-efflux Madin-Darby canine kidney (MDCKII-LE) cell line (Pfizer, Groton, CT). Cells were grown in minimum essential medium alpha-nucleosides (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, 1% minimum nonessential amino acids solution, 1% GlutaMAX, and 1% penicillin-streptomycin prior to seeding into Millipore 96-well cell-culture insert plates (EMD Millipore Corporation). MDCKII-LE cells were cultured on the insets with 100 μL of medium per well on the apical side and 36 mL for all 96 wells on the basolateral side. Donor solutions were prepared from HBSS containing 20 mM HEPES (pH 6.5). Stock solutions of test compounds, prepared at 5 mM in DMSO, were used to prepare donor solutions of 6 μM compound. Receiver solutions were prepared from HBSS containing 20 mM HEPES and 0.4% (w/v) BSA (pH 7.4). Prior to the assay, the cell-culture medium was removed, and the cells were preincubated with HBSS for 10 min. To start the assay, 100 μL of donor solution and 300 μL of receiver solution were added to the apical and basolateral chambers, respectively. After 90 min incubations, aliquots were taken from the receiver chambers to determine the translocated amount of compound. Samples were taken from the donor chambers before and after incubation to determine the initial concentration (C0) and recovery values. An internal standard solution, 0.5 μg/mL CP-628374 (MW = 687) in 100% methanol, was added to the receiver and donor samples. The samples were analyzed by LC-MS/MS to determine the peak area for the test compound and the internal standard. All incubations
Apparent permeability (Papp) values were calculated according to the following equation: Papp = (dx/dt)/C0*A where dx is the amount of compound in the receiver compartment, dt is the incubation time, C0 is the initial concentration in the donor compartment, and A is the area of the filter of the transwell plate.
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