| Assay Detail |
MDCK-LE (low efflux) cells were cultured at 37 °C under a 5% CO2 atmosphere, at 95% relative humidity in DMEM containing 10% FBS, penicillin-streptomycin (100 μg/mL), and 2 mM Ala-Gln. Cells were passaged every 3-4 days. For assay purposes, cells were seeded at a density of approximately 265,000 cells/cm^2 of a 96-well Transwell plate (Corning Life Sciences, Acton, MA) and cultured in the same media noted above for a period of 4 days. Papp values were determined as Papp= VA/(S[D0]) * A120 / t, Percent recovery values were determined as %recovery = 100 * ((A120 + D120)/D0) where VA is the volume of the acceptor (mL), S is the surface area of the membrane, D0 is the donor solution concentration at t=0, D120 is the donor solution concentration at t=120, A120 is the acceptor solution concentration at t=120, and t = time (seconds).
The determination of the apparent permeability (Papp) was performed in the A → B (apical to basal) direction where each compound was assayed in triplicate. The zwitterion bestatin, a poorly permeably compound, was used as a marker of monolayer integrity. To initiate the assay, media was aspirated, and the cells and basal chambers were washed three times with Hank's Balanced Salt Solution (HBSS) containing 10 mM HEPES (pH 7.4). Compound test solutions were prepared in triplicate in HBSS containing 10 mM HEPES (pH 7.4) and 0.02% bovine serum albumin (BSA) to a final concentration of 10 μM and centrifuged for 2 min at 4000g and then applied to the donor compartment at time zero. Additionally, at time zero, a 37 °C solution without test articles [HBSS + 10 mM HEPES (pH 7.4) plus 0.02% BSA] was added to the receiver chamber of the Transwell plate. A time zero sample of the donor solution was also sampled for further analysis. The assay was conducted for a period of 120 min at 37 °C without shaking. At the time of assay termination, samples were taken from each donor compartment and each acceptor compartment of the Transwell plate. To each of the 0 and 120 min samples was added an internal standard solution containing glyburide in water: acetonitrile, 50:50 (v/v). Concentration curves were prepared using a Labcyte Echo in the same matrix noted above. Samples and concentration curve samples were centrifuged for 10 min at 4000g and subsequently analyzed by mass spectroscopy.
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