| Assay Detail |
Caco-2 cells were cultured on a 96-well Transwell (Corning Inc., New York) for 3 weeks, fasted state simulated intestinal fluids (FaSSIF)/Dulbecco’s Modified Eagle Medium (DMEM) buffer (1% DMSO) containing the compound was added to the apical side and DMEM was added to the basal side, and this was preincubated at 5% CO2, 37 °C, and 80 rpm for 20–24 h. After preincubation, the preincubation solutions on the apical side and on the basal side were removed by aspiration and washed with Hanks’ balanced salt solution (HBSS) buffer. Then, FaSSIF/HBSS buffer (pH 6.0) containing the compound and 1% DMSO was added to the apical side, and HBSS buffer containing 4% BSA (pH 7.4) was added to the basal side, and membrane permeability assays were initiated. Each well was shaken under conditions of 5% CO2, 37 °C, and 80 rpm, and 180 min after initiation, samples on the basal side were collected, and the amount of compound was determined by LC-MS. From the amount of compounds, the apparent permeability coefficient (Papp) was calculated. Papp = (dQ/dt) x [1/(AxC0)] where (dQ/dt) is the amount of compound transported to the acceptor side per time (nmol/s), A is the surface area of the membrane (cm^2), and C0 is the initial compound concentration in the donor side (μM).
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