| Assay Detail |
Caco-2 cells were cultured on a 96-well Transwell® (Corning Inc., NY, US) for 3 weeks. Fasted State Simulated Intestinal Fluids (FaSSIF)/Dulbecco’s Modified Eagle Medium (DMEM) buffer (1% DMSO) containing the compound were added to the Apical side and DMEM was added to the Basal side, and this was pre-incubated at 5% CO2, 37 °C, and 80 rpm for 20-24 h. After pre-incubation, the pre-incubation solutions on the Apical side and Basal side were removed by aspiration and washed by 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 compounds was determined by LC-MS. From the amount of compounds, the apparent permeability coefficient (Papp) was calculated. Papp = (dQ / dt) * [1 / (A * C0)]. where (dQ / dt) is the amount of compound transported to the acceptor side per time (nmol / sec), A is the surface area of the membrane (cm2), and C0 is the initial compound concentration in the donor side (μM).
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