| Assay Detail |
24-well Transwell plates with 0.4 µm polycarbonate membrane supports (Corning cat. # 3413) were used in the PAMPA. A 1.8% (w/v) solution of lecithin in dodecane was prepared fresh. The solution (15 µL) was applied to each membrane and allowed to adsorb for 15 minutes. The acceptor wells were prepared by adding 600 µL of PBS (pH 7.4) to each well. The donor wells were prepared by adding 200 µL of each compound (50 µM in 1% DMSO/PBS (pH 7.4)). The lid was placed on the plate and the system was incubated for 16 hours in a sealed chamber with wet paper towels to prevent evaporation. Once the assay was complete, a sample from each donor well (150 µL) and acceptor well (400 µL) was collected. The samples were then analysed using LC/MS in selected ion monitoring (SIM) mode to detect the parent masses and quantified using a standard curve of known compound concentrations. The permeability values (Papp x 10^-6 cm/s) were calculated using the following equation: Papp = (V_B / (A x C_i)) X C_B / delta_t.
Where V_B = volume of acceptor well (0.6 cm^3), A = membrane area (0.33 cm^2), C_i = concentration added to donor well (50 µM), C_B = concentration determined by LC/MS in the acceptor well and t = time (57,600 seconds). A minimum of 2 data points was collected for each compound.
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