| Assay Detail |
In order to determine the passive membrane diffusion rates, PAMPA assay was performed with a Corning Gentest Pre-coated PAMPA Plate System. The acceptor plate was prepared by adding 200 μL of 0.1 M phosphate buffer (pH 7.4) supplemented with 5% DMSO to each well, and then 300 μL of a solution of 1 μM test compounds in 0.1 M phosphate buffer (pH 6.4) with 5% DMSO was added to the donor wells. The acceptor plate was then placed on top of the donor plate and incubated at 37 °C for 4 h without agitation. After the incubation, the plates were separated and the solutions from each well of both the acceptor plate and the donor plate were transferred to 96-well plates and mixed with acetonitrile. The final concentrations of compounds in the donor and acceptor wells, as well as the concentrations of the initial donor solutions, were analyzed using LC-MS/MS. The permeability of the compounds was calculated as described previously.5,6 Antipyrine (100 or 1 μM), metoprolol (500 or 1 μM), and sulfasalazine (500 or 1 μM) were used as reference compounds. The permeabilities of antipyrine, metoprolol, and sulfasalazine were 15.4 ± 2.7, 1.9 ± 0.5, and 0.055 ± 0.0058 x 10^-6 cm/s, respectively. Two technical replicates were performed.
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