Parallel Artificial Membrane Permeability Assay

A parallel artificial membrane permeability assay (PAMPA) [28 (link),29 (link)] was conducted on a Biomek FX lab automation workstation (Beckman Coulter, Inc.) with PAMPA evolution 96 command software (pION Inc.) as follows: 3 μL of 10 mM test compound stock was mixed with 600 μL of PBS (pH 7.4) to make diluted test compound. Then 150 μL of diluted test compound was transferred to a UV plate (pION Inc.), and the UV spectrum was read as the reference plate. The membrane on a preloaded PAMPA sandwich (pION Inc.) was painted with 4 μL of GIT lipid (pION Inc.). The acceptor chamber was then filled with 200 μL of acceptor solution buffer (pION Inc.), and the donor chamber was filled with 180 μL of diluted test compound. The PAMPA sandwich was assembled, placed on the Gut-Box controlled environment chamber and stirred for 30 min. The aqueous boundary layer was set to 40 μm for stirring. The UV spectrum (250–500 nm) of the donor and the acceptor were read. The permeability coefficient was calculated using PAMPA evolution 96 command software (pION Inc.) based on the AUC of the reference plate, the donor plate, and the acceptor plate. All compounds were tested in triplicate.