| Assay Detail |
Pion Stirwell PAMPA sandwich plates were prepared with an artificial membrane solution of 2.5% lecithin in dodecane (5 µL per well) and an acceptor well solution of PBS buffer (200 µL per well, pH 7.4). Donor solutions of macrocycle in PBS (500 µM, 5% DMSO) were prepared and added to donor wells (200 µL per well). Acceptor wells were then placed on top of the donor wells and incubated for 4 h. Concentrations of macrocycle in each acceptor well were then measured by UV integration on an LCMS and expressed apparent permeability using equation 1. Papp = CA/CD * V/At [1]. Here CA is the final concentration in the acceptor well measured by UV integration, CD is the initial concentration in the donor well measured by UV integration, V is the volume of the donor well (0.2 cm^3), A is the area of the lipid membrane (0.3 cm^2) and t is the incubation time (14400 s).
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