Immobilon p membrane supports

PAMPA was performed using a 96-well filter donor plate with 0.45 μm hydrophobic Immobilon-P membrane supports (Millipore MAIPNTR10) and a 96-well Teflon acceptor plate (Millipore MSSACCEPTOR). The donor plate was prepared by applying 5 μL of 1% (w/v) soybean lecithin (90%, Alfa Aesar) in n-dodecane to each filter. The acceptor plate was prepared by addition of 300 μl of pH = 7.4 phosphate-buffered saline (PBS) with 5% DMSO to each well. Donor solutions composed of PBS with 5% DMSO, 10 μM carbamazepine standard, and 10 μM compound 1, 2 or 8 were prepared with and without additional KCl (0.27 mM or 100.27 mM K⁺ concentration) before addition of 150 μl to each donor well, with all conditions run in quadruplicate. The donor plate was then loaded into the acceptor plate and was left in a sealed, damp chamber at 25 °C for approximately 17 hours (exact times recorded and used in calculation of P_e). The plates were then separated, and 100 μl of solution from each well was transferred to another plate for LC-MS analysis. Permeabilities were calculated identically to a previous study.^{80 (link)}