PAMPA Assay for Permeability Profiling

The reference compounds, well-permeable caffeine and low-permeable norfloxacin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Used for this study, pre-coated PAMPA Plate System Gentest™ was provided by Corning (Tewksbury, MA, USA). The system consisted of 96-well receiver filter plate pre-coated with structured layers of phospholipids and a donor microplate. The assay was performed as described previously [17 (link),45 (link)]. In brief, the tested compound KSK-59 and the reference compounds were diluted in the PBS buffer (pH 7.4) and applied into the plate at the final concentration of 200 μM. After 5 h of incubation at RT° the UPLC-MS spectrometry (Waters ACQUITY™ TQD system with the TQ Detector, Waters, Milford, MA, USA) method was used to estimate the quantity of compounds that penetrated from donor to acceptor wells. The permeability coefficients (Pe, cm/s) were calculated using the formulas provided by Corning.