3.2. Parallel Artificial Membrane Permeability ASSAY (PAMPA BBB)

BBB permeability of the compounds was investigated by using a PAMPA method (parallel artificial membrane permeability assay). The PAMPA system consisted of a 96-well microfilter plate and a 96-well filter plate and was divided into two chambers: a donor at the bottom and an acceptor at the top, separated by a 120-μm-thick microfilter disc coated with BBB lipid solution (Pion, Inc.). The solutions of each compound were prepared in dimethyl sulfoxide (DMSO) at 4 mg/mL concentration and then diluted with Prisma buffer (pH = 7.4) to obtain the donor drug solution with the final nominal concentration of 20 µg/mL. The donor solutions were placed on the donor plate. Acceptor plate contained Brain Sink Buffer (BSB). The plates were put together and incubated at 37 °C for 180 min in a humidity-saturated atmosphere. The concentrations of the compounds were determined with a UV-reader (Multiskan GO, Thermo Scientific) at 254 nm in the donor and acceptor compartments.

The permeability values (Pe) were calculated by using the following equation:

where V_D: donor volume, V_A: acceptor volume, C_equilibrium: equilibrium concentration Cequilibrium=CD×VD+ CA×VAVD+VA,CD: donor concentration, C_A: acceptor concentration, S: membrane area, and t: incubation time (in seconds). High BBB permeation (CNS+) was expected for compounds with Pe > 5.19, whereas low BBB permeation (CNS-) was expected for compounds with Pe < 2.07 [21].