The parallel artificial membrane permeability assay (PAMPA) has been used as the non-cell-based in vitro assay to predict BB penetration in a coated 96-well membrane filter . The filter membrane of the donor plate was coated with PBL (Polar Brain Lipid, Avanti, USA) in dodecane (4 µL of 20 mg/mL PBL in dodecane), and the acceptor well was filled with 300 µL of PBS buffer (pH 7.4; VA). Tested compounds were dissolved first in DMSO and then diluted with PBS (pH 7.4) to achieve the final concentration of 25-100 µM in the donor well. The concentration of DMSO did not exceed 0.5% (v/v) in the donor solution. 300 µL of the donor solution (VD) was added to the donor wells, and the donor filter plate was carefully put on the acceptor plate so that coated membrane was "in touch" with both the donor solution and acceptor buffer. The test compound diffused from the donor well through the polar brain lipid membrane (Area = 0.28cm 2 ) to the acceptor well. The concentration of the tested compound in both the donor and acceptor wells was assessed after 3, 4, 5, and 6 h of incubation, respectively, in quadruplicate using a UV plate reader Spark (Tecan Group Ltd, Männedorf, Switzerland) at the maximum absorption wavelength of each compound. Also prepared were solutions at the theoretical equilibrium of the given compound (i.e., the theoretical concentration if the donor and acceptor compartment were combined). The concentration of the compounds in the donor and acceptor well and the equilibrium concentration were calculated from the standard curve and expressed as the permeability (Pe) according to equation ( ):