4.2.9. In Vitro Blood–Brain Barrier Permeation Assay (PAMPA-BBB)

Prediction of the brain permeation of compounds 2, 5, 8, 11, and 12 was evaluated using a parallel artificial membrane permeation assay (PAMPA-BBB), following a well-established procedure.^40,50 Briefly, a semiautomated pipetting system (BenchSmart 96, Mettler Toledo) and a microplate spectrophotometer (SpectraMax Plus 384 microplate reader, Molecular Devices) were employed for pipetting and UV reading, respectively. All commercial drugs and reagents were purchased from Sigma-Aldrich. The porcine brain lipid (PBL) was acquired from Avanti Polar Lipids, while Millex filter units (PVDF membrane, pore size 0.45 μm) were obtained from Millipore. For the assay, the 96-well acceptor microplate (PTFE, Millipore) was filled with 300 μL of PBS:ethanol (70:30), whereas the artificial membrane of the donor microplate (PVDF membrane, pore size 0.45 μm, Millipore) was coated with 4 or 5 μL of PBL dissolved in dodecane (20 mg/L). All compounds were first dissolved in DMSO and then diluted with PBS/EtOH (70:30, pH 7.4) to reach the final concentration in the range 40–100 μM in the donor well, filtered through a Millex filter, and then added to the donor microplate wells (200 μL). The donor filter microplate was carefully placed onto the acceptor microplate so as to form a sandwich, which was left undisturbed for 16 h at 25 °C into a sealed container with wet paper towels to avoid evaporation. After incubation, the donor microplate was cautiously removed and the concentrations of the tested compounds in the acceptor and donor microplate wells were determined via UV–vis spectroscopy. Every sample was analyzed at five wavelengths in four wells and in three independent runs. Accordingly, the results given in Table 1 are reported as average values ± standard deviation. P_e was calculated by the following formula: P_e = {−V_dV_a/[(V_d + V_a)At]} × ln(1 – drug_acceptor/drug_equilibrium), where V_d and V_a are the volumes of the donor and acceptor wells, respectively, A is artificial membrane area, t is the permeation time, drug_acceptor is the absorbance obtained in the acceptor well, and drug_equilibrium is the theoretical equilibrium absorbance. Twenty known commercial drugs of known BBB permeability (Table S1, Figure S1) were used as quality control standards to validate and normalize the analysis set. Donepezil and quercetin were further tested as CNS+ and CNS– positive and negative controls, respectively (Table 1). Upon completion of the PAMPA-BBB assay for each present and standard compound, lipid membrane integrity was verified based on the transport of Lucifer Yellow (Sigma-Aldrich), a fluorescent molecule with very poor membrane permeability which, in the presence of a uniform and integral lipid membrane, should effectively be completely rejected.⁶⁷ The Lucifer Yellow test was performed following Millipore protocol lit. no. PC1545EN00 (https://www.sigmaaldrich.com/technical-documents/protocols/biology/membrane-integrity-test-for-lipid-pampa-artificial-membranes.html). As the relevant fluorescence readings (SpectraMax Gemini XPS microplate reader, Molecular Devices) were comparable to background readings of buffer only (5% DMSO in PBS), the membrane integrity after all PAMPA-BBB assays was confirmed.