Parallel artificial membrane permeability assay adapted for BBB (PAMPA-BBB)

PAMPA-BBB experiments were conducted using the PAMPA Explorer kit (pION Inc., Billerica, MA, USA) using previously reported protocols^24,28,29. Compounds (25 μM, 200 μl) in pH 7.4 Prisma HT buffer (pION) were added to the wells of a donor plate (number of replicates=12). The polyvinylidene fluoride (PVDF, 0.45 μM) filter membrane on the acceptor plate was coated with BBB-1 lipid formulation (5 μl, pION). The acceptor plate was placed on top of the donor plate. Brain sink buffer (BSB, 200 μl, pION) was added to each well of the acceptor plate and was incubated for 4 h at ambient temperature without stirring. UV–vis spectra of the solutions in the reference, acceptor, and donor plates were measured using a microplate reader. The PAMPA Explorer software v. 3.5 (pION) was used to calculate the −logP_e values for compounds. CNS±designations were assigned by comparison with compounds that were identified in previous reports^50,51,52.