Parallel Artificial Membrane Permeability Assay (PAMPA)

The PAMPA assay was used to exclude passive diffusion capacity across the BBB. The standard parameter that quantifies transport independently of time and concentrations is effective permeability, Pe=−218.3tLog(1−2CA(t)CD(t0))×10−6cm/s; where t is the running time (4 h), C_A (t) is the concentration of the compound in the acceptor well at time t, and C_D (t₀) is the compound concentration in the donor well before running the PAMPA assay (t₀ = 0 h). Transport (%) values were obtained by dividing the amount of peptide in the acceptor well at time t, C_A (t), by the amount in the donor well at time zero, C_D (t₀), multiplied by 100. The protocol followed was the same as that previously used by Arranz-Gibert et al.¹. Permeability is considered excellent with values >4.0 × 10⁻⁶) cm/s, uncertain between 2.0 × 10⁻⁶ and 4.0 × 10⁻⁶ cm/s, and poor with values below 2.0 × 10⁻⁶) cm/s⁴¹.