2.6.3. In Vitro Parallel Artificial Membrane Permeability Assay (PAMPA)

Gastrointestinal permeability through passive diffusion was evaluated in a cell-free permeation model. Samples of the GEN-AAs co-amorphous systems were prepared in the same way as for the solubility test, and GEN was dissolved in dimethyl sulfoxide. The experiment was performed in two 96-well plates. Each well of the donor plate was filled with a sample mixed with donor solution with pH adjusted to 6.8, and an Acceptor Sink Buffer was added to each well of the acceptor plate. The microfilter discs that separate the wells were impregnated with GIT-0 lipid. The acceptor plate was placed into the donor plate, creating a PAMPA sandwich, which was incubated for 3 h at 37 °C. The developed and validated HPLC method was used to analyze GEN concentrations, and the following equations were used to calculate the apparent permeability coefficient (P_app):

where C_A is the concentration in the acceptor well, C_D is the concentration in the donor well, V_D is the donor volume, V_A is the acceptor volume, S is the membrane area, and t is the time (in seconds).

Substances found as low permeable have a P_app below 0.1 × 10⁻⁶ cm·s⁻¹, compounds considered to be medium permeable have 0.1 × 10⁻⁶ cm·s⁻¹ ≤ P_app < 1 × 10⁻⁶ cm·s⁻¹, and those whose P_app is ≥ 1 × 10⁻⁶ cm·s⁻¹ are classified as highly permeable [41].