3.3.3. Parallel Artificial Membrane Permeability Assay (PAMPA)

The PAMPA was performed using a 96-well precoated Corning Gentest PAMPA plate at a pH of 7.4. The PAMPA plate system consists of a donor plate and an acceptor plate. When both plates are coupled, each well is divided into two chambers that are separated by a lipid–oil–lipid trilayer constructed in a porous filter. Stock solutions (10 mM) of each compound were prepared in DMSO and then further diluted to a concentration of 200 µM in phosphate-buffered saline (PBS) at pH 7.4. The compound dissolved in PBS was then added to the donor plate and pure PBS was added to the acceptor plate. Four replicates of each compound and negative control (PBS) were transferred into different wells of the donor plate. Both plates were coupled and left at room temperature for 5 h. Then, the plates were separated, and the solutions of each donor well and acceptor well were transferred to 96-well UV-Star Microplates (Greiner Bio-One). The UV absorbance of compounds in donor wells and acceptor wells were analyzed by a SpectraMax M3 UV plate reader (Molecular Devices). The concentrations were received from a calibration curve for each substance. The plates were analyzed at a wavelength where the R² value of the calibration curve was higher than 0.99 [34]. The effective permeability (P_e) was calculated as shown in the following Equations (1)–(3):

where:

P_e—effective permeability;

S—filter area (0.3 cm²);

V_D—donor well volume;

V_A—acceptor well volume;

t—incubation time (14,400 s);

c_A(t)—concentration of compound in acceptor well at time t;

c_equ—equilibrium concentration.

where:

c_D(t) —concentration of compound in donor well at time t.

Recovery of compounds from donor and acceptor wells (mass retention) was calculated as shown in the equation below. Data were only accepted when recovery exceeded 70%.

where:

R—mass retention (%);

c_D(t)—concentration of compound in donor well at time t;

c_A(t)—concentration of compound in acceptor well at time t;

c₀—initial concentration of compound in donor well;

V_D—donor well volume;

V_A—acceptor well volume.