PAMPA assay

The PAMPA was used to determine compound permeability by passive diffusion. The assay used an artificial membrane consisting of 2% phosphatidyl choline in dodecane (Sigma-Aldrich, Dorset, UK). The donor plate was a MultiScreen-IP Plate with 0.45-μm hydrophobe Immobilon-P membrane (Millipore, UK), and the acceptor plate was a MultiScreen 96-well Transport Receiver Plate (Millipore, UK). The permeability was measured at three different pH levels (pH 5, 6.5, and pH 7.4) in buffer containing 1% bovine serum albumin (Sigma-Aldrich, Dorset, UK). A 10 mM DMSO stock solution of test compound was used to prepare the 10 μM PAMPA donor solutions and calibration curves in each of the three buffers.

Six microliters of the membrane solution was added to each well of the donor plate. Buffer donor solutions (200 μl) were added to the appropriate wells of the PAMPA donor plate. Three hundred microliters per well of blank PBS (pH 7.4) was added to the PAMPA acceptor plate.

The donor and acceptor plates were then sandwiched together, covered with a lid, and incubated at 30°C in a humid environment for 16 hours. After the incubation period, the plates were removed from the incubator and the sandwich was dismantled. Samples were then transferred into a fresh plate and centrifuged. All sample supernatants were diluted and analyzed using a Waters (Milford, MA, US) TQ-S LC-MS/MS system.

Permeability values (cm/s) were calculated using the following equation

where V_D is the volume of donor, V_A is the volume of acceptor, and area is the surface area of the membrane × porosity.