A permeability assay adapted for the assessment of very poorly permeable compounds in the bRo5 space

Poorly permeable compounds in the beyond rule of 5 (bRo5) space often demonstrate acceptor well concentrations below the lower limit of quantification or limited resolution between apparent permeability (P_app) values. To address the acceptor well concentration limitation and better resolve differences between poorly permeable molecules, a straightforward solution was proposed, extend the incubation time from 2-h to 24-h to enable acceptor well concentrations to achieve quantifiable values. MDCK cells with knock out of the endogenous canine Mdr1 gene (MDCK-KO) were seeded on 96-Tanswell plates and grown to confluence over three days at which point the 24-h assay was initiated. The longer incubation time enabled receiver compartment concentrations to exceed limits of quantification enabling P_app determination in a test set of approximately 30 molecules. The approximately 10-fold increase in incubation time enabled P_app value determination of up to an order of magnitude lower compared to a 2-h incubation. The assay demonstrates good reproducibility and resolution between poorly permeable compounds. In summary, a straightforward modification of a traditional Transwell permeability assay has produced an assay suitable for the measurement and resolution of very poorly permeable compounds.