Wild-type MDCK (Madin-Darby canine kidney) cells, when transfected with either the Multidrug Resistance gene-1 (MDR1; P-gp) or Breast Cancer Resistance gene (BCRP), are used as a model of brain penetration and to understand the impact of drug transporters on drug uptake into the brain. When cultured as a monolayer, MDCK cells differentiate to form tight junctions between cells that mimic the blood-brain barrier.
Differentiated and polarized MDCK cells, wild-type and MDR1 (BCRP transfected cells are also available), are plated on a 96-transwell permeable system as a single monolayer to allow for automated high throughput screening of compounds. Drug transport is assessed in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)) across the cell monolayer (Figure 1). The buffer used for the assay does not include HEPES, to minimise the inhibitory effect on uptake transporters (Luo et al., 2010).
Test compound concentrations are quantified using a calibration curve following analysis by LC-MS/MS, and the apparent permeability coefficient (Papp) and efflux ratio of the compound across the monolayer are calculated. The efflux ratio is used as an indicator of active efflux.
Figure 1 The schematic illustrates the Transwell of PrediPort™-WT/MDR1/BCRP Multiwell Insert