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.
Sygnature’s MDCK wildtype/MDR1 assay uses the PrediPort™-WT/MDR1 Kit from ReadyCell S.L. (Barcelona, Spain), a ready-to-use cell-based assay for rapid in-vitro assessment of drug’s permeability and MDR1 substrate assessment. 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, so as 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
|Compound requirements||10 mM DMSO, 100µL|
|Test Article Concentrations||10 µM|
|Buffer||HBSS buffer at pH7.4 or 6.5-7.4 (apical-basolateral – recommended for acidic compounds); alternate pH’s on request|
|Incubation Time||2 hours at 37°C in a CO2 incubator|
|Controls||Digoxin, Quinidine or other MDR1 efflux markers (please request) and propranolol (high permeability marker)|
|Cell monolayer integrity marker||Lucifer yellow|
|Data Delivery||Papp (AtoB and BtoA)
The permeability coefficient (Papp) is calculated from the following equation:
Where dQ/dt is the amount of compound in basal (A-B) or apical (B-A) compartment as a function of time (nmol/s). C0 is the initial concentration in the donor (apical or basal) compartment (Mean of T=0) (nmol/mL) and A is the area of the transwell (cm2).
The efflux ratio is calculated in wild type and MDR1-MDCK as:
A net efflux ratio is also calculated:
Figure 2 Comparison of efflux ratio in wild type MDCKII and human MDR1 transfected MDCKII cells for propranolol (negative MDR1 efflux substrate) and quinidine (positive MDR1 efflux substrate).
Figure 3 Net efflux ratio (MDCKWT/MDCKMDR1) for propranolol (negative MDR1 efflux substrate) and quinidine (positive MDR1 efflux substrate).
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Luo et al. (2010) Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids. Mol Pharm; 7(2); 412-420.