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MDCK-WT/MDR1 Permeability

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

 

Protocol

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
Replicates Triplicate
Incubation Time 2 hours at 37°C in a CO2 incubator
Analysis Method LC-MS/MS
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)

Efflux ratio

Mass Balance

 

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:

 

 

Results

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).

 

About Us

The DMPK & Physical Sciences department at Sygnature Discovery is dedicated to understanding and optimising the absorption, distribution, metabolism and excretion of drug candidates by working in close partnership with clients and other departments within Sygnature to provide successful optimisation strategies.

We have extensive know-how and expertise to provide well validated, state-of-the-art assays and a comprehensive applied consultancy service for interpretation of the in vitro ADME and in vivo PK data.

Our corporate vision is to accelerate the discovery of new medicines, from the laboratory into development to treat patients.

Our DMPK mission is to deliver tailored DMPK expertise through innovation, quality and commitment.

 

References

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.

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