MDCK Permeability Assay

Domainex’s MDCK assay provides a robust in vitro model for evaluating passive permeability and transporter-mediated efflux, critical parameters in predicting oral absorption, brain penetration and pharmacokinetic behaviour of test compounds. 

Utilising differentiated Madin-Darby Canine Kidney (MDCK) cells engineered to express clinically relevant transporters such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), this assay enables precise characterisation of compound permeability and identification of efflux liabilities before they become costly setbacks. 

Similar to the Caco-2 permeability assay, the MDCK cell layer separates apical and basal compartments, allowing the assessment of both apical-to-basal (A-B) and basal-to-apical (B-A) transport (Table 1). This bidirectional setup enables both evaluation of passive diffusion and identification which compounds are substrates for efflux transporters, providing critical insights into potential bioavailability issues and drug-drug interaction risks. The MDCK assay offers additional advantages, including lower costs and faster turnaround times compared to Caco-2 assessment, making it an especially valuable tool for early ADME profiling and lead optimisation. 

Domainex’s Standard Experimental Procedure: 

Compound is dosed on one side of the MDCK cell barrier, either apical or basal. The plate is then incubated for 120 minutes at 37 °C. A sample is taken from both sides of the MDCK cell monolayer, and levels of compound are quantified via Ultra-High Performance Liquid Chromatography (UHPLC)-Mass Spectrometry (MS) analysis. 

Prior to incubation, the integrity of the MDCK cell monolayer is determined via Trans-Epithelial Electrical Resistance (TEER) measurement. Following incubation, the integrity of the MDCK cell monolayer is confirmed via a Lucifer Yellow Paracellular Permeability assay. Example data is shown in Figure 1. 

Data Analysis: 

Supernatants from both apical and basal compartments are analysed using Waters Acquity UPLC TQ-S, TQ-S-micro or TQ-XS instruments. Triple quadrupole mass spectrometers operated in multiple reaction monitoring (MRM) mode, provide accurate quantification with excellent sensitivity, selectivity and reproducibility.

  

Figure 1: Typical result for A-B permeability of compounds through an MDCK-MDR1 cell barrier (A) and efflux ratios (ER) of the same compounds (B).

Figure 1: Typical result for A-B permeability of compounds through an MDCK-MDR1 cell barrier (A) and efflux ratios (ER) of the same compounds (B). 

Deliverables: 

The results are reported in Excel file format as Papp(A-B, 10-6 cm *s-1), Papp(B-A, 10-6 cm * s-1), efflux ratio (ER) and compound mass balance (%). Any relevant comments about compound stability/solubility/binding are also included in the report.  

Turnaround time for MDCK Permeability Assay is available on request. 

FeatureMDCKCaco-2PAMPA
Application
  • Predicts passive permeability
  • Assesses efflux 
    (P-gp, BCRP)
  • Early ADME screening
  • Predict intestinal absorption
  • Transporter studies (P-gp, BCRP)
  • Regulatory support
  • Predict passive diffusion only
  • High-throughput screening
Advantages
  • Fast and cost-effective
  • Can be engineered for efflux transporters
  • Physiologically relevant human intestinal model
  • Regulatory acceptance
  • Comprehensive permeability data
  • High throughput
  • Low cost
  • No cell culture required
Limitations
  • Non-human origin (canine)
  • Less physiologically relevant than Caco-2
  • Limited transporter diversity
  • Time-consuming (21-day differentiation)
  • Higher cost
  • Limited throughput
  • No active transport or efflux
  • Cannot predict transporter interactions
  • Less predictive for complex molecules

Table 1: Comparison of MDCK, Caco-2, and PAMPA assays for permeability and efflux assessment 

MDCK services table
*Other options available on request