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Permeability - MDCKII-MDR1

Background:
The permeability of a compound will influence its ability to cross the intestinal membrane and its oral bioavailability.  MDCKII-MDR1 cells are Madin-Darby canine kidney epithelial cells, over-expressing  the human multi-drug resistance (MDR1) gene, coding for P-glycoprotein (P-gp). They represent a good model for fast in vitro screening of passive permeability and for estimating the potential for human  P-gp to transport the compounds of interest. 

Transport of a compound through the MDCKII-MDR1 cell monolayer is used for measuring the rate of membrane transport, expressed as apparent (Papp) permeability. Resulting Papp values from both transport directions (apical to basolateral side, and basolateral to apical side) are used to calculate an efflux ratio. The inclusion of a P-gp inhibitor confirms whether membrane transport is mediated by P-gp.
   Assay description

   Cells
      MDCKII-MDR1

   Direction
      apical to basolateral  (A2B)
      basolateral to apical (B2A)
      with or without P-gp inhibitor (elacridar)

   Compound concentration
      10µM (1% DMSO)

   Compound requirements
      50µl of 10mM stock solution or
      1-2 mg of dry matter

   Incubation details
      medium: Dulbecco’s PBS (pH 7.4)
      calibration curve: optional

   Detection method
      LC-MS/MS with internal standard

   Results
      Papp(A2B),  Papp(B2A)
      efflux ratio, recovery

Assay controls
   reference compounds:  amprenavir and propranolol (Figure 1)
   cell monolayer integrity control: lucifer yellow
   data obtained for 10 commercial compounds (Figure 2)
Figure 1.
Data  for reference compounds obtained on MDCKII-MDR1 cells:
(a) Permeability values, Papp(A2B), (b) Efflux ratio
                   

Figure 2.
In-house data obtained for 10 commercial compounds in three separate experiments on MDCKII-MDR1 cell monolayer:
(a) Permeability values, Papp(A2B), (b) Efflux ratio

Assay details adjustable to client’s and/or project specific requests

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