Development of a new permeability assay using low-efflux MDCKII cells

J Pharm Sci. 2011 Nov;100(11):4974-85. doi: 10.1002/jps.22674. Epub 2011 Jul 15.


Permeability is an important property of drug candidates. The Madin-Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P-glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII-LE (low efflux), has been developed by selecting a subpopulation of low-efflux cells from MDCKII-WT using an iterative fluorescence-activated cell sorting technique with calcein-AM as a Pgp and efflux substrate. MDCKII-LE cells are a subpopulation of MDCKII cells with over 200-fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII-WT. MDCKII-LE cells showed less functional efflux activity than MDCKII-WT based on efflux ratios. Notably, MDCKII-MDR1 showed about 1.5-fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII-LE cells offer clear advantages over the MDCKII-WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII-LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII-LE permeability assay (4-in-1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost-effective.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / physiology
  • Animals
  • Cell Line
  • Cell Separation
  • Chromatography, Liquid
  • Dogs
  • Flow Cytometry
  • Humans
  • Intestinal Absorption
  • Permeability*
  • Real-Time Polymerase Chain Reaction
  • Tandem Mass Spectrometry


  • ATP Binding Cassette Transporter, Subfamily B, Member 1