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

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. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4974-4985, 2011