Pharmacokinetics, Pharmacodynamics and Drug Metabolism
Development of a new permeability assay using low‐efflux MDCKII cells

https://doi.org/10.1002/jps.22674Get rights and content

ABSTRACT

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

Section snippets

INTRODUCTION

A successful drug discovery and development program involves selecting the right disease target, identifying the best chemical matter, and executing a flawless development program. High‐throughput absorption, distribution, metabolism, excretion and toxicity (ADME/TOX) screening is an integrated part of the drug discovery process to enhance the success of drug candidates in the clinic.1., 2., 3., 4.

The ADME/TOX assay panels are most typically comprised solubility, permeability, lipophilicity,

Materials

The MDCKII‐WT and MDCKII–MDR1 cells were obtained from Professor Piet Borst, Netherlands Cancer Institute, Amsterdam, the Netherlands. Minimum essential medium (MEM) with l‐glutamine, ribo/deoxyribo nucleosides, heat‐inactivated fetal bovine serum (FBS), non‐essential amino acids (NEAAs), penicillin (10,000 U/mL)–streptomycin 10,000 µg/mL, l‐glutamine 200 mM solution and 0.25% trypsin –EDTA (ethylenediaminetetraacetic acid), Hank's balanced salt solution (HBSS) with CaCl2,d‐glucose, HEPES

Isolation and Characterization of MDCKII‐LE Cells

The MDCKII‐LE cells were isolated from MDCKII‐WT cells by an iterative and functional FACS based on the efflux of calcein‐AM as a Pgp substrate. The Pgp‐related properties of the MDCKII‐LE cells were characterized using a number of techniques, including distribution of fluorescent intensity with flow cytometry; canine Pgp mRNA level by real‐time PCR; canine Pgp quantification with LC–MS/MS and functional activity based on efflux ratios in the bidirectional monolayer transport study.

Functional Characterization by Flow Cytometry

The

CONCLUSIONS

A new cell line, MDCKII‐LE, has been developed from MDCKII‐WT for permeability measurement. The MDCKII‐LE cells are a subpopulation of MDCKII‐WT cells with a distinct experimental advantage: low expression of endogenous canine Pgp, resulting in near‐negligent functional efflux activity for all but the strongest Pgp substrates. MDCKII‐LE cells express over 200‐fold less canine Pgp than MDCKII‐WT based on mRNA level using real‐time PCR and greater than fivefold lower Pgp protein level by LC–MS/MS

ACKNOWLEDGEMENTS

The authors would like to thank the following colleagues for their contributions: Youping Huang, Mathew Pletcher, Kevin Whalen, Veronica Zelesky, Hua Gao, and Dennis Pereira.

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