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