title: Development of a new permeability assay using low-efflux MDCKII cells.
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.
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, Connecticut, USA. li.di@pfizer.com
authors.forename: Li
authors.initials: L
authors.lastname: Di
authors.forename: Carrie
authors.initials: C
authors.lastname: Whitney-Pickett
authors.forename: John P
authors.initials: JP
authors.lastname: Umland
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Xun
authors.initials: X
authors.lastname: Zhang
authors.forename: David F
authors.initials: DF
authors.lastname: Gebhard
authors.forename: Yurong
authors.initials: Y
authors.lastname: Lai
authors.forename: James J
authors.initials: JJ
authors.lastname: Federico
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Davidson
authors.forename: Russ
authors.initials: R
authors.lastname: Smith
authors.forename: Eric L
authors.initials: EL
authors.lastname: Reyner
authors.forename: Caroline
authors.initials: C
authors.lastname: Lee
authors.forename: Bo
authors.initials: B
authors.lastname: Feng
authors.forename: Charles
authors.initials: C
authors.lastname: Rotter
authors.forename: Manthena V
authors.initials: MV
authors.lastname: Varma
authors.forename: Sarah
authors.initials: S
authors.lastname: Kempshall
authors.forename: Katherine
authors.initials: K
authors.lastname: Fenner
authors.forename: Ayman F
authors.initials: AF
authors.lastname: El-Kattan
authors.forename: Theodore E
authors.initials: TE
authors.lastname: Liston
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Troutman
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology; D000818:Animals; D002460:Cell Line; D002469:Cell Separation; D002853:Chromatography, Liquid; D004285:Dogs; D005434:Flow Cytometry; D006801:Humans; D007408:Intestinal Absorption; D010539:Permeability; D060888:Real-Time Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1