Original scientific paper

https://doi.org/10.2478/acph-2022-0003

Isolation of MDCK cells with low expression of mdr1 gene and their use inmembrane permeability screening

ANA BOKULIĆ ; Fidelta Ltd., 10000 Zagreb, Croatia
JASNA PADOVAN ; Fidelta Ltd., 10000 Zagreb, Croatia
DARIJA STUPIN-POLANČEC DARIJA STUPIN-POLANČEC ; Fidelta Ltd., 10000 Zagreb, Croatia
ASTRID MILIĆ ; Fidelta Ltd., 10000 Zagreb, Croatia

Abstract

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P-glycoprotein activity. Clone isolation via the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery.

Keywords

permeability; MDCK; efflux ratio; clone isolation; P-glycoprotein; qRT-PCR; LC-MS/MSX

Hrčak ID:

262126

URI

https://hrcak.srce.hr/262126

Publication date:

30.6.2022.

Visits: 1.614 *