<abstract xmlns="http://www.w3.org/1999/xhtml">

<p>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 <italic>via</italic> 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.</p>
</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.

Isolation of MDCK cells with low expression of <italic>mdr1</italic> gene and their use in membrane permeability screening

Acta Pharmaceutica

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Isolation of MDCK cells with low expression of  gene and their use in membrane permeability screening

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most...

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

Article Category: Original research paper

Published Online: Dec 30, 2021

Page range: 275 - 288

Accepted: Mar 29, 2021

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

Keywords
permeability, MDCK, efflux ratio, clone isolation, P-glycoprotein, qRT-PCR, LC-MS/MS

© 2022 Ana Bokulić., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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

Article Category: Original research paper

Published Online: Dec 30, 2021

Page range: 275 - 288

Accepted: Mar 29, 2021

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

Keywordspermeability, MDCK, efflux ratio, clone isolation, P-glycoprotein, qRT-PCR, LC-MS/MS

© 2022 Ana Bokulić., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Keywords
permeability, MDCK, efflux ratio, clone isolation, P-glycoprotein, qRT-PCR, LC-MS/MS