期刊
ACTA PHARMACEUTICA
卷 72, 期 2, 页码 275-288出版社
SCIENDO
DOI: 10.2478/acph-2022-0003
关键词
permeability; MDCK; efflux ratio; clone isolation; P-glycoprotein; qRT-PCR; LC-MS; MS
In this study, a subclonal line with low P-glycoprotein expression was obtained through an isolation procedure, and MDCK clones with substantially lower P-glycoprotein efflux were successfully identified, demonstrating their usefulness as a tool for assessing passive permeability in early drug discovery.
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.
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