We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems.
hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu).
MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration.
cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.
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Active pharmaceutical ingredient
Breast cancer resistance protein
Central nervous system
Count per minute
- CT :
Dulbecco’s modified eagle medium
Dulbecco’s phosphate buffered saline
European medicines agency
Fetal bovine serum
U.S. Food and drug administration
Glyceraldehyde 3-phosphate dehydrogenase
Hank’s balanced salt solution
Madin-Darby canine kidney
Multidrug resistance associated protein
Non-essential amino acids
Net efflux ratio
Organic anion-transporting polypeptide
Organic cation transporter
- Papp :
Apparent permeability coefficient
Reverse transcription-polymerase chain reaction
Zinc finger nuclease
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The authors would like to thank Juliane Hoeckels-Messemer, Sylvia Hellwig and Patricia Muschong of AbbVie for their technical support.
Dong Ye, Manuel Weinheimer, Loic Laplanche, Mario Mezler are employees of AbbVie and may own AbbVie stock. Zhizhou Fang was an employee of AbbVie at the time when he contributed to the work being discussed, and he currently works at Merck Healthcare KGaA, Germany. Anna Harder was a master student of AbbVie for this work at the time and currently works at Braun Melsungen AG, Germany. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, writing and approval of the publication. All authors declare no competing financial interest in this work.
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Dong Ye, Anna Harder, Zhizhou Fang. The first draft of the manuscript was written by Dong Ye and all authors commented on previous versions of the manuscript. All authors actively contributed to the final text, read and approved the final manuscript.
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Ye, D., Harder, A., Fang, Z. et al. Characterization and Validation of Canine P-Glycoprotein-Deficient MDCK II Cell Lines for Efflux Substrate Screening. Pharm Res 37, 194 (2020). https://doi.org/10.1007/s11095-020-02895-9
- Brain penetration
- Efflux ratio (ER)
- Madin-Darby canine kidney type II (MDCKII) cells
- Net efflux ratio (NER)
- Zinc finger nucleases (ZFNs)