Characterization and Validation of Canine P-Glycoprotein-Deficient MDCK II Cell Lines for Efflux Substrate Screening

Abstract

Purpose

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.

Method

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).

Results

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.

Conclusion

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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Abbreviations

ABC:

ATP-binding cassette

API:

Active pharmaceutical ingredient

BCRP:

Breast cancer resistance protein

CNS:

Central nervous system

cP-gp:

Canine P-glycoprotein

Cpm:

Count per minute

CT :

Cycle threshold

DMEM:

Dulbecco’s modified eagle medium

DMSO:

Dimethyl sulfoxide

DPBS:

Dulbecco’s phosphate buffered saline

EDTA:

Ethylenediaminetetraacetic acid

EMA:

European medicines agency

ER:

Efflux ratio

FBS:

Fetal bovine serum

FDA:

U.S. Food and drug administration

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

HBSS:

Hank’s balanced salt solution

MDCK:

Madin-Darby canine kidney

MDR:

Multidrug resistance

mRNA:

Messenger RNA

MRP:

Multidrug resistance associated protein

NEA:

Non-essential amino acids

NER:

Net efflux ratio

OATP:

Organic anion-transporting polypeptide

OCT:

Organic cation transporter

Papp :

Apparent permeability coefficient

RT-PCR:

Reverse transcription-polymerase chain reaction

P-gp:

P-glycoprotein

WT:

Wild type

ZFN:

Zinc finger nuclease

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Acknowledgement

The authors would like to thank Juliane Hoeckels-Messemer, Sylvia Hellwig and Patricia Muschong of AbbVie for their technical support.

Disclosures

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

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Key Words

  • Brain penetration
  • Efflux ratio (ER)
  • Madin-Darby canine kidney type II (MDCKII) cells
  • Net efflux ratio (NER)
  • Zinc finger nucleases (ZFNs)