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Functional Evaluation of P-gp and Bcrp at the Murine Blood-Cerebrospinal Fluid Barrier

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Abstract

Purpose

The brain is protected from circulating metabolites and xenobiotics by the blood–brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Previous studies report that P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are expressed apically or subapically at the blood-CSF barrier (BCSFB), implying a paradoxical function to mediate blood-to-CSF transport of xenobiotics. As evidence of P-gp and Bcrp activity at the BCSFB is limited, the goal of this study is to investigate functional activity of P-gp and Bcrp at the murine BCSFB using a live tissue imaging approach.

Methods

The choroid plexuses (CP) forming the BCSFB were freshly isolated from mouse brain ventricles and incubated with fluorescent probes calcein-AM and BODIPY FL-Prazosin. Using quantitative fluorescence microscopy, the functional contributions of Bcrp and P-gp were examined using inhibitors and mice with targeted deletion of the Abcb1a/b or Abcg2 gene.

Results

Apical transport of calcein-AM in choroid plexus epithelial (CPE) cells is sensitive to inhibition by elacridar and Ko143 but is unaffected by P-gp deletion. In wild-type mice, elacridar increased CPE accumulation of BODIPY FL-Prazosin by 220% whereas deletion of Bcrp increased BODIPY FL-Prazosin accumulation by 43%. There was no change in Mdr1a/1b mRNA expression in CP tissues from the Bcrp−/− mice.

Conclusions

This study demonstrated functional activity of Bcrp at the BCSFB apical membrane and provided evidence supporting an additional contribution by P-gp. These findings contribute to the understanding of transport mechanisms that regulate CSF drug concentrations, which may benefit future predictions of CNS drug disposition, efficacy, and toxicity.

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Data Availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

Abbreviations

BBB:

Blood-brain barrier

BCSFB:

Blood-CSF barrier

BCRP/Bcrp:

Breast cancer resistance protein

CP:

Choroid plexus

CPE cells:

Choroid plexus epithelial cells

CPEI:

Choroid plexus efflux index

CNS:

Central nervous system

CSF:

Cerebrospinal fluid

P-gp/MDR1:

P-glycoprotein

References

  1. Feigin VL, Vos T, Nichols E, Owolabi MO, Carroll WM, Dichgans M, et al. The global burden of neurological disorders: translating evidence into policy. Lancet Neurol. 2020;19(3):255–65.

    PubMed  Google Scholar 

  2. Gribkoff VK, Kaczmarek LK. The need for new approaches in CNS drug discovery: Why drugs have failed, and what can be done to improve outcomes. Neuropharmacology. 2017;120:11–9.

    CAS  PubMed  Google Scholar 

  3. Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KLR, Chu X, et al. Membrane transporters in drug development. Nat Rev Drug Discov. 2010;9(3):215–36.

    CAS  PubMed  Google Scholar 

  4. Agarwal S, Hartz AMS, Elmquist WF, Bauer B. Breast cancer resistance protein and P-glycoprotein in brain cancer: two gatekeepers team up. Curr Pharm Des. 2011;17(26):2793–802.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Morris ME, Rodriguez-Cruz V, Felmlee MA. SLC and ABC transporters: expression, localization, and species differences at the blood-brain and the blood-cerebrospinal fluid barriers. AAPS J. 2017;19(5):1317–31.

    PubMed  Google Scholar 

  6. Rao VV, Dahlheimer JL, Bardgett ME, Snyder AZ, Finch RA, Sartorelli AC, et al. Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood–cerebrospinal-fluid drug-permeability barrier. Proc Natl Acad Sci. 1999;96(7):3900– 3905.

  7. Tachikawa M, Watanabe M, Hori S, Fukaya M, Ohtsuki S, Asashima T, et al. Distinct spatio-temporal expression of ABCA and ABCG transporters in the developing and adult mouse brain. J Neurochem. 2005;95(1):294–304.

    CAS  PubMed  Google Scholar 

  8. Lun MP, Monuki ES, Lehtinen MK. Development and functions of the choroid plexus-cerebrospinal fluid system. Nat Rev Neurosci. 2015;16(8):445–57.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Sun A, Wang J. Choroid plexus and drug removal mechanisms. AAPS J. 2021;23(3):61.

    CAS  PubMed  Google Scholar 

  10. Shen H, Smith DE, Keep RF, Xiang J, Brosius FC. Targeted disruption of the PEPT2 gene markedly reduces dipeptide uptake in choroid plexus. J Biol Chem. 2003;278(7):4786–91.

    CAS  PubMed  Google Scholar 

  11. Shen H, Smith DE, Keep RF, Brosius FC. Immunolocalization of the proton-coupled oligopeptide transporter PEPT2 in developing rat brain. Mol Pharm. 2004;1(4):248–56.

    CAS  PubMed  Google Scholar 

  12. Ocheltree SM, Shen H, Hu Y, Keep RF, Smith DE. Role and relevance of peptide transporter 2 (PEPT2) in the kidney and choroid plexus: in vivo studies with glycylsarcosine in wild-type and PEPT2 knockout mice. J Pharmacol Exp Ther. 2005;315(1):240–7.

    CAS  PubMed  Google Scholar 

  13. Sweet DH, Miller DS, Pritchard JB, Fujiwara Y, Beier DR, Nigam SK. Impaired organic anion transport in kidney and choroid plexus of organic anion transporter 3 (Oat3 (Slc22a8)) knockout mice. J Biol Chem. 2002;277(30):26934–43.

    CAS  PubMed  Google Scholar 

  14. Duan H, Wang J. Impaired monoamine and organic cation uptake in choroid plexus in mice with targeted disruption of the plasma membrane monoamine transporter (Slc29a4) gene. J Biol Chem. 2013;288(5):3535–44.

    CAS  PubMed  Google Scholar 

  15. Zhuang Y, Fraga CH, Hubbard KE, Hagedorn N, Panetta JC, Waters CM, et al. Topotecan central nervous system penetration is altered by a tyrosine kinase inhibitor. Can Res. 2006;66(23):11305–13.

    CAS  Google Scholar 

  16. Baehr C, Reichel V, Fricker G. Choroid plexus epithelial monolayers—a cell culture model from porcine brain. Cerebrospinal Fluid Res. 2006;3(1):13.

    PubMed  PubMed Central  Google Scholar 

  17. Kläs J, Wolburg H, Terasaki T, Fricker G, Reichel V. Characterization of immortalized choroid plexus epithelial cell lines for studies of transport processes across the blood-cerebrospinal fluid barrier. Cerebrospinal Fluid Res. 2010;7(1):11.

  18. Bernd A, Ott M, Ishikawa H, Schroten H, Schwerk C, Fricker G. Characterization of efflux transport proteins of the human choroid plexus papilloma cell line HIBCPP, a functional in vitro model of the blood-cerebrospinal fluid barrier. Pharm Res. 2015;32(9):2973–82.

    CAS  PubMed  Google Scholar 

  19. Hu T, Zha W, Sun A, Wang J. Live tissue imaging reveals distinct transcellular pathways for organic cations and anions at the blood-cerebrospinal fluid barrier. Mol Pharmacol. 2022;101(5):334–42.

  20. Sun A, Wang J. Evaluation of blood-CSF barrier transport by quantitative real time fluorescence microscopy. Pharm Res. 2022;39(7):1469–80.

    CAS  PubMed  Google Scholar 

  21. Schinkel AH, Mayer U, Wagenaar E, Mol CAAM, van Deemter L, Smit JJM, et al. Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci. 1997;94(8):4028–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Jonker JW, Buitelaar M, Wagenaar E, van der Valk MA, Scheffer GL, Scheper RJ, et al. The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc Natl Acad Sci. 2002;99(24):15649–54.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82.

    CAS  PubMed  Google Scholar 

  24. Cui YJ, Cheng X, Weaver YM, Klaassen CD. Tissue distribution, gender-divergent expression, ontogeny, and chemical induction of multidrug resistance transporter genes (Mdr1a, Mdr1b, Mdr2) in mice. Drug Metab Dispos. 2009;37(1):203–10.

    CAS  PubMed  Google Scholar 

  25. Holló Z, Homolya L, Davis CW, Sarkadi B. Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochim Biophys Acta (BBA) - Biomembrane. 1994;1191(2):384–8.

    Google Scholar 

  26. Glavinas H, von Richter O, Vojnits K, Mehn D, Wilhelm I, Nagy T, et al. Calcein assay: a high-throughput method to assess P-gp inhibition. Xenobiotica. 2011;41(8):712–9.

    CAS  PubMed  Google Scholar 

  27. Mao Q, Unadkat JD. Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport—an update. AAPS J. 2015;17(1):65–82.

    CAS  PubMed  Google Scholar 

  28. Dey S, Ramachandra M, Pastan I, Gottesman MM, Ambudkar SV. Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. Proc Natl Acad Sci. 1997;94(20):10594–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. de Bruin M, Miyake K, Litman T, Robey R, Bates SE. Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter. MXR Cancer Lett. 1999;146(2):117–26.

    PubMed  Google Scholar 

  30. Mao Q. Role of the breast cancer resistance protein (ABCG2) in drug transport. AAPS J. 2005;7(1):E118–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, et al. The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci. 2000;113(11):2011–21.

    CAS  PubMed  Google Scholar 

  32. Leggas M, Adachi M, Scheffer GL, Sun D, Wielinga P, Du G, et al. Mrp4 confers resistance to topotecan and protects the brain from chemotherapy. Mol Cell Biol. 2004;24(17):7612–21.

  33. Wijnholds J, de Lange ECM, Scheffer GL, van den Berg DJ, Mol CAAM, van der Valk M, et al. Multidrug resistance protein 1 protects the choroid plexus epithelium and contributes to the blood-cerebrospinal fluid barrier. J Clin Invest. 2000;105(3):279–85.

  34. Olson DP, Taylor BJ, Ivy SP. Detection of MRP functional activity: Calcein AM but not BCECF AM as a multidrug resistance-related protein (MRP1) substrate. Cytometry. 2001;46(2):105–13.

    CAS  PubMed  Google Scholar 

  35. Hyafil F, Vergely C, Du Vignaud P, Grand-Perret T. In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative. Can Res. 1993;53(19):4595–602.

    CAS  Google Scholar 

  36. Allen JD, van Loevezijn A, Lakhai JM, van der Valk M, van Tellingen O, Reid G, et al. Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C1. Mol Cancer Ther. 2002;1(6):417–25.

    CAS  PubMed  Google Scholar 

  37. Weidner LD, Zoghbi SS, Lu S, Shukla S, Ambudkar SV, Pike VW, et al. The inhibitor Ko143 is not specific for ABCG2. J Pharmacol Exp Ther. 2015;354(3):384–93.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Mairinger S, Zoufal V, Wanek T, Traxl A, Filip T, Sauberer M, et al. Influence of breast cancer resistance protein and P-glycoprotein on tissue distribution and excretion of Ko143 assessed with PET imaging in mice. Eur J Pharm Sci. 2018;115:212–22.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Polli JW, Olson KL, Chism JP, St. John-Williams L, Yeager RL, Woodard SM, et al. An unexpected synergist role of P-glycoprotein and breast cancer resistance protein on the central nervous system penetration of the tyrosine kinase inhibitor lapatinib (N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}meth. Drug Metab Dispos. 2009;37(2):439–442.

    Google Scholar 

  40. de Vries NA, Zhao J, Kroon E, Buckle T, Beijnen JH, van Tellingen O. P-glycoprotein and breast cancer resistance protein: two dominant transporters working together in limiting the brain penetration of topotecan. Clin Cancer Res. 2007;13(21):6440–9.

    PubMed  Google Scholar 

  41. Chen Y, Agarwal S, Shaik NM, Chen C, Yang Z, Elmquist WF. P-glycoprotein and breast cancer resistance protein influence brain distribution of dasatinib. J Pharmacol Exp Ther. 2009;330(3):956–63.

    CAS  PubMed  Google Scholar 

  42. Agarwal S, Sane R, Gallardo JL, Ohlfest JR, Elmquist WF. Distribution of gefitinib to the brain is limited by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2)-mediated active efflux. J Pharmacol Exp Ther. 2010;334(1):147–55.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Lagas JS, van Waterschoot RAB, Sparidans RW, Wagenaar E, Beijnen JH, Schinkel AH. Breast cancer resistance protein and P-glycoprotein limit sorafenib brain accumulation. Mol Cancer Ther. 2010;9(2):319–26.

    CAS  PubMed  Google Scholar 

  44. Lee YJ, Kusuhara H, Jonker JW, Schinkel AH, Sugiyama Y. Investigation of efflux transport of dehydroepiandrosterone sulfate and mitoxantrone at the mouse blood-brain barrier: a minor role of breast cancer resistance protein. J Pharmacol Exp Ther. 2005;312(1):44–52.

    CAS  PubMed  Google Scholar 

  45. Kodaira H, Kusuhara H, Ushiki J, Fuse E, Sugiyama Y. Kinetic analysis of the cooperation of P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp/Abcg2) in limiting the brain and testis penetration of erlotinib, flavopiridol, and mitoxantrone. J Pharmacol Exp Ther. 2010;333(3):788–96.

    CAS  PubMed  Google Scholar 

  46. Uchida Y, Ohtsuki S, Katsukura Y, Ikeda C, Suzuki T, Kamiie J, et al. Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors. J Neurochem. 2011;117(2):333–45.

    CAS  PubMed  Google Scholar 

  47. Uchida Y, Zhang Z, Tachikawa M, Terasaki T. Quantitative targeted absolute proteomics of rat blood-cerebrospinal fluid barrier transporters: comparison with a human specimen. J Neurochem. 2015;134(6):1104–15.

    CAS  PubMed  Google Scholar 

  48. Spector R, Robert Snodgrass S, Johanson CE. A balanced view of the cerebrospinal fluid composition and functions: focus on adult humans. Exp Neurol. 2015;273:57–68.

    CAS  PubMed  Google Scholar 

  49. Jani M, Ambrus C, Magnan R, Jakab KT, Beéry E, Zolnerciks JK, et al. Structure and function of BCRP, a broad specificity transporter of xenobiotics and endobiotics. Arch Toxicol. 2014;88(6):1205–48.

    CAS  PubMed  Google Scholar 

  50. Ghersi-Egea JF, Vasiljevic A, Blondel S, Strazielle N. Neuroprotective mechanisms at the blood-CSF barrier of the developing and adult brain BT - role of the choroid plexus in health and disease. In: Praetorius J, Blazer-Yost B, Damkier H, editors. New York. Springer, US; 2020. p. 193–207.

    Google Scholar 

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Acknowledgements

The authors would like to thank Nathaniel Peters at the UW Keck Microscopy Center for his help and insights in confocal microscopy practice.

Funding

This work was supported in part by the National Institutes of Health grant R21AG071827.

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Authors and Affiliations

  1. Department of Pharmaceutics, University of Washington, H272 Health Sciences Building, Seattle, WA, 98195-7610, USA

    Austin Sun & Joanne Wang

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  1. Austin Sun
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Contributions

Original study conception and design was completed by Joanne Wang and Austin Sun. Experiments were performed by Austin Sun, and data analysis was performed by Austin Sun and Joanne Wang. The initial draft of the manuscript was written by Austin Sun and critically revised by Joanne Wang.

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Correspondence to Joanne Wang.

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Sun, A., Wang, J. Functional Evaluation of P-gp and Bcrp at the Murine Blood-Cerebrospinal Fluid Barrier. Pharm Res 40, 2667–2675 (2023). https://doi.org/10.1007/s11095-023-03598-7

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