Skip to main content

Advertisement

SpringerLink
Log in

Development of a sensitive and quantitative capillary LC-UV method to study the uptake of pharmaceuticals in zebrafish brain

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

The present study explores the potential of 10-day-old zebrafish (Danio rerio) as a predictive blood-brain-barrier model using a set of 7 pharmaceutical agents. For this purpose, zebrafish were incubated with each of these 7 drugs separately via the route of immersion and the concentration reaching the brain was determined by applying a brain extraction procedure allowing isolation of the intact brain from the head of the zebrafish larvae. Sample analysis was performed utilizing capillary ultra-high performance liquid chromatography (cap-UHPLC) on a Pepmap RSLC C18 capillary column (150 mm × 300 μm, dp = 2 μm) coupled to a variable wavelength UV detector. Gradient separation was performed in 28 min at a flow rate of 5 μL/min and the optimal injection volume was determined to be 1 μL. The brain extraction procedure was established for the zebrafish strain TG898 exhibiting red fluorescence of the brain, allowing control of the integrity of the extracted parts. Quantitative experiments carried out on pooled samples of six zebrafish (n = 6) demonstrated the selective semipermeable nature of the blood-brain barrier after incubating the zebrafish at the maximum tolerated concentration for the investigated pharmaceuticals. The obtained brain-to-trunk ratios ranged between 0.3 for the most excluded compound and 1.2 for the pharmaceutical agent being most accumulated in the brain of the fish.

Workflow of brain extraction to study the uptake of pharmaceuticals in the brain of zebrafish larvae

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Engelhardt B. Development of the blood-brain barrier. Cell Tissue Res. 2003;314:119–29. https://doi.org/10.1007/s00441-003-0751-z.

    Article  CAS  Google Scholar 

  2. Pardridge WM. The blood-brain barrier: bottleneck in brain drug development. NeuroRx. 2005;2:3–14. https://doi.org/10.1602/neurorx.2.1.3.

    Article  Google Scholar 

  3. Reichel A. Addressing central nervous system (CNS) penetration in drug discovery: basics and implications of the evolving new concept. Chem Biodivers. 2009;6:2030–49. https://doi.org/10.1002/cbdv.200900103.

    Article  CAS  Google Scholar 

  4. Hitchcock SA. Blood-brain barrier permeability considerations for CNS-targeted compound library design. Curr Opin Chem Biol. 2008;12:318–23. https://doi.org/10.1016/j.cbpa.2008.03.019.

    Article  CAS  Google Scholar 

  5. Gabathuler R. Approaches to transport therapeutic drugs across the blood-brain barrier to treat brain diseases. Neurobiol Dis. 2010;37:48–57. https://doi.org/10.1016/j.nbd.2009.07.028.

    Article  CAS  Google Scholar 

  6. Nau R, Sorgel F, Eiffert H. Penetration of drugs through the blood-cerebrospinal fluid/blood-brain barrier for treatment of central nervous system infections. Clin Microbiol Rev. 2010;23:858–83. https://doi.org/10.1128/CMR.00007-10.

    Article  CAS  Google Scholar 

  7. Banks WA. Characteristics of compounds that cross the blood-brain barrier. BMC Neurol. 2009;9:S3. https://doi.org/10.1186/1471-2377-9-S1-S3.

  8. Pajouhesh H, Lenz GR. Medicinal chemical properties of successful central nervous system drugs. J Am Soc Exp Neurother. 2005;2:541–53. https://doi.org/10.1602/neurorx.2.4.541.

    Google Scholar 

  9. Schinkel AH, Wagenaar E, Mol CAAM, Van Deemter L. P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J Clin Invest. 1996;97:2517–24. https://doi.org/10.1172/JCI118699.

    Article  CAS  Google Scholar 

  10. Nielsen PA, Andersson O, Hansen SH, Simonsen KB, Andersson G. Models for predicting blood-brain barrier permeation. Drug Discov Today. 2011;16:472–5. https://doi.org/10.1016/j.drudis.2011.04.004.

    Article  CAS  Google Scholar 

  11. Mensch J, L LJ, Sanderson W, Melis A, Mackie C, Verreck G, et al. Application of PAMPA-models to predict BBB permeability including efflux ratio, plasma protein binding and physicochemical parameters. Int J Pharm. 2010;395:182–97. https://doi.org/10.1016/j.ijpharm.2010.05.037.

    Article  CAS  Google Scholar 

  12. Di L, Kerns EH, Fan K, McConnell OJ, Carter GT. High throughput artificial membrane permeability assay for blood-brain barrier. Eur J Med Chem. 2003;38:223–32.

    Article  CAS  Google Scholar 

  13. Syvänen S, Lindhe Ö, Palner M, Lindhe O, Kornum BR, Rahman O, et al. Species differences in blood-brain barrier transport of three PET radioligands with emphasis on P-glycoprotein transport. Drug Metab Dispos. 2008;37:635–43. https://doi.org/10.1124/dmd.108.024745.

    Article  Google Scholar 

  14. Lundquist S, Renftel M, Brillault J, Fenart L, Cecchelli R, Dehouck MP. Prediction of drug transport through the blood-brain barrier in vivo: a comparison between two in vitro cell models. Pharm Res. 2002;19:976–81.

    Article  CAS  Google Scholar 

  15. Roux F, Couraud P-O. Rat brain endothelial cell lines for the study of blood-brain barrier permeability and transport functions. Cell Mol Neurobiol. 2005;25:41–58. https://doi.org/10.1007/s10571-004-1376-9.

    Article  Google Scholar 

  16. Andersson O, Hansen SH, Hellman K, Olsen LR, Andersson G, Badolo L, et al. The grasshopper: a novel model for assessing vertebrate brain uptake. J Pharmacol Exp Ther. 2013;346:211–8. https://doi.org/10.1124/jpet.113.205476.

    Article  CAS  Google Scholar 

  17. DeSalvo MK, Mayer N, Mayer F, Bainton RJ. Physiologic and anatomic characterization of the brain surface glia barrier of drosophila. Glia. 2011;59:1322–40. https://doi.org/10.1002/glia.21147.PHYSIOLOGIC.

    Article  Google Scholar 

  18. Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013;496:498–503. https://doi.org/10.1038/nature12111.

    Article  CAS  Google Scholar 

  19. Barros TP, Alderton WK, Reynolds HM, Roach AG, Berghmans S. Zebrafish: an emerging technology for in vivo pharmacological assessment to identify potential safety liabilities in early drug discovery. Br J Pharmacol. 2008;154:1400–13. https://doi.org/10.1038/bjp.2008.249.

    Article  CAS  Google Scholar 

  20. Fleming A, Diekmann H, Goldsmith P. Functional characterisation of the maturation of the blood-brain barrier in larval zebrafish. PLoS One. 2013;8:1–12. https://doi.org/10.1371/journal.pone.0077548.

    Article  Google Scholar 

  21. Escher BI, Ashauer R, Dyer S, Hermens JLM, Lee JH, Leslie HA, et al. Crucial role of mechanisms and modes of toxic action for understanding tissue residue toxicity and internal effect concentrations of organic chemicals. Integr Environ Assess Manag. 2011;7:28–49. https://doi.org/10.1002/ieam.100.

    Article  CAS  Google Scholar 

  22. Tufi S, Leonards P, Lamoree M, De Boer J, Legler J, Legradi J. Changes in neurotransmitter profiles during early zebrafish (Danio rerio) development and after pesticide exposure. Environ Sci Technol. 2016;50:3222–30. https://doi.org/10.1021/acs.est.5b05665.

    Article  CAS  Google Scholar 

  23. Berghmans S, Butler P, Goldsmith P, Waldron G, Gardner I, Golder Z, et al. Zebrafish based assays for the assessment of cardiac, visual and gut function—potential safety screens for early drug discovery. J Pharmacol Toxicol Methods. 2008;58:59–68. https://doi.org/10.1016/j.vascn.2008.05.130.

    Article  CAS  Google Scholar 

  24. Beker van Woudenberg A, Snel C, Rijkmans E, De Groot D, Bouma M, Hermsen S, et al. Zebrafish embryotoxicity test for developmental (neuro)toxicity: demo case of an integrated screening approach system using anti-epileptic drugs. Reprod Toxicol. 2014;49:101–16. https://doi.org/10.1016/j.reprotox.2014.07.082.

    Article  CAS  Google Scholar 

  25. Jones HS, Trollope HT, Hutchinson TH, Panter GH, Chipman JK. Metabolism of ibuprofen in zebrafish larvae. Xenobiotica. 2012;42:1069–75. https://doi.org/10.3109/00498254.2012.684410.

    Article  CAS  Google Scholar 

  26. Kirla KT, Groh KJ, Steuer AE, Poetzsch M, Banote RK, Stadnicka-Michalak J, et al. Zebrafish larvae are insensitive to stimulation by cocaine: importance of exposure route and toxicokinetics. Toxicol Sci. 2016;154:183–93. https://doi.org/10.1093/toxsci/kfw156.

    Article  CAS  Google Scholar 

  27. Chen F, Gong Z, Kelly BC. Rapid analysis of pharmaceuticals and personal care products in fish plasma micro-aliquots using liquid chromatography tandem mass spectrometry. J Chromatogr A. 2015;1383:104–11. https://doi.org/10.1016/j.chroma.2015.01.033.

    Article  CAS  Google Scholar 

  28. Kühnert A, Vogs C, Altenburger R, Küster E. The internal concentration of organic substances in fish embryos—a toxicokinetic approach. Environ Toxicol Chem. 2013;32:1819–27. https://doi.org/10.1002/etc.2239.

    Article  Google Scholar 

  29. Brox S, Ritter AP, Küster E, Reemtsma T. A quantitative HPLC-MS/MS method for studying internal concentrations and toxicokinetics of 34 polar analytes in zebrafish (Danio rerio) embryos. Anal Bioanal Chem. 2014;406:4831–40. https://doi.org/10.1007/s00216-014-7929-y.

    Article  CAS  Google Scholar 

  30. Kantae V, Krekels EHJ, Ordas A, González O, van Wijk RC, Harms AC, et al. Pharmacokinetic modeling of paracetamol uptake and clearance in zebrafish larvae: expanding the allometric scale in vertebrates with five orders of magnitude. Zebrafish. 2016;13:504–10. https://doi.org/10.1089/zeb.2016.1313.

    Article  CAS  Google Scholar 

  31. Kalueff AV, Stewart AM, Gerlai R, Court P. Zebrafish as an emerging model for studying complex brain disorders. Trends Pharmacol Sci. 2014;35:63–75. https://doi.org/10.1016/j.tips.2013.12.002.Zebrafish.

    Article  CAS  Google Scholar 

  32. Jeong JY, Kwon HB, Ahn JC, Kang D, Kwon SH, Park JA, et al. Functional and developmental analysis of the blood-brain barrier in zebrafish. Brain Res Bull. 2008;75:619–28. https://doi.org/10.1016/j.brainresbull.2007.10.043.

    Article  CAS  Google Scholar 

  33. Zhuo H, Jin H, Peng H, Huang H. Distribution, pharmacokinetics and primary metabolism model of tramadol in zebrafish. Mol Med Rep. 2016;14:5644–52. https://doi.org/10.3892/mmr.2016.5956.

    Article  CAS  Google Scholar 

  34. Kim SS, Im SH, Yang JY, Lee Y-R, Kim GR, Chae JS, et al. Zebrafish as a screening model for testing the permeability of blood–brain barrier to small molecules. Zebrafish. 2017;14:322–30. https://doi.org/10.1089/zeb.2016.1392.

    Article  CAS  Google Scholar 

  35. Kulkarni P, Chaudhari GH, Sripuram V, Banote RK, Kirla KT, Sultana R, et al. Oral dosing in adult zebrafish: proof-of-concept using pharmacokinetics and pharmacological evaluation of carbamazepine. Pharmacol Rep. 2014;66:179–83. https://doi.org/10.1016/j.pharep.2013.06.012.

    Article  CAS  Google Scholar 

  36. Hrdina PD, Dubas TC. Brain distribution and kinetics of desipramine in the rat. Can J Physiol Pharrnacol. 1980;59:163–7.

    Article  Google Scholar 

  37. Bicker J, Alves G, Fortuna A, Falcao A. Blood-brain barrier models and their relevance for a successful development of CNS drug delivery systems: a review. Eur J Pharm Biopharm. 2014;87:409–32. https://doi.org/10.1016/j.ejpb.2014.03.012.

    Article  CAS  Google Scholar 

  38. Benya TJ, Wagner JG. Rapid equilibration of warfarin between rat tissue and plasma. J Pharmacokinet Biopharma. 1975;3:237-255.

  39. DeVane CL, Boulton DW, Miller LF, Miller RL. Pharmacokinetics of trazodone and its major metabolite m-chlorophenylpiperazine in plasma and brain of rats. Int J Neuropsychopharmacol. 1999;2:17–23. https://doi.org/10.1017/S1461145799001303.

    Article  CAS  Google Scholar 

  40. O’Byrne PM, Williams R, Walsh JJ, Gilmer JF. Part two: evaluation of N-methylbupropion as a potential bupropion prodrug. Pharmaceuticals. 2014;7:676–94. https://doi.org/10.3390/ph7060676.

    Article  Google Scholar 

  41. Hendrickx S, Uğur DY, Yilmaz IT, Şener E, Van Schepdael A, Adams E, et al. A sensitive capillary LC-UV method for the simultaneous analysis of olanzapine, chlorpromazine and their FMO-mediated N-oxidation products in brain microdialysates. Talanta. 2017;162:268–77. https://doi.org/10.1016/j.talanta.2016.09.053.

    Article  CAS  Google Scholar 

  42. Kislyuk S, Kroonen J, Adams E, Augustijns P, De Witte P, Cabooter D. Development of a sensitive and quantitative UHPLC-MS/MS method to study the whole-body uptake of pharmaceuticals in zebrafish. Talanta. 2017;174:780–8. https://doi.org/10.1016/j.talanta.2017.06.075.

    Article  CAS  Google Scholar 

  43. EMA. Guideline on bioanalytical method validation. EMEA, Comm Med Prod Hum Use. 2012;44:1–23.

    Google Scholar 

  44. Lee M, Hong S, Kim N, Shin KS, Kang SJ. Tricyclic antidepressants amitriptyline and desipramine induced neurotoxicity associated with Parkinson’s disease. Mol Cells. 2015;38:734–40. https://doi.org/10.14348/molcells.2015.0131.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

ARIADME, a European FP7 ITN Community’s Seventh Framework Program under Grant Agreement No. 607517, is acknowledged for the financial support.

Author information

Authors and Affiliations

  1. Pharmaceutical Analysis, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven (KU Leuven), Herestraat 49, 3000, Leuven, Belgium

    Stanislav Kislyuk, Wannes Van den Bosch, Erwin Adams & Deirdre Cabooter

  2. Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, University of Leuven (KU Leuven), Herestraat 49, 3000, Leuven, Belgium

    Peter de Witte

Authors
  1. Stanislav Kislyuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wannes Van den Bosch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Erwin Adams
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter de Witte
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Deirdre Cabooter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Deirdre Cabooter.

Ethics declarations

All animal experiments were approved by the Animal Ethical Committee of the University of Leuven (approval no. P007/2016) and conducted in accordance with the Belgian and European laws, guidelines, and policies for animal experimentation, housing, and care (Belgian Royal Decree of 29 May 2013 and European Directive 2010/63/EU on the protection of animals used for scientific purposes of 20 October 2010).

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

ESM 1

(PDF 994 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kislyuk, S., Van den Bosch, W., Adams, E. et al. Development of a sensitive and quantitative capillary LC-UV method to study the uptake of pharmaceuticals in zebrafish brain. Anal Bioanal Chem 410, 2751–2764 (2018). https://doi.org/10.1007/s00216-018-0955-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-018-0955-4

Keywords

Search

Navigation