title: Computational and functional analysis of biopharmaceutical drugs in zebrafish: Erythropoietin as a test model.
abstract: The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly due to the zebrafish advantages when compared to other animal models. Erythropoietin is a glycoprotein hormone that acts principally on erythroid progenitors, stimulating their survival, proliferation and differentiation. Recombinant human erythropoietin (rhEPO) has been widely used in medicine to treat anemia and it is one of the best-selling biotherapeutics worldwide. The recombinant molecule, industrially produced in CHO cells, has the same amino acid sequence of endogenous human erythropoietin, but differs in the glycosylation pattern. This may influence efficacy and safety, particularly immunogenicity, of the final product. We employed the zebrafish embryo as a vertebrate animal model to perform in vivo pharmacological assays. We conducted a functional analysis of rhEPO alpha Eprex(®) and two biosimilars, the erythropoietin alpha Binocrit(®) and zeta Retacrit(®). By in silico analysis and 3D modeling we proved the interaction between recombinant human erythropoietin and zebrafish endogenous erythropoietin receptor. Then we treated zebrafish embryos with the 3 rhEPOs and we investigated their effect on erythrocytes production with different assays. By real time-PCR we observed the relative upregulation of gata1 (2.4 ± 0.3 fold), embryonic α-Hb (1.9 ± 0.2 fold) and β-Hb (1.6 ± 0.1 fold) transcripts. A significant increase in Stat5 phosphorylation was also assessed in embryos treated with rhEPOs when compared with the negative controls. Live imaging in tg (kdrl:EGFP; gata1:ds-red) embryos, o-dianisidine positive area quantification and cyanomethemoglobin content quantification revealed a 1.8 ± 0.3 fold increase of erythrocytes amount in embryos treated with rhEPOs when compared with the negative controls. Finally, we verified that recombinant human erythropoietins did not cause any inflammatory response in the treated embryos. Our data showed that zebrafish embryo can be a valuable tool to study in vivo effects of complex pharmacological compounds, such as recombinant human glycoproteins, allowing to perform fast and reproducible pharmacological assays with excellent results.
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. Electronic address: michela.guarienti@unibs.it.
authors.forename: Michela
authors.initials: M
authors.lastname: Guarienti
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. Electronic address: edoardo.giacopuzzi@unibs.it.
authors.forename: Edoardo
authors.initials: E
authors.lastname: Giacopuzzi
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. Electronic address: alessandra.gianoncelli@unibs.it.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Gianoncelli
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. Electronic address: sandra.sigala@unibs.it.
authors.forename: Sandra
authors.initials: S
authors.lastname: Sigala
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. Electronic address: pierfranco.spano@unibs.it.
authors.forename: Pierfranco
authors.initials: P
authors.lastname: Spano
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy; Agenzia Italiana del Farmaco, Via del Tritone 181, 00187 Roma, Italy. Electronic address: sergio.pecorelli@unibs.it.
authors.forename: Sergio
authors.initials: S
authors.lastname: Pecorelli
authors.affiliation: Agenzia Italiana del Farmaco, Via del Tritone 181, 00187 Roma, Italy. Electronic address: l.pani@aifa.gov.it.
authors.forename: Luca
authors.initials: L
authors.lastname: Pani
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy. Electronic address: maurizio.memo@unibs.it.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Memo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D059451:Biosimilar Pharmaceuticals / Q000494:pharmacology*; D019295:Computational Biology / Q000379:methods; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism; D000068817:Epoetin Alfa / Q000494:pharmacology; D004921:Erythropoietin / Q000378:metabolism*; D050982:GATA1 Transcription Factor / Q000378:metabolism; D006801:Humans; D023421:Models, Animal; D008969:Molecular Sequence Data; D017467:Receptors, Erythropoietin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D015854:Up-Regulation / Q000187:drug effects; D015027:Zebrafish / Q000378:metabolism*
chemical_list: D059451:Biosimilar Pharmaceuticals; D050982:GATA1 Transcription Factor; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D000068817:Epoetin Alfa
keywords: 3D modeling; Biosimilars; Erythropoietin; Functional analysis; Inflammatory response; Zebrafish