Computational and functional analysis of biopharmaceutical drugs in zebrafish: Erythropoietin as a test model

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• 作者：Michela Guarienti^a ; ^{michela.guarienti@unibs.it" class="auth_mail" title="E-mail the corresponding author} ; Edoardo Giacopuzzi^a ; ^{edoardo.giacopuzzi@unibs.it" class="auth_mail" title="E-mail the corresponding author} ; Alessandra Gianoncelli^a ; ^{alessandra.gianoncelli@unibs.it" class="auth_mail" title="E-mail the corresponding author} ; Sandra Sigala^a ; ^{sandra.sigala@unibs.it" class="auth_mail" title="E-mail the corresponding author} ; Pierfranco Spano^a ; ^{pierfranco.spano@unibs.it" class="auth_mail" title="E-mail the corresponding author} ; Sergio Pecorelli^a ; ^b ; ^{sergio.pecorelli@unibs.it" class="auth_mail" title="E-mail the corresponding author} ; ^{s.pecorelli@aifa.gov.it" class="auth_mail" title="E-mail the corresponding author} ; Luca Pani^b ; ^{l.pani@aifa.gov.it" class="auth_mail" title="E-mail the corresponding author} ; Maurizio Memo^a ; ^{maurizio.memo@unibs.it" class="auth_mail" title="E-mail the corresponding author}
• 关键词：hpf ; hours post fertilization ; dpf ; days post fertilization ; CHO ; Chinese hamster ovary ; EPO ; erythropoietin ; EPOR ; erythropoietin receptor ; rhEPO ; recombinant human erythropoietin ; hepo ; human erythropoietin gene ; hEPO ; human erythropoietin protein ; hEPOR ; human erythropoietin receptor gene ; hEPOR ; human erythropoietin receptor protein ; zepo ; zebrafish erythropoietin gene ; zEpo ; zebrafish erythropoietin protein ; zepor ; zebrafish erythropoietin receptor gene ; zEpor ; zebrafish erythropoietin rece
• 刊名：Pharmacological Research
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：102
• 期：Complete
• 页码：12-21
• 全文大小：2353 K

文摘

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.