Neuroprotective activity of a new erythropoietin formulation with increased penetration in the central nervous system

ETCHEVERRIGARAY, MARINA; CEAGLIO, NATALIA; MATTIO, MÓNICA; OGGERO EBERHARDT, MARCOS; AMADEO, GABRIEL IGNACIO; FORNO, ÁNGELA GUILLERMINA; PEROTTI, NORMA; KRATJE, RICARDO

Viena

Congreso; 22nd ESACT Meeting; 2011

European Society for Animal Cell Technology

<!-- /* Font Definitions */ @font-face {font-family:"MS Mincho"; panose-1:2 2 6 9 4 2 5 8 3 4; mso-font-alt:"ＭＳ 明朝"; mso-font-charset:128; mso-generic-font-family:roman; mso-font-format:other; mso-font-pitch:fixed; mso-font-signature:1 134676480 16 0 131072 0;} @font-face {font-family:"@MS Mincho"; panose-1:2 2 6 9 4 2 5 8 3 4; mso-font-charset:128; mso-generic-font-family:roman; mso-font-format:other; mso-font-pitch:fixed; mso-font-signature:1 134676480 16 0 131072 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; mso-hyphenate:none; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"MS Mincho"; mso-fareast-language:AR-SA;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Apart from its hematopoietic effect, erythropoietin (EPO) is a molecule with high neuroprotective potential. However, its prolonged application may cause serious adverse effects due to the erythropoiesis stimulation. Therefore, it is necessary to develop EPO derivates with neuroprotective properties but low hematopoietic activity. For this purpose we developed an alternative purification process for the recombinant hormone produced in CHO cells (rhEPO). The process was designed to obtain a combination of EPO glycoforms called neuroepoetin (rhNEPO). rhNEPO isoforms composition is less acidic (pI 4.2-6.1) and has lower sialic acid content (8.0 ± 1.0 mol/mol protein). This resulted in a very low in vivo erythropoietic activity in normocytemic mice (5,034 ± 2,166 IU/mg vs. 131,976 ± 52,324 IU/mg in the case of rhEPO). Despite this, no changes were observed in the affinity with its receptor in in vitro UT-7 cells assays. Additionally, we evaluated the neuroprotective action of both molecules in SH-SY5Y and PC‑12 cells (their neural phenotype differentiated with NGF) by subjecting them to different apoptotic stimuli. In proliferation assays using SH-SY5Y cells both variations showed similar neuroprotective action. On the other hand, rhNEPO revealed higher anti-apoptotic activity (evaluated using TUNEL technique) in PC-12 cells. When an intravenous pharmacokinetic study was performed, rhNEPO showed higher clearance than rhEPO (31.6 ± 1.9 ml.h-1 and 3.3 ± 0.3 ml.h-1, respectively).  However, rhNEPO was detected early in cerebrospinal fluid after 5 minutes of injection, while rhEPO was detected only after 30 minutes.These results encourage the study of rhNEPO as a potential drug for the treatment of neurological diseases due to its negligible hematopoietic activity, its neuroprotective capacity and its fast passage through the blood-brain barrier.