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

Abstract

BackgroundApart from its hematopoietic effect, erythropoietin(EPO) is a molecule with high neuroprotective potential.However, its prolonged application may cause seriousadverse effects due to the erythropoiesis stimulation.Therefore, an EPO derivative with neuroprotective prop-erties but low hematopoietic activity, designated as neu-ropoietin (rhNEPO), was developed in our lab using analternative purification process of the recombinanthuman erythropoietic counterpart (rhEPO) produced inCHO cells [1]. The in vitro cytoprotective activity ofrhNEPO on neural phenotype cells and its brain uptakefrom blood are herein analyzed.ResultsIn vitro citoprotective activity of rhNEPO was analyzedon rat pheochromocytoma cells (PC-12) differentiated toneural phenotype with neural growth factor (NGF).Apoptosis was triggered by NGF and serum withdrawalfrom cell cultures. Thus, nuclear DNA fragmentationwas analyzed by colorimetric TUNEL detection. One-way analysis of variance was carried out followed byDunnett´s multiple comparison test. Probabilities lowerthan 0.05 were considered significant (p 0.05), confirming their prop-erties to protect PC-12 cells from apoptosis. Therefore,this novel combination of erythropoietin glycoforms(rhNEPO) with lower sialic acid content and antennaritythan rhEPO [1] preserved its binding receptor capacityexerting an in vitro neuroprotective activity even betterthan the mentioned counterpart. Also, rhAEPO showedan in vitro activity that is similar to that of rhNEPO,having both derivatives the lowest content of carbohy-drates. It is well known that the affinity of EPO analo-gues for EPO receptor is inversely related to thesialylation of their attached carbohydrate [2] and thatremoval of sialic acid turns it into a molecule with avery short half-life with almost no erythropoietic activ-ity. This is the case of rhAEPO that explains its rapidhepatic clearance from blood [3]. For that reason,rhNEPO emerges as a neuroprotective candidate dis-playing higher in vitro activity than rhEPO.Taking into account the cytoprotective activity ofrhNEPO on neural phenotype cells, cerebrospinal fluid(CSF) and blood pharmacokinetics of rhEPO andrhNEPO were evaluated in rats following intravenousadministration of a single dose of each protein, aimingto evaluate their CSF uptake from plasma.The distribution and the elimination half-lives ofrhNEPO in blood were significantly shorter than thecorresponding ones for rhEPO. Differences in the sialic