Neuroepoetin: novel combination of basic erythropoietin glycoforms with in vitro neuroprotective activity

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

Banff, Alberta, Canada

Congreso; Cell Culture Engineering XII; 2010

Engineering Conferences International

Erythropoietin (EPO) is a highly glycosylated protein which was first characterized as an hematopoietic growth factor and that has been clinically used for decades for the treatment of anemia. However, the observation that EPO and its receptor are expressed in the central nervous system has led to the discovery of a neuroprotective activity of the hormone. Nonetheless, EPO hematopoietic activity in the treatment of neurologic pathologies may result in undesired side effects, such as polycythaemia, arterial hypertension and thrombotic phenomena. To circumvent this problem, a novel combination of EPO glycoforms, named as neuroepoetin (rhNEPO), was obtained through an alternative purification of the recombinant hormone produced in CHO cells. Particularly, a fraction that is currently discarded during the purification process of the hematopoietic rhEPO was subjected to two additional ion-exchange chromatographic steps in order to get a variant with a more basic isoform composition (pI range 4.2-6.1 in isoelectric focusing assays) and less average sialic acid content (6.9 moles of sialic acid per mol of protein compared to 11.7 corresponding to rhEPO, as determined by the periodate-resorcinol method). Evaluation of in vivo biological activity in normocytemic mice demonstrated a highly reduced erythropoietic activity of rhNEPO (6,200 UI/mg compared to 132,000 UI/mg corresponding to rhEPO). Contrarily, in vitro activity of neuroepoetin using UT-7 cells was comparable to that of rhEPO, indicating that the binding affinity towards the receptor is not altered. Neuroprotective action of both molecules was evaluated in rat PC-12 cells differentiated to neural phenotype with NGF, and human neuroblastoma SH-SY5Y cells subjected to different apoptotic stimuli. In this experiments, rhNEPO showed a higher antiapoptotic activity on PC-12 cells compared to rhEPO after serum and NGF withdrawal, as evidenced by TUNEL (60% and 25% of protection, respectively). Besides, both EPO variants evidenced a significant neuroprotective action on SH-SY5Y cells treated with 25 nM staurosporine, without significant differences among them. Pharmacokinetic analysis showed an increased apparent clearance of rhNEPO with respect to rhEPO after intraperitoneal inoculation in rats (1.84 ± 0.16 versus 0.14 ± 0.01 ml.h-1). However, rhNEPO was rapidly absorbed and detected after 1 h in cerebrospinal fluid, while rhEPO was not detected until 4 h after injection, although it was eliminated more slowly. Taken together, these results encourage the study of neuroepoetin as a potential drug for the treatment of neurological diseases, in which a highly neuroprotective drug with low side effects and rapid passage through blood-brain-barrier is desirable.