P.1.g.031 The neuroprotective and anti-amnestic effects of carbamylated erythropoietin derivates in ischemic brain injury

Abstract

Purpose: To compare the neuroprotection of carbamylated erythropoietin (CEPO) and EPO fusion protein containing TR domain from glycoprotein MUC1 (CEPO-TR), and EPO fusion protein with modified Fc fragments of IgG1 (CEPO-Fc) against ischemic brain injury, including behavioral disturbances in a bilateral focal ischemic infarction of the rat prefrontal cortex. Methods: The carbamylated erythropoietin and erythropoietin derivates were produced by treatment of purified proteins with potassium cyanate in borate buffer. The resulting carbamylated erythropoietin and derivates exhibit no erythropoietic activity in UT-7/EPOR cell viability assay. Bilateral focal ischemic infarction of the prefrontal cortex (areas Frl and Fr2) was induced by the method of photochemical thrombosis in rats. After passive avoidance training and testing rats were treated, respectively, with following regimens: saline, 50mg/kg CEPO, 50mg/kg CEPO-TR, 50mg/kg CEPO-Fc. The substance was injected intraperitoneally in 1 h after operation. Neurological deficit scores and infarct volume were assessed at 4 and 7 days after operation. Functional state of CNS was determined by conditioned passive avoidance response (PA), i.e. by the latency of transition from light compartment to dark compartment (in sec). Morphometric measurements of the areas and volumes of the ischemic focus on serial slices were carried out on animal brain fixed by plunging into formalinethanol-acetic acid mixture (2:7:1). The data were statistically processed using Statistica 6.0 software. Summary of results: Additional oligosaccharides, linked to the erythropoietin, prolong its half-life and increase bioactivity in vivo. For this purpose we, for the first time, used TR domain from glycoprotein MUC1, bearing 5 additional sites for O-glycosylation. Our result indicated that ligation of TR-domain to the coding sequence of EPO did not affect secretion of the chimeric protein into the medium, receptor binding affinity in vitro bioactivity, compared with EPO wild type. However, both the in vitro potency and half-life in circulation of EPO bearing TR or Fc fragments were significantly enhanced. After photothrombosis and injection of saline, the latency of entry into the dark compartment decreased from 300 s to 76 s. Treatment of rats with CEPO, CEPOTR, and CEPO-Fc after photothrombosis restored latency of PA to 243, 258, 227 s, respectively, on day 4. Only injection of CEPOTR retains passive avoidance latency 207 s on the day 7. Ischemic damage volume in animals treated with CEPO-TR on 7th day after operation was 15.8±5.4 against 24.22±4.7 with control injection of saline (P< 0.05). This phenomenon on 7th day was observed only with CEPO-TR. Protection efficiency coefficient, calculated from these experiments represented 34%. Another erythropoietin derivate did not give statistically significant results. Conclusion: Treatment of rats with CEPO and CEPO fusion proteins after photothrombosis of cortex resulted in restoration of passive avoidance response and diminishing of volume of ischemic damage. Those, our study indicates that CEPO-Fc and CEPO-TR display neuroprotectionand anti-amnestic activity, while CEPO-TR demonstrates prolongation ability.