Detection of a nonerythropoietic erythropoietin, Neuro‐EPO, in blood after intranasal administration in rat

Abstract

Nonerythropoietic erythropoietins (EPOs) are investigated for their high antioxidant properties. A new drug candidate under clinical investigation to treat brain diseases is Neuro‐EPO, produced by selecting EPO isoforms with low sialic acid content. Intranasal administration allows to bypass the blood–brain barrier to get a fast and concentrated delivery to the brain. The aims of this project were to characterize Neuro‐EPO with anti‐doping methods used to detect conventional recombinant EPOs (isoelectric focusing [IEF] and sodium dodecyl sulfate–polyacrylamide gel electrophoresis [SDS‐PAGE]) and to evaluate the window of detection of Neuro‐EPO in brain and blood (plasma) after a single intranasal administration in rats. Neuro‐EPO drug analyzed by IEF‐PAGE presented a very basic profile completely detected only when using a 2–8 or 2–10 pH gradient instead of the conventional 2–6 pH gradient. Its profile consisted in six main bands that did not interfere with endogenous EPO profile from human or rat. After SDS‐PAGE, a broad band was detected for Neuro‐EPO in the same area as endogenous EPO, making Neuro‐EPO identification very difficult by this approach. Therefore, IEF was the method for identification chosen after administration in rats. Neuro‐EPO was clearly identified in blood 2 and 6 h after the delivery. Fainter signals were obtained between 12 and 48 h, but some characteristic very basic bands remained detectable. Surprisingly, brain extracts did not show the presence of Neuro‐EPO even 2 h after administration, indicating a fast degradation or elimination from the brain to the bloodstream. This experiment indicated that detection of Neuro‐EPO after intranasal delivery should be possible for a few days.

Erythropoietin and derivatives: Potential beneficial effects on the brain

“…The authors then proposed that the neuroprotective effect shown by nasal application of Neuro-EPO may block metabolic pathways that are affected in neurodegenerative diseases.A phase I study (Trial registration: Cuban Public Registry of Clinical Trials RPCEC00000157, June 10, 2013) for intranasal Neuro-EPO administration carried out in 25 healthy volunteers allowed to detect few and mild adverse effects that could be completely resolved and suggested safety of the treatment(Santos Morales et al, 2017), although the study needs to be expanded in terms of periods and doses applied. In a recent report, Neuro-EPO was characterized by immune-purification/concentration of Epo from tissues followed by electrophoresis techniques and specific Epo immunodetection(Martin et al, 2020). The very low sialic acid content at the end of the N-glycan protein structure agreed with the finding of a pattern consisting of five very basic isoforms that differed from endogenous and recombinant erythropoietins.…”