Low Sialic Acid Content Research Articles

NeuroEPO plus (NeuralCIM®) in mild-to-moderate Alzheimer’s clinical syndrome: the ATHENEA randomized clinical trial

BackgroundNeuroEPO plus is a recombinant human erythropoietin without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. NeuroEPO plus prevents oxidative damage, neuroinflammation, apoptosis and cognitive deficit in an Alzheimer’s disease (AD) models. The aim of this study was to assess efficacy and safety of neuroEPO plus.MethodsThis was a double-blind, randomized, placebo-controlled, phase 2–3 trial involving participants ≥ 50 years of age with mild-to-moderate AD clinical syndrome. Participants were randomized in a 1:1:1 ratio to receive 0.5 or 1.0 mg of neuroEPO plus or placebo intranasally 3 times/week for 48 weeks. The primary outcome was change in the 11-item cognitive subscale of the AD Assessment Scale (ADAS-Cog11) score from baseline to 48 weeks (range, 0 to 70; higher scores indicate greater impairment). Secondary outcomes included CIBIC+, GDS, MoCA, NPI, Activities of Daily Living Scales, cerebral perfusion, and hippocampal volume.ResultsA total of 174 participants were enrolled and 170 were treated (57 in neuroEPO plus 0.5 mg, 56 in neuroEPO plus 1.0 mg and 57 in placebo group). Mean age, 74.0 years; 121 (71.2%) women and 85% completed the trial. The median change in ADAS-Cog11 score at 48 weeks was −3.0 (95% CI, −4.3 to −1.7) in the 0.5 mg neuroEPO plus group, −4.0 (95% CI, −5.9 to −2.1) in the 1.0 mg neuroEPO plus group and 4.0 (95% CI, 1.9 to 6.1) in the placebo group. The difference of neuroEPO plus 0.5 mg vs. placebo was 7.0 points (95% CI, 4.5–9.5) P = 0.000 and between the neuroEPO plus 1.0 mg vs. placebo was 8.0 points (95% CI, 5.2–10.8) P = 0.000. NeuroEPO plus treatment induced a statistically significant improvement in some of clinical secondary outcomes vs. placebo including CIBIC+, GDS, MoCA, NPI, and the brain perfusion.ConclusionsAmong participants with mild-moderate Alzheimer’s disease clinical syndrome, neuroEPO plus improved the cognitive evaluation at 48 weeks, with a very good safety profile. Larger trials are warranted to determine the efficacy and safety of neuroEPO plus in Alzheimer’s disease.Trial registrationhttps://rpcec.sld.cu Identifier: RPCEC00000232.

The Effect of Neuroepo on Cognition in Parkinson's Disease Patients Is Mediated by Electroencephalogram Source Activity.

We report on the quantitative electroencephalogram (qEEG) and cognitive effects of Neuroepo in Parkinson’s disease (PD) from a double-blind safety trial (https://clinicaltrials.gov/, number NCT04110678). Neuroepo is a new erythropoietin (EPO) formulation with a low sialic acid content with satisfactory results in animal models and tolerance in healthy participants and PD patients. In this study, 26 PD patients were assigned randomly to Neuroepo (n = 15) or placebo (n = 11) groups to test the tolerance of the drug. Outcome variables were neuropsychological tests and resting-state source qEEG at baseline and 6 months after administering the drug. Probabilistic Canonical Correlation Analysis was used to extract latent variables for the cognitive and for qEEG variables that shared a common source of variance. We obtained canonical variates for Cognition and qEEG with a correlation of 0.97. Linear Mixed Model analysis showed significant positive dependence of the canonical variate cognition on the dose and the confounder educational level (p = 0.003 and p = 0.02, respectively). Additionally, in the mediation equation, we found a positive dependence of Cognition with qEEG for (p = < 0.0001) and with dose (p = 0.006). Despite the small sample, both tests were powered over 89%. A combined mediation model showed that 66% of the total effect of the cognitive improvement was mediated by qEEG (p = 0.0001), with the remaining direct effect between dose and Cognition (p = 0.002), due to other causes. These results suggest that Neuroepo has a positive influence on Cognition in PD patients and that a large portion of this effect is mediated by brain mechanisms reflected in qEEG.

Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis

NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [12C6]/[13C6] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS). A superior amount of low sialylated glycans and less branched structures were detected in NeuroEPO plus compare to other commercial rhEPOs. At the intact glycoprotein level, NeuroEPO plus glycoforms were separated by capillary zone electrophoresis with ultraviolet detection (CE-UV), optimizing the composition and pH of the separation electrolyte. Moreover, an isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) method was also optimized for the simultaneous analysis of this basic rhEPO and conventional acidic rhEPO products. The proposed glycomic and intact glycoprotein methods provide a robust and reliable analytical platform for NeuroEPO plus characterization and for its future implementation as biopharmaceutical in neurodegenerative diseases.

Correlative N-glycan and charge variant analysis of cetuximab expressed in murine, chinese hamster and human expression systems

A well-defined and controlled glycosylation pattern is important to maintain quality and safety of therapeutic proteins. Glycosylation is strongly dependent on the host cell line used for recombinant protein expression. Cetuximab, which is produced in mouse myeloma cells has been shown to harbour Fab glycans, which contain non-human like features and hence, can potentially cause an immunogenic response in patients. In light of the advent of biosimilar and biobetter development, we produced cetuximab variants in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. A combination of orthogonal chromatographic modes such as hydrophilic interaction, size exclusion and strong cation exchange chromatography with various detection strategies was employed to characterise the three different cetuximab variants and to compare the in-house produced HEK and CHO variants with the reference drug product. While Fc galactosylation and sialic acid content of the drug product and the HEK variant were highly similar, the CHO product showed lower galactosylation on Fc glycans and a comparatively low sialic acid content in the Fab region. The elevated high-mannose content of CHO cetuximab also suggests potential rapid clearence from circulation. The combination of multiple chromatographic separation modes has proven powerful for the characterisation of expression system dependent protein quality attributes such as N-glycosylation.

Short-term Tolerance of Nasally-administered NeuroEPO in Patients with Parkinson Disease.

No neuroprotective treatment has been able to successfully halt the progression of Parkinson disease or prevent development of associated complications. Recombinant erythropoetin (EPO), an erythropoiesis-stimulating agent originally indicated in anemia, produced and manufactured in Cuba (iorEPOCIM, CIMAB S.A, Havana, Cuba) has neuroprotective properties. NeuroEPO is a new nasal formulation of recombinant EPO with a low content of sialic acid and without hematopoietic effects. It has neuroprotective effects in animal models. Evaluate short-term tolerance of intranasal NeuroEPO in patients with Parkinson disease. As part of a monocentric randomized placebo-controlled double-blind study (registered at www.clinicaltrials.gov number NCT04110678), 26 patients with Parkinson disease (stages 1 and 2 on Hoehn & Yahr Scale), were randomly divided into two groups: NeuroEPO (n = 15) and placebo (n = 11), both treated intranasally either with the drug (1 mL, at a concentration of 1 mg/mL of NeuroEPO) or placebo once a week for 5 weeks. At each application, we recorded any adverse events and blood pressure. To assess potential hematopoietic effects of the drug, hematological and biochemical variables were evaluated one week before and one week after the intervention. There were no significant differences (p = 0.22) between the two groups in terms of frequency of adverse events (20.0% in NeuroEPO and 9.1% in placebo groups). Three patients in NeuroEPO presented nausea, and one vomited (possibly due to the patient's positioning during drug application). One patient in placebo group reported polyuria and nasal irritation. In both groups, the adverse events were mild, brief, required no treatment and did not present sequelae. Nasally administered NeuroEPO for five weeks in patients with Parkinson disease stages 1 and 2 on Hoehn & Yahr Scale is well tolerated.

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

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.

Presumable role of outer membrane proteins of Salmonella containing sialylated lipopolysaccharides serovar Ngozi, sv. Isaszeg and subspecies arizonae in determining susceptibility to human serum.

BackgroundThe O48 group comprises Salmonella bacteria containing sialic acid in the lipopolysaccharide (LPS). Bacteria with sialylated surface structures are described as pathogens that avoid immunological response of the host by making similar their surface antigens to the host’s tissues (molecular mimicry). It is known that the smooth-type LPS of Salmonella enterica and outer membrane proteins (OMP) PgtE, PagC and Rck mediate serum resistant phenotype by affecting complement system (C). The aim of this study was to investigate C3 component activation by Salmonella O48 LPS and OMP.FindingsIn the present study, we examined C3 component deposition on the three Salmonella O48 strains: S. enterica subspecies enterica serovar Ngozi, S. enterica subsp. enterica sv. Isaszeg, and S.enterica subsp. arizonae containing sialic acid in the O-specific part of LPS. The greatest C3 deposition occurred on Salmonella sv. Isaszeg cells (p < 0.005) as well as on their LPS (low content of sialic acid in LPS) (p < 0.05) after 45 min of incubation in 50% human serum. Weaker C3 deposition ratio on the Salmonella sv. Ngozi (high content of sialic acid in LPS) and Salmonella subsp. arizonae (high content of sialic acid in LPS) cells correlated with the lower C3 activation on their LPS. Immunoblotting revealed that OMP isolated from the tested strains also bound C3 protein fragments.ConclusionsWe suggest that activation of C3 serum protein is dependent on the sialic acid contents in the LPS as well as on the presence of OMP in the range of molecular masses of 35–48 kDa.

Modified (desialylated) low-density lipoprotein measured in serum by lectin-sorbent assay

Modified low-density lipoprotein (LDL) with a low sialic acid content was found in the blood of patients with coronary atherosclerosis. This desialylated lipoprotein causes lipid accumulation in arterial smooth-muscle cells and stimulates cell proliferation and production of the extracellular matrix, i.e., induces all atherogenic manifestations at the cellular level. We have developed a lectin-sorbent assay for the determination of desialylated LDL in sera. The assay is based on the binding of desialylated LDL by immobilized Ricinus communis agglutinin with subsequent measurement of lipoprotein through use of anti-apolipoprotein (apo) B antibody. The assay is sensitive to desialylated apo B concentrations as low as 5 micrograms/L. Comparison between the lectin-sorbent assay and a lectin chromatographic technique showed a good correlation. This determination of modified desialylated LDL in human serum with high accuracy and reproducibility may help establish the diagnostic value of this lipoprotein as a risk factor of atherosclerosis.

Developing the IVIG biomimetic, hexa-Fc, for drug and vaccine applications.

The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements. Using quality-by-design (QbD) approaches, we generated hexameric-Fc (hexa-Fc), a ~20 nm oligomeric Fc-based scaffold that we here show binds low-affinity inhibitory receptors (FcRL5, FcγRIIb, and DC-SIGN) with high avidity and specificity, whilst eliminating significant clinical limitations of monomeric Fc-fusions for vaccine and/or cancer therapies, in particular their poor ability to activate complement. Mass spectroscopy of hexa-Fc reveals high-mannose, low-sialic acid content, suggesting that interactions with these receptors are influenced by the mannose-containing Fc. Molecular dynamics (MD) simulations provides insight into the mechanisms of hexa-Fc interaction with these receptors and reveals an unexpected orientation of high-mannose glycans on the human Fc that provides greater accessibility to potential binding partners. Finally, we show that this biosynthetic nanoparticle can be engineered to enhance interactions with the human neonatal Fc receptor (FcRn) without loss of the oligomeric structure, a crucial modification for these molecules in therapy and/or vaccine strategies where a long plasma half-life is critical.

Low density lipoprotein-containing circulating immune complexes: role in atherosclerosis and diagnostic value.

It has been suggested that low density lipoprotein-containing circulating immune complexes (LDL-CIC) play a role in atherogenesis and are involved in the formation of early atherosclerotic lesion. These complexes, as well as anti-LDL autoantibodies, have been found in the blood and in the atherosclerotic lesions of patients with different cardiovascular diseases, as well as in the blood of animals with experimental atherosclerosis. It can be suggested that the presence of anti-LDL antibodies in the blood is a result of immune response induced by lipoprotein modification. LDL-CIC differs from native LDL in many aspects. It has much lower sialic acid content, smaller diameter, and higher density and is more electronegative than native LDL. Fraction of LDL-CICs is fundamental to the serum atherogenicity manifested at the cellular level. LDL-CIC, unlike native LDL, is able to induce intracellular accumulation of neutral lipids, especially esterified cholesterol, in cells cultured from uninvolved human aortic intima and in macrophage cultures. After removal of LDL-CIC, the CHD patient's sera lose their atherogenic properties. Titer of LDL-CIC in blood serum significantly correlates with progression of atherosclerosis in human in vivo and has the highest diagnostic value among other measured serum lipid parameters. Elevated CIC-cholesterol might well be a possible risk factor of coronary atherosclerosis.

Recombinant Human Factor IX Produced from Transgenic Porcine Milk

Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

The Impact of Sialic Acids on the Pharmacokinetics of a PEGylated Erythropoietin

Erythropoietin (EPO) is an important molecule in the erythropoiesis and various forms of EPO have been marketed in managing anemia in humans. Long acting EPOs for less frequent dosing have been generated either by increasing the number of glycosylation sites of the EPO molecule or by linking it to a polyethylene glycol (PEG). We have generated recombinant human EPO (rhEPO) using glycoengineered Pichia pastoris strains and evaluated the pharmacokinetics (PK) in rats of this molecule linked to a 40 kDa PEG (PEGylated rhEPO), in relation to its glycosylation patterns. As expected, the PEGylated rhEPO exhibited a significant improvement in half-life of serum when compared with the non-PEGylated version. Interestingly, the PK properties of the PEGylated rhEPO molecule were also significantly influenced by the glycosylation profile. Specifically, PEGylated rhEPO with a significantly higher sialic acid content in the biantennary structure (high A2) exhibited lower systemic clearance and higher systemic exposure than those with a lower sialic acid content (low A2) following either intravenous or subcutaneous administrations. These results suggest that A2 content may be one of the important criteria for release in manufacturing PEGylated rhEPO to ensure consistent PK.

Short-Term Intra-Nasal Erythropoietin Administration with Low Sialic Acid Content is without Toxicity or Erythropoietic Effects

The objective of this investigation was to assess the toxicological potential of nasal formulation of erythropoietin with low sialic acid content (Neuro EPO) after 28 days of intra-nasal dosing in rats besides to evaluate the immunogenicity and erythropoietic effect of the test substance. Healthy Wistar rats of both sexes were used for 28 days subacute toxicity and immunogenicity assays. Doses evaluated were 3450, 4830 and 6900 UI/kg/day. The toxicological endpoints examined included animal body weight, food consumption, hematological and biochemical patterns, antibodies determination, selected tissue weights and histopathological examination. Reversibility of toxic effects was evaluated at high dose 14 days after treatment period. Female B6D2F1 mice were used for evaluated erythropoietic effect of the nasal formulation. Hematological endpoints were examined every week during 28 days of intra-nasal dosing of 6900 UI/kg/day. Variations of hematological patterns were not observed after 28 days of intranasal dosing. A slight increase in glucose level of treated animals within the normal range was observed. This effect was not dose related and was reversible. Antibody formation was not observed in any of the test doses. Histopathological examination of organs and tissues did not reveal treatment induced changes. The administration of Neuro EPO in normocythaemic mice did not produce erythropoietic effect. These results suggest that Neuro EPO could be used as a neuroprotective agent, without significant systemic haematological side effects.

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

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

Absence of hematological side effects in acute and subacute nasal dosing of erythropoietin with a low content of sialic acid

The use of human recombinant erythropoietin (EPO) as a neuroprotective agent is limited due to its hematological side effects. An erythropoietin along with a low content of sialic acid (rhEPO b), similar to that produced in the brain during hypoxia, may be used as a neuroprotective agent without risk of thrombotic events. The objective of this investigation was to assess the toxicological potential of a nasal formulation with rhEPO b in acute, subacute and nasal irritation assays in rats. Healthy Wistar rats (Cenp:Wistar) were used for the assays. In an irritation test, animals received 15 μl of rhEPO b into the right nostril. Rats were sacrificed after 24 h and slides of the nasal mucosa tissues were examined. Control and treated groups showed signs of a minimal irritation consisting of week edema and vascular congestion in all animals. In the acute toxicity test, the dose of 47,143 UI/kg was administered by nasal route. Hematological patterns, body weight, relative organ weight, and organ integrity were not affected by single dosing with rhEPO b. In the subacute toxicity test, Wistar rats of both sexes received 6,600 UI/kg/day for 14 days. The toxicological endpoints examined included animal body weight, food consumption, hematological and biochemical patterns, selected tissue weights, and histopathological examinations. An increase of lymphocytes was observed in males that was considered to reflect an immune response to treatment. Histopathological examination of organs and tissues did not reveal treatment-induced changes. The administration of rhEPO b at daily doses of 6,600 UI/kg during 14 days did not produce hematological side effects. These results suggest that rhEPO b could offer the same neuroprotection as EPO, without hematological side effects.

Effect observed after 28 days nasal dosing of erythropoietin with a low content of sialic acid

Effect observed after 28 days nasal dosing of erythropoietin with a low content of sialic acid

Treatment with Nasal Neuro-EPO Improves the Neurological, Cognitive, and Histological State in a Gerbil Model of Focal Ischemia

Vascular illness of the brain constitutes the third cause of death and the first cause of disability in Cuba and many other countries. Presently, no medication has been registered as a neuroprotector. Neuroprotection with intranasal Neuro-EPO (EPO, erythropoietin) has emerged as a multifunctional therapy that plays a significant role in neural survival and functional recovery in an animal model of stroke. On the other hand, there is limited access to the brain through the blood brain barrier (BBB) for intravenously applied EPO, and the high EPO dosages needed to obtain a protective effect increase the danger of elevated hematocrit levels and practically exclude chronic or subchronic treatment with EPO. A promising approach has been recently developed with a nonerythropoietic variant of EPO, Neuro-EPO, with low sialic acid content, a very short plasma half-life, and without erythropoietic activity, probably similar to endogenous brain EPO. The objective of this work was to determine the neuroprotective effect of intranasal Neuro-EPO in comparison with the human recombinant EPO injected intraperitoneally in the acute phase of cerebral ischemia, employing the common carotid artery occlusion model in gerbils. Neuro-EPO has demonstrated a better neuroprotective effect, evidenced through increased viability, improvements of the neurological state and cognitive functions, as well as protection of the CA3 region of the hippocampus, temporal cortex, and the thalamus. In conclusion, the intranasal application of Neuro-EPO has a better neuroprotective effect than intraperitoneal EPO, evidenced by the significant improvement of neurological, cognitive, and histological status in the animal model of stroke employed.

Nonhematopoietic Variants of Erythropoietin in Ischemic Stroke: Need for Step-Wise Proof-of-Concept Studies

Neuroprotective, but not hematopoietic, variants of erythropoietin (EPO), such as Neuro-EPO, are promising candidates for treatment in the acute and subacute stroke phase. Characterized by its low sialic acid content and therefore exhibiting a very short plasma half-life, Neuro-EPO can probably not be administered systemically via the blood. As such, alternate routes of delivery are required. In their paper that now appears in TheScientificWorldJOURNAL, Rodríguez Cruz and colleagues provide evidence that Neuro-EPO promotes neurological recovery in the ischemic gerbil brain in a way that is similarly potent, if not superior, to systemically administered EPO. In view of the potential clinical use of Neuro-EPO, stringent proof-of-concept studies are urgently needed to define (1) how intranasally delivered Neuro-EPO reaches the brain, (2) which concentrations are achieved in the ischemic and nonischemic brain tissue of rodents and nonhuman primates, and (3) which are the mechanisms via which Neuro-EPO protects from injury. Only with such information should decisions be made whether intranasal Neuro-EPO may be evaluated in human patients.

Crude urinary human chorionic gonadotropin contains variant forms of HCG with low sialic acid content that exhibit an increased thyrotropic activity in CHO cells expressing the human TSH receptor.

Hyperthyroidism occurs in association with pregnancy or trophoblastic tumours. This is due to the secretion of thyroid stimulators by trophoblastic cells, most likely hCG or a variant form of hCG. In the present studies we sought to identify hCG variants with enhanced thyrotropic activity contained in crude hCG extract from pregnancy urine (hCGc). Such studies seem now feasible, because highly sensitive assays employing CHO cells transfected with the recombinant human TSH receptor recently became available. Initially, we found the activity of hCGc to both inhibit the binding of 125I-bTSH to CHO-TSHr cells and to stimulate the cAMP release by the cells to be increased, compared to highly purified hCG (hCGp), which was tested in comparable immunological concentrations. We then processed hCGc on a DEAE-52 anionexchange column to separate materials of interest, termed hCGv, from hCGp. HCGv was further purified by gel chromatography, and found to be enriched in terms of both, its holo-hCG immunoactivity and its TSH binding inhibiting activity, compared to hCGc where it was derived from. It also proved more potent than hCGp to bind to recombinant hTSH receptor and to stimulate adenylate cyclase activity in CHO-TSHr cells. Enzymatic desialylation was able to increase the potency of both hCGv and hCGp, and rendered the two desialylated hCG forms nearly equipotent. Isoelectric focusing and direct measurement of sialic acid contents revealed hCGv to be less sialylated than hCGp.(ABSTRACT TRUNCATED AT 250 WORDS)

The Nasal Route as a Potential Pathway for Delivery of Erythropoietin in the Treatment of Acute Ischemic Stroke in Humans

Intranasal delivery provides a practical, noninvasive method of bypassing the blood-brain barrier (BBB) in order to deliver therapeutic agents to the brain. This method allows drugs that do not cross the BBB to be delivered to the central nervous system in a few minutes. With this technology, it will be possible to eliminate systemic administration and its potential side effects. Using the intranasal delivery system, researchers have demonstrated neuroprotective effects in different animal models of stroke using erythropoietin (EPO) as a neuroprotector or other different types of EPO without erythropoiesis-stimulating activity. These new molecules retain their ability to protect neural tissue against injury and they include Asialoerythropoietin (asialoEPO) carbamylated EPO (CEPO), and rHu-EPO with low sialic acid content (Neuro-EPO). Contrary to the other EPO variants, Neuro-EPO is not chemically modified, making it biologically similar to endogenous EPO, with the advantage of less adverse reactions when this molecule is applied chronically. This constitutes a potential benefit of Neuro-EPO over other variants of EPO for the chronic treatment of neurodegenerative illnesses. Nasal administration of EPO is a potential, novel, neurotherapeutic approach. However, it will be necessary to initiate clinical trials in stroke patients using intranasal delivery in order to obtain the clinical evidence of its neuroprotectant capacity in the treatment of patients with acute stroke and other neurodegenerative disorders. This new therapeutic approach could revolutionize the treatment of neurodegenerative disorders in the 21 century.