Erythropoietin Research Articles

Overview of preclinical and phase II clinical studies on Pegmolesatide’s long-term erythropoiesis stimulating effect via EPOR-mediated signal transduction

IntroductionAnemia is a prevalent complication of chronic kidney disease (CKD), primarily due to insufficient erythropoietin (EPO). Pegmolesatide (by Hansoh Pharma) is currently the only marketed long-acting EPO mimetic peptide (EMP) for the treatment of anemia in both dialysis and non-dialysis CKD patients. This paper aimed to explore the long-acting erythropoiesis stimulating molecular mechanism of Pegmolesatide.MethodsIn vitro assays were utilized to assess Pegmolesatide erythropoietin receptor (EPOR) affinity, competitive binding, cell proliferation/survival, apoptosis, cell surface receptor expression, and signal transduction. Pharmacokinetics (PK) and Pharmacodynamics (PD) parameters were evaluated in BALB/c mice following single administration. Furthermore, two Phase II clinical trials in dialysis and non-dialysis chronic kidney disease (CKD) patients with anemia, respectively CTR20140533 and CTR20140539, assessed PK-PD and safety following repeated administration.ResultsIn vitro Pegmolesatide demonstrated enhanced binding stability and prolonged residency at EPOR, surpassing erythropoiesis-stimulating agents (ESAs) rHuEPO and Darbepoetin. This sustained EPOR binding facilitated heightened endogenous EPOR expression post-drug withdrawal, maintaining downstream signal transduction pathways (JAK2/STAT5, ERK1/2 MAPK) for erythropoiesis. Pegmolesatide promoted UT-7 cell proliferation & survival and suppressed apoptosis. Following a single 0.08 mg/kg dose of Pegmolesatide in BALB/c mice, reticulocyte count, red blood cells, hemoglobin, and hematocrit persisted at elevated levels 4-6 days after administration. In the two clinical Phase II studies dose-dependent increases in hemoglobin and prolonged response duration were independently observed. Pegmolesatide showed significant PK-PD dual prolongation effects and was well tolerated. Adverse events were mild and manageable, with no reports of severe anaphylaxis.DiscussionPreclinical and clinical evidence signifies that Pegmolesatide is a unique, potent PEGylated EPO-memetic peptide (EMP) with a prolonged PD efficacy and PK half-life and a good safety-tolerability profile. To elucidate further, future studies will address the endocytosis, intracellular degradation, and ligand release of EPOR subsequent to Pegmolesatide binding, thereby supplementing our understanding of the molecular mechanism at play.Trial registration: Phase IIa clinical study of Pegmolesatide on renal anemia, CTR20140533. Initial Public Notice 14 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml. Phase II clinical study of Pegmolesatide on renal anemia, CTR20140539. Initial Public Notice 26 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml.

Induction of erythropoietin by dietary medium-chain triacylglycerol in humans.

Erythropoietin (EPO) is pivotal in regulating red blood cell (erythrocyte) concentrations and is primarily synthesized in the kidney. Recent research has unveiled a possible link between elevated circulating concentrations of ketone bodies (KB) and circulating EPO concentrations; however, it is not known whether nutritionally induced endogenous ketogenesis can be a stimulus to induce EPO in humans. Therefore, this study aimed to assess whether acute and chronic intake of medium-chain fatty acid-containing triacylglycerol (MCT), which rapidly enhances endogenous circulating KB, would elevate circulating EPO concentrations in humans, as indicated by prior work with exogenous KB administration. The study followed a crossover design involving 16 young men undergoing two 8-day MCT or energy-matched long-chain fatty acid-containing triacylglycerol (LCT) interventions in a randomized order. Five-hour test days were performed before and after each intervention, in which circulating KB and EPO concentrations as well as hematological parameters were assessed. Acute intake of MCT yielded a 222% sustained 5-h elevation in KB concentrations compared with LCT-with notable peak values of 0.7 ± 0.1 mmol·L-1 (312% above basal values). Remarkably, within just 8 days of daily MCT intake an impressive 38% increase in basal, fasting plasma EPO concentrations (7.19 ± 1.14 to 9.91 ± 1.25 mIU·mL-1) was demonstrated. In conclusion, this study unveils a novel physiological stimulus of circulating EPO concentrations in humans, potentially offering a new dietary approach to counter anemia in cardiovascular diseases.NEW & NOTEWORTHY This study is the first to assess the effects of nutritionally induced ketogenesis by acute and subchronic intake of medium-chain fatty acids on plasma erythropoietin concentrations. Medium-chain fatty acid intake increases postprandial ketone body concentrations and within only 8 days of daily intake substantially enhances basal plasma erythropoietin concentrations in young men. We therefore reveal a dietary stimulus of endogenous circulating erythropoietin concentrations in humans, with the potential to counter anemia in cardiovascular diseases.

Differential protein and mRNA cargo loading into engineered large and small extracellular vesicles reveals differences in in vitro and in vivo assays.

Extracellular vesicles (EV) represent an advanced platform for genetic material and protein delivery, particularly when they are loaded through the so-called endogenous loading method. This study investigates the differences between large EV (lEV) and small EV (sEV) obtained from genetically engineered C2C12 myoblasts overexpressing two different model biomolecules. Erythropoietin (EPO) is a secretory protein with anti-inflammatory, angiogenic and hematopoietic effects, while TGL is a chimeric cytosolic protein containing green fluorescent protein (GFP) and luciferase, used for imaging. We compared these EV subtypes in terms of protein and nucleic acid loading, intercellular cargo transfer capacity, and subsequent functional effects both in vitro and in vivo. Our results demonstrated that lEV exhibited higher protein and mRNA cargo content than sEV, which also translated into increased intercellular cargo transfer capacity, even when dosing according to the constitutive sEV and lEV secretion ratio (10,1). Indeed, we showed that, although receptor cells successfully internalized both EV subtypes, cells treated with lEV displayed stronger intracellular luciferase signals and higher EPO protein secretion compared to those treated with sEV. In terms of functional effects, both EV subtypes exerted anti-inflammatory and antioxidant effects in lipopolysaccharide-activated macrophages, as well as angiogenic effects in human umbilical vein endothelial cells. Finally, in vivo studies evidenced that subcutaneously administered lEV led to a more significant increase in hematocrit levels and red blood cell counts than sEV. Taken together, these findings suggest that the protein and mRNA cargo differ between endogenously loaded EV subtypes, and that this variation in cargo loading leads to differences in their functional outcomes. Therefore, the choice of EV subtype could be critical for optimizing EV-based delivery strategies for biologic drugs.

Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis

Blood transfusion plays a vital role in modern medicine, but frequent shortages occur. Ex vivo manufacturing of red blood cells (RBCs) from universal donor cells offers a potential solution, yet the high cost of recombinant cytokines remains a barrier. Erythropoietin (EPO) signaling is crucial for RBC development, and EPO is among the most expensive media components. To address this challenge, we develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) using design-build-test cycles and genome editing. By integrating synEPOR at the endogenous EPOR locus in O-negative induced pluripotent stem cells, we achieve equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production using small molecules compared to EPO-supplemented cultures. This approach dramatically reduces culture media costs. Our strategy not only addresses RBC production challenges but also demonstrates how protein and genome engineering can introduce precisely regulated cellular behaviors, potentially improving scalable manufacturing of a wide range of clinically relevant cell types.

Bacopa monnieri Extract Diminish Hypoxia-Induced Anxiety by Regulating HIF-1α Signaling and Enhancing the Antioxidant Defense System in Hippocampus.

Hypoxia is a significant stressor, and stabilized hypoxia-inducible factor-1α (HIF-1α) regulates the expression of numerous genes, leading to various biochemical, molecular, physiological and genomic changes. The body's oxygen-sensing system activates gene expression to protect brain tissues from hypoxia. Gamma-aminobutyric acid, an inhibitory neurotransmitter, regulates brain excitability during hypoxia through the activation of HIF-1 α. Herbal medicines have been widely used for managing various toxicological effects and disorders including hypoxia; however, the data on safety, efficacy and the molecular mechanisms that increase vulnerability or lethality against hypoxia are still lacking and urgently need to be investigated. The Current study aims to investigate how Bacopa monnieri extract (BME), specially CDRI-08 affects the hippocampus of mice subjected to conditions that simulate hypoxia. The pre and co-treatment of mice involved administrating BME (200mg/kg BW) for 14days, followed by exposure to CoCl2 (40mg/kg BW). BME decreased the levels of reactive oxygen species (ROS) and lipid peroxidation, while it increased the Gamma-aminobutyric acid receptor subunit-ɑ1 (GABAAR-ɑ1) level as well as the activity of antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). Furthermore BME reduced the levels of HIF-1α and its downstream targets glucose transporter-1 (GLUT-1) and erythropoietin (EPO) in the DG, CA1, and CA3 regions of hippocampus. Additionally, results obtained from the open field, elevated zero maze and plus maze tests indicate that BME restores anxiety caused by hypoxia. Together, these findings suggested that BME mitigates the harmful effects of oxidative stress and altered hypoxia related signaling in hippocampus; and may provide a basis for its therapeutic use in the recovery from hypoxia-led anxiety.

Blood management protocol for baseline anemic patients undergoing hip arthroplasty.

Patients undergoing total hip arthroplasty (THA) with preoperative anemia are at higher risk for transfusion. Blood-conserving interventions can reduce perioperative transfusions. This retrospective study evaluates the efficacy and safety of a patient blood management (PBM) protocol in elective primary THA patients with preoperative anemia. We analyzed data from a prospectively collected database of THAs performed consecutively from January 2013 to October 2023. The patients were grouped based on baseline hemoglobin (Hb) levels, as follows: <12g/dL (group 1) and ≥ 12g/dL (group 2). The PBM protocol included optimized preoperative epoetin (EPO) for Hb < 11g/dL, bleeding reduction measures, and restrictive transfusion thresholds. Blood loss was measured using a bleeding index (BI-7), accounting for Hb decrease from admission to the 7th postoperative day and total transfused units. Multiple linear regression was used to assess the differences in BI-7. Of the 1,442 patients, 104 (7%) had Hb < 12g/dL (group 1). Among these, 46 (45%) received EPO, with none requiring transfusion in the first week. The mean adjusted BI-7 was 2.3g/dL (95% CI, 2.1 to 2.6) in group 1 and 2.7g/dL (95% CI, 2.6 to 2.7) in group 2, showing a significant difference (-0.4g/dL; 95% CI, -0.6 to -0.1; p < 0.001). THA patients with baseline Hb < 12g/dL had a zero-transfusion rate and significantly lower blood loss than those with Hb ≥ 12g/dL. Optimizing preoperative Hb levels above 12 g/dL is not necessary for patients undergoing standard THA. Optimizing preoperative Hb may not be necessary for patients with baseline anemia due to their low blood loss. The PBM protocol was effective in conserving blood, time, and resources, offering a viable alternative for blood conservation in elective hip arthroplasty.

Erythropoietin decreases apoptosis and promotes Schwann cell repair and phagocytosis following nerve crush injury in mice.

After peripheral nerve trauma, insufficient clearance of phagocytic debris significantly hinders nerve regeneration. Without sufficient myelin debris clearance, Schwann cells (SCs) undergo increased apoptosis, impairing functional recovery. There is no treatment for peripheral nerve crush injury (PNCI). Erythropoietin (EPO) is an FDA-approved drug for anemia, which may help in the treatment of PNCI by transdifferentiating resident SCs into repair SCs (rSCs) and enhancing phagocytosis to facilitate the removal of cellular debris. For the first time, we conducted bulk RNA sequencing on mice with calibrated sciatic nerve crush injuries (SNCIs) on days 3, 5, and 7 post-SNCI to uncover transcriptomic changes with and without EPO treatment. We found EPO altered several biological pathways and associated genes, particularly those involved in cell apoptosis, differentiation, proliferation, phagocytosis, myelination, and neurogenesis. We validated the effects of EPO on SNCI on early (days 3/5) and intermediate (day 7) post-SNCI, and found EPO treatment reduced apoptosis (TUNEL), and enhanced SC repair (c-Jun and p75-NTR), proliferation (Ki67), and the phagocytosis of myelin debris by rSCs at crush injury sites. This improvement corresponded with an enhanced sciatic functional index (SFI). We also confirmed these findings in-vitro . EPO significantly enhanced SC repair during early de-differentiation, marked by high c-Jun and p75-NTR protein levels, and later re-differentiation with high EGR2 and low c-Jun and p75-NTR levels. These changes occurred under lipopolysaccharide (LPS) stress at 24 and 72h, respectively, compared to LPS treatment alone. Under LPS stress, EPO also significantly increased rSCs proliferation and phagocytosis of myelin or dead SCs. In conclusion, our findings support EPO may enhance the function of rSCs in debris clearance as a basis for its possible use in treating nerve trauma.

Ras Guanine Nucleotide-Releasing Protein-4 Inhibits Erythropoietin Production in Diabetic Mice with Kidney Disease by Degrading HIF2A.

In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination-proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

Influence of Functional Variations in Genes of Neurotrophins and Neurotransmitter Systems on the Development of Retinopathy of Prematurity

Retinal neurodevelopment, vascularization, homeostasis, and stress response are influenced by factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH), and erythropoietin (EPO). As retinopathy of prematurity (ROP) is a neurovascular retinal disease, this study analyzed the contributions of NGF (rs6330), BDNF (rs7934165), TH (rs10770141), and EPO (rs507392) genetic functional polymorphisms to the modulation of hematological and biochemical parameters of the first week of life and their association with ROP development. A multicenter cohort of 396 preterm infants (gestational age &lt; 32 weeks or birth weight &lt; 1500 g) was genotyped using MicroChip DNA and iPlex MassARRAY® platform. Multivariate regression followed univariate assessment of ROP risk factors. NGF (GG) genotype was associated with a higher ROP risk (OR = 1.79), which increased further (OR = 2.38) when epistatic interactions with TH (allele C) and BDNF (allele G) were present. Significant circulating biomarker differences, including bilirubin, erythrocytes, monocytes, neutrophils, lymphocytes, and platelet markers, were found between ROP and non-ROP groups, with variations depending on the polymorphism. These findings suggest that NGF (rs6330) and its interactions with related genes contribute to ROP risk, providing valuable insights into the genetic and biological mechanisms underlying the disease and identifying potential predictive biomarkers.

Conventional and complementary alternative medicine therapies for renal anemia: a literature review.

Renal anemia stems mainly from chronic inflammation with elevated hepcidin levels, iron deficiency, and reduced red blood cell lifespan. Inadequate erythropoietin (EPO) production, worsened kidney function, leads to symptoms such as low energy, fatigue, and impaired physical function, significantly affecting patients' quality of life. We conducted a comprehensive search across electronic databases including PubMed, Embase, Cochrane Library, Chinese National Knowledge Infrastructure, Airiti library, and Wanfang, to compile recent clinical trials and pilot studies on conventional and complementary alternative medicine approaches for renal anemia. This discussion focuses on the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) axis theory, from lab research to clinical applications. It explores non-extracorporeal treatments for renal anemia, including pharmaceutical interventions, dietary strategies, and complementary and alternative medicine (CAM). The article details the effects of Roxadustat, Ferumoxytol, and Epodion. Clinical studies show that modulating the gut microbiome can reduce inflammation and improve renal anemia. Clinical trials suggest that CAM therapy can improve renal anemia through mechanisms such as enhanced iron metabolism, anti-inflammatory effects, reduced hepcidin levels, and increased EPO and HIF expressions. By synthesizing this information, the review aims to furnish valuable insights and treatment recommendations aimed at ameliorating renal anemia in individuals grappling with chronic kidney disease.

Quantum Dot Erythropoietin Bioconjugates Enhance EPO-Receptor Clustering on Transfected Human Embryonic Kidney Cells.

Erythropoietin (EPO)-induced cellular signaling through the EPO receptor (EPOR) is a fundamental pathway for the modulation of cellular behavior and activity. In our previous work, we showed in primary human astrocytes that the multivalent display of EPO on the surface of semiconductor quantum dots (QDs) mediates augmented JAK/STAT signaling, a concomitant 1.8-fold increase in the expression of aquaporin-4 (AQPN-4) channel proteins, and a 2-fold increase in the AQPN-4-mediated water transport activity. Our hypothesis is that this enhanced signaling involves the simultaneous ligation and clustering of EPOR by QD-EPO conjugates. Here, we utilized a human embryonic kidney (HEK 293T/17) cell line transfected with EPOR fused to enhanced green fluorescent protein (eGFP) to visualize EPOR clustering. We demonstrate that QDs displaying five copies of EPO (bearing a C-terminal 6-histidine tract) on the nanoparticle surface induce a 1.8-fold increase in EPOR clustering compared to monomeric EPO at the same concentration. Our findings confirm the critical role played by the multivalent display of EPO in mediating clustering of the EPOR. More generally, these results illustrate the capability of nanoparticle-growth factor bioconjugates to control the activity of cognate receptors and the important role played by multivalent display in the modulation of selective cellular delivery and signaling.

Therapeutic effects of erythropoietin-alginate/chitosan hydrogel on impaired male rat fertility after spinal cord injury.

Infertility is a significant issue in spinal cord injury (SCI) patients. Men with SCI often experience erectile and ejaculatory dysfunctions, and low sperm quality leading to impaired fertility. In this study, we investigated the effectiveness of Erythropoietin (EPO)alginate/chitosan (CH-AL) hydrogel on SCI-induced male rat infertility. Twenty-four male Wistar rats were divided into three groups: Sham, SCI-induced group without any further intervention (Negative control, NC), and a treatment group receiving 10,000 IU/kg EPO-CH/AL hydrogel (EPO group), immediately following SCI induction. Sperm parameters, oxidative stress markers, apoptosis, and histopathological changes in animal testes were analyzed. Sperm viability and motility significantly decreased in the NC group compared to the sham group (p < 0.01). Treatment with 10,000 IU/kg EPO-CH/AL hydrogel significantly improved sperm viability and motility compared to the NC group (p < 0.05). Malondialdehyde (MDA) and Superoxide dismutase (SOD) levels were elevated and reduced, respectively, in the NC group compared to the sham group. Treatment with EPO-CH/AL hydrogel group, significantly decreased MDA level and increased SOD levels compared to the NC group (p < 0.05). Apoptosis analysis revealed upregulation of Bcl-2 and downregulation of Bax genes in EPO-CH/AL hydrogel compared to the NC group (p < 0.05). Histological analysis showed that the EPO-CH/AL hydrogel improved depletion of germ cell population and Luminal spaces compared to NC group but did not fully restore normal histology. EPO-CH/AL hydrogel effectively improves sperm viability and motility, reduces oxidative stress, and mitigates apoptosis, demonstrating its potential as a therapeutic strategy for SCI-induced infertility.

Epoetin alpha in multiple myeloma: literature review and our own experience

Background. Anemia is the main symptom of multiple myeloma (MM) both at the time of disease onset and during tumor progression. Previously, the main method of anemia treatment was blood transfusion therapy. Currently, blood transfusions are supplemented by erythropoietin (EPO) administration. Safety and effectiveness of the drug have been proven in multiple trials including trials involving oncohematological patients.Aim. To present the results of using epoetin alpha (Eralfon) in patients with MM complicated by dialysis-dependent myeloma cast nephropathy in real clinical practice; to analyze the literature data on the use of EPO for the treatment of anemia in MM patients.Materials and methods. A retrospective analysis of a series of clinical observations was carried out: 4 patients with newly diagnosed MM at the ages between 52 and 60 years who underwent treatment at the Department of Hematology and Chemotherapy of Paraproteinemic Hemablastoses with a Bone Marrow and Hematopoietic Stem Cell Transplantation Block. All patients were diagnosed with myeloma cast nephropathy with significantly decreased glomerular filtration rate of 7–15 mL/min requiring renal replacement therapy. At the time of disease diagnosis, median hemoglobin level was 75 g/L, median creatinine level was 517.5 µmole/L. Endogenous EPO level was measured in all patients prior to epoetin alpha prescription: it varied between 2.31 and 149.6 IU/mL. Epoetin alpha (Eralfon) was prescribed at dose 12 000 IU – 0.3 mL subcutaneously 3 times a week. A review of the literature data on the use of EPO in patients with MM was conducted.Results. All patients at MM onset were dependent on renal replacement therapy and blood transfusion, therefore epoetin alpha was prescribed immediately. In case of renal function recovery and end of dialysis at target hemoglobin levels, administration of the drug was ceased. If dependence on renal replacement therapy persisted, epoetin alpha treatment continued as synthetic function of EPO-producing cells was compromised. In all clinical cases, epoetin alpha therapy was effective.Conclusion. Clot formation prevention should be kept in mind during epoetin alpha therapy. Decreased requirement for blood transfusions, improved quality of life with favorable safety profile of the drug make epoetin alpha an indispensable part of accompanying therapy in patients with MM and anemia.

A Recombinant Antibody Against ALK2 Promotes Tissue Iron Redistribution and Contributes to Anemia Resolution in a Mouse Model of Anemia of Inflammation.

Patients with chronic inflammation are burdened with anemia of inflammation (AI), where inflammatory cytokines inhibit erythropoiesis, impede erythropoietin production, and limit iron availability by inducing the iron regulator hepcidin. High hepcidin hinders iron absorption and recycling, thereby worsening the impaired erythropoiesis by restricting iron availability. AI management is important as anemia impacts quality of life and potentially affects morbidity and mortality. The bone morphogenetic protein (BMP)-SMAD pathway is crucial for hepcidin regulation. Here, we characterized a research antibody against BMP receptor ALK2, RKER-216, and investigated its mechanism in suppressing hepcidin and improving anemia in acute/chronic inflammation. Additive effects of RKER-216 and recombinant human erythropoietin (rhEPO) on erythropoiesis and iron utilization were also explored. We showed that RKER-216 neutralized ALK2 activity by competing with the binding of BMP6. RKER-216 reduced hepcidin transcription in Hep3B cells, and a subcutaneous dose of RKER-216 at 3 mg/kg suppressed serum hepcidin and increased circulating iron for 3-4 days in wildtype mice. Moreover, RKER-216 decreased hepcidin by inhibiting SMAD1/5/9 signaling in lipopolysaccharide-mediated inflammation and liberated iron from the recycling pathway to alleviate anemia in mice with adenine-induced chronic kidney disease (CKD), a mouse model of AI. Finally, RKER-216 reversed iron-restricted erythropoiesis in CKD mice and supplied the iron requirement for complete resolution of anemia when coupled with rhEPO in addressing AI. Our data support that ALK2 is a key hepcidin regulator and that a neutralizing ALK2 antibody has the potential to restore iron homeostasis as monotherapy or in combination with rhEPO to ameliorate AI.

In Silico and Experimental Evidence for the Stabilization of rhEPO by Glycine, Glutamic Acid and Lysine.

The available literature indicates that amino acids can stabilize proteins. Our experimental data demonstrated that lysine and glutamic acid can stabilize recombinant human erythropoietin (rhEPO) at 40°C for at least 1 month, as measured by RP-UPLC. Studies with different excipient concentrations demonstrated optimal concentrations of these amino acids within 10-12 mM. The results suggest that a lower concentration of amino acids may not be sufficient to stabilize formulations, while a higher concentration of amino acids can lead lower stability. In silico studies highlighted the importance of the FA4G4S4 model in experimental glycosylation determination, particularly in glycoprotein analysis. We obtained insights into the interactions between the glycosylated ligands of rhEPO and amino acids, as well as their impact on protein behavior and stability. We observed different interactions between the amino acids glycine, glutamic acid, and lysine and the rhEPO protein using this model in docking experiments. They also made it easier to find specific interaction areas by analyzing ligand‒protein interaction fingerprints (PLIFs). This demonstrated how the ligands bind to the proteins or remain outside their vicinity. Furthermore, this study revealed specific places where ligands and rhEPO residues can interact, which helps us learn more about how they stabilize rhEPO.

Donkey-Hide Gelatin-Derived Carbon Dots Activate Erythropoiesis and Eliminate Oxidative Stress for Aplastic Anemia Treatment.

Aplastic anemia (AA) is a life-threatening hematologic disease with limited therapeutic options. Stalled erythropoiesis and oxidative stress-induced hemocyte apoptosis are the main pathological features of AA, yet therapeutic agents that address these issues remain elusive. In this study, we report distinctive donkey-hide gelatin-derived carbon dots (G-CDs) that enable erythropoiesis activation and oxidative stress elimination to tackle refractory AA. We demonstrate that G-CDs can promote the proliferation and erythroid differentiation of hematopoietic stem cells as well as erythrocyte maturation, activating the whole process of erythropoiesis. Moreover, G-CDs display multienzyme-like activities and dramatically alleviate the oxidative stress of bone marrow and peripheral blood via catalytic scavenging of multiple reactive oxygen species, reconstructing the hematopoietic microenvironment. Intravenously or orally administered to AA mice induced by chemotherapy drugs, G-CDs significantly boost the level of red blood cells and hemoglobin and lead to the complete recovery of hematopoietic function, showing better therapeutic performance than clinically approved erythropoietin (EPO) without adverse effects. By collaboratively addressing the issues of stalled erythropoiesis and oxidative stress, the G-CDs-based intervention strategy may offer a powerful paradigm for clinical AA management.

ASSESSMENT OF IRON PROFILE AND ITS CORRELATION WITH MICRO R% AND OTHER RED CELL PARAMETERS IN PATIENTS WITH CHRONIC RENAL FAILURE ON DIALYSIS AND RECEIVING ERYTHROPOIETIN INJECTION

Objectives: The objectives of the study are to correlate Micro R% with red cell indices and to assess their efficiency in detecting iron deficiency in chronic kidney disease (CKD) patients receiving erythropoietin (EPO). Methods: Red cell indices such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), MCH concentration, and red cell distribution width generated from automated analyzers frequently help in the diagnosis of iron deficiency. Micro R% is a research-only parameter in an automated analyzer while doing a complete blood count. This study is done to correlate and compare hemoglobin and biochemical parameters in healthy controls and CKD patients and to correlate the Micro R% with MCV and serum iron in detecting iron deficiency in CKD patients receiving EPO. Results: Red cell indices showed a good correlation with serum iron studies. Micro R% is a reliable parameter for the identification of iron deficiency anemia and it correlates with other red cell indices and with serum iron studies. Conclusion: Identification of iron deficiency in patients with chronic renal failure on dialysis helps us to start the treatment earlier. Anemia-related complications can be prevented and we can give a better quality of life in these patients.

A novel serous ovarian carcinoma model induced by DMBA: Results from OncoTherad® (MRB-CFI-1) immunotherapy preclinical testing.

The term ovarian carcinoma (OC) refers to a heterogeneous collection of five distinct diseases known as histotypes. While histotype-specific treatment is still a clinical challenge in OC, well-characterized models are required for testing new therapeutic strategies. We employed OncoTherad® (MRB-CFI-1), an interferon (IFN-γ)-stimulating nano-immunotherapy mediated by Toll-like receptors (TLR) 2/4, in association or not with Erythropoietin (EPO) in a chemically-induced ovarian cancer model. Besides characterization of the therapies effects, we also assessed whether the animal model was representative of human OC by providing histotype classification. Thirty-five Fischer rats were distributed into five groups: Control (Sham surgery); Cancer (7,12-dimethylbenzoanthracene - DMBA injection in the ovarian bursa, 1.25 mg/kg); OncoTherad® (20 mg/kg intraperitoneal); EPO (8.4 µg/kg intraperitoneal); and OncoTherad+EPO (same doses). Ovaries were formalin-fixed into paraffin-embedded blocks. TLR pathway and the inflammatory response profile were evaluated by immunohistochemistry (IHC). After DNA extraction and tissue microarray construction, we assessed typical gene mutations directly (Sanger sequencing) or indirectly (IHC surrogates) and examined biomarkers of different OC histotypes. OC induction decreased TLR2, TLR4, and proinflammatory cytokines. OncoTherad® alone or associated with EPO modulated the OC microenvironment to a cytotoxic immune profile through stimulation of the TLR4-mediated non-canonical pathway. EPO stimulated TLR2-mediated canonical pathway and notably increased Tregs. The features analyzed favored interpretation of our DMBA-induced tumor model as predominantly low-grade, serous carcinoma-like, in which treatments with OncoTherad® and EPO showed immunomodulatory properties related to the reduction of ovarian lesions.

Supplemental Iron and Recombinant Erythropoietin for Anemia in Infants Born Very Preterm: A Survey of Clinical Practice in Europe

To survey practices of iron and recombinant human erythropoietin (rhEpo) administration to infants born preterm across Europe. Over a 3-month period, we conducted an online survey in 597 neonatal intensive care units (NICUs) of 18 European countries treating infants born with a gestational age of<32weeks. We included 343 NICUs (response rate 56.3%) in the survey. Almost all NICUs (97.7%) routinely supplement enteral iron, and 74.3% of respondents to all infants born <32weeks of gestation. We found that 65.3% of NICUs routinely evaluate erythropoiesis and iron parameters beyond day 28 after birth. Most NICUs initiate iron supplementation at postnatal age of 2 weeks and stop after 6 months (34.3%) or 12months (34.3%). Routine use of rhEpo was reported in 22.2% of NICUs, and in individual cases in 6.9%. RhEpo was mostly administered subcutaneously (70.1%) and most frequently at a dose of 250 U/kg 3 times a week (44.3%), but the dose varied greatly between centers. This survey highlights wide heterogeneity in evaluating erythropoietic activity and iron deficiency in infants born preterm. Variation in iron supplementation during infancy likely reflects an inadequate evidence base. Current evidence on the efficacy and safety profile of rhEpo is only poorly translated into clinical practice. This survey demonstrates a need for standards to optimize patient blood management in anemia of prematurity.

Analytical characterization of aberrant trisulfide bond formation in therapeutic proteins and their impact on product quality.

Post translational modifications (PTMs) of proteins play an integral role in maintaining the overall structure and function of proteins including their proper folding, binding, and potency. However, not all PTMs play a positive role in protein drugs as some can lead to product-related impurities that negatively impact protein function. One example of a PTM is trisulfide formation, which appears as a product related species in multiple biologic drug products. The impacts of trisulfide formation on protein structure, stability, potency, and safety remains under investigation. Herein, we investigated and report the impact of aberrant trisulfides on erythropoietin (EPO) and somatropin (growth hormone/GH) therapeutic proteins. Utilizing LC-MS we show that one EPO product contains measurable basal levels of trisulfide bonds in its formulation and exposure to H2S induced aberrant trisulfides in all products investigated. We report that exposure to H2S produces moderate effects on protein stability via thermal melting monitored by circular dichroism, protein purity utilizing size exclusion chromatography, and particle content using micro-flow imaging. No changes were observed in protein folding via circular dichroism, immunogenicity screening via a THP1-blue assay, or receptor binding activity via biolayer interferometry. Together, these data provide evidence on the effects of aberrant trisulfide formation on overall product quality.