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We describe herein the case of a 65-year-old patient on chronic hemodialysis with a medical history of idiopathic thrombocytopenia who experienced numerous episodes of severe thrombocytopenia with no specific diagnosis. Further analysis of the evolution of the platelet count showed that cyclic thrombocytopenia occurred after each injection of recombinant erythropoietin (rHu-EPO). Exploration of the involved mechanisms revealed the presence of a rHu-EPO-dependent anti-GPIV/IIIb antibody associated with a significant increase in GPIV/IIIb expression on her platelets after the addition of rHu-EPO. EPO was discontinued and the patient was treated with roxadustat with favorable results on her hemoglobin and platelet counts., (© 2022. The Author(s) under exclusive licence to Italian Society of Nephrology.)

Several factors may affect erythropoietin (EPO) sugar structures including designing cell culture procedure, pH, concentration of additives, dissolved oxygen, and other physicochemical parameters. In this study, we investigated the influence of changes in effective parameters and compounds on the growth rate of Chinese hamster ovary cell (CHO) cells producing recombinant EPO. Cell culture was performed at different temperature, buffering conditions, and varied concentrations of additives such as pyruvic acid, insulin, GlutaMAX, and sodium butyrate. Results indicated that the optimal temperature and pH were 37 °C and 7.2, respectively. Also, optimal concentrations for pyruvic acid, butyrate, glutamate, and insulin were obtained to be 20 mM, 1 mM, 2 mM, and 40 μg/mL, respectively. Then, cell culture was performed in microcarrier-coated spinner flasks under the optimized condition. The results showed recombinant human EPO (rhEPO) production with adequate purity. Optimization of physicochemical conditions and culture media are important factors to improve the quantity and quality of protein products. This study showed that cell growth and recombinant EPO protein production significantly increased under the optimized conditions. The results of this research can also be used in scale-up to increase the efficiency of EPO production. Abbreviations: EPO: erythropoietin; CHO cell: Chinese hamster ovary cell; rhEPO: recombinant human EPO; DMEM: modified eagle's medium; FBS: fetal bovine serum; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IGF-1: insulin-like growth factor 1.

Comprehensive characterization of the N-glycome of a therapeutic is challenging because glycans may harbor numerous modifications (e.g., phosphorylation, sulfation, sialic acids with possible O-acetylation). The current report presents a comparison of two chromatographic platforms for the comprehensive characterization of a recombinant human erythropoietin (rhEPO) N-glycome. The two platforms include a common workflow based on 2-AB-derivatization and hydrophilic interaction chromatography (HILIC) and a native N-linked glycan workflow employing high performance anion exchange (HPAE) chromatography. Both platforms were coupled to an Orbitrap mass spectrometer, and data dependent HCD fragmentation allowed confident structural elucidation of the glycans. Each platform identified glycans not revealed by the other, and both exhibited strengths and weaknesses. The reductive amination based HILIC workflow provided better throughput and sensitivity, had good isomer resolution, and revealed the presence of O-acetylated sialic acids. However, it exhibited poor performance toward phosphorylated glycans and did not reveal the presence of sulfated glycans. Furthermore, reductive amination introduced dehydration artifacts and modified the glycosylation profile in the rhEPO glycome. Conversely, HPAE provided unbiased charge classification (sialylation levels), improved isomer resolution, and revealed multiple phosphorylated and sulfated structures, but delivered lower throughput, had artifact peaks due to epimer formation, and loss of sialic acid O-acetylation. The MS 2 based identification of phosphorylated and sulfated glycans was not possible in HILIC mode due to their poor solubility caused by the high acetonitrile concentrations employed at the beginning of the gradient. After analyzing the glycome by both approaches and determining the glycans present, a glycan library was created for site specific glycopeptide analyses. Glycopeptide analyses confirmed all the compositions annotated by the combined use of 2-AB- and native glycan workflows and provided site specific location of the glycans. These two platforms were complementary and in combination delivered a more thorough and comprehensive characterization of the rhEPO N-glycome, supporting regulatory conformance for the pharmaceutical industry.

The effect of recombinant erythropoietin (rhEPO) on renal and systemic hemodynamics was evaluated in a randomized double-blinded, cross-over study. Sixteen healthy subjects were tested with placebo, or low-dose rhEPO for 2 weeks, or high-dose rhEPO for 3 days. Subjects refrained from excessive salt intake, according to instructions from a dietitian. Renal clearance studies were done for measurements of renal plasma flow, glomerular filtration rate (GFR) and the segmentel tubular handling of sodium and water (lithium clearance). rhEPO increased arterial blood pressure, total peripheral resistance, and renal vascular resistance, and decreased renal plasma flow in the high-dose rhEPO intervention and tended to decrease GFR. In spite of the decrease in renal perfusion, rhEPO tended to decrease reabsorption of sodium and water in the proximal tubule and induced a prompt decrease in circulating levels of renin and aldosterone, independent of changes in red blood cell mass, blood volumes, and blood pressure. We also found changes in biomarkers showing evidence that rhEPO induced a prothrombotic state. Our results suggest that rhEPO causes a direct downregulation in proximal tubular reabsorption that seems to decouple the activity of the renin-angiotensin-aldosterone system from changes in renal hemodynamics. This may serve as a negative feed-back mechanism on endogenous synthesis of EPO when circulating levels of EPO are high. These results demonstrates for the first time in humans a direct effect of rhEPO on renal hemodynamics and a decoupling of the renin-angiotensin-aldosterone system., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Background: Oxidative degradation of human recombinant erythropoietin (hrEPO) may occur in manufacturing process or therapeutic applications. This unfavorable alteration may render EPO inefficient or inactive. We investigated the effect of methionine/54 oxidative changes on the amino acid sequences, glycoform distribution and biological activity of hrEPO., Methods: Mass spectrometry was applied to verify the sequence and determine the methionine oxidation level of hrEPO. Isoform distribution was studied by capillary zone electrophoresis method. In vivo normocythemic mice assay was used to assess the biological activity of three different batches (A, B, and C) of the proteins., Results: Nano-LC/ESI/MS/MS data analyses confirmed the amino acid sequences of all samples. The calculated area percent of three isoforms (2-4 of the 8 obtained isoforms) were decreased in samples of C, B, and A with 27.3, 16.7, and 6.8% of oxidation, respectively. Specific activities were estimated as 53671.54, 95826.47, and 112994.93 mg/mL for the samples of A, B, and C, respectively., Conclusion: The observed decrease in hrEPO biological activity, caused by increasing methionine oxidation levels, was rather independent of its amino acid structure and mainly associated with the higher contents of acidic isoforms.

Background: Adequately powered studies directly comparing hard clinical outcomes of darbepoetin alfa (DPO) versus epoetin alfa (EPO) in patients undergoing dialysis are lacking., Study Design: Observational, registry-based, retrospective cohort study; we mimicked a cluster-randomized trial by comparing mortality and cardiovascular events in US patients initiating hemodialysis therapy in facilities (almost) exclusively using DPO versus EPO., Setting & Participants: Nonchain US hemodialysis facilities; each facility switching from EPO to DPO (2003-2010) was matched for location, profit status, and facility type with one EPO facility. Patients subsequently initiating hemodialysis therapy in these facilities were assigned their facility-level exposure., Intervention: DPO versus EPO., Outcomes: All-cause mortality, cardiovascular mortality; composite of cardiovascular death, nonfatal myocardial infarction (MI), and nonfatal stroke., Measurements: Unadjusted and adjusted HRs from Cox proportional hazards regression models., Results: Of 508 dialysis facilities that switched to DPO, 492 were matched with a similar EPO facility; 19,932 (DPO: 9,465 [47.5%]; EPO: 10,467 [52.5%]) incident hemodialysis patients were followed up for 21,918 person-years during which 5,550 deaths occurred. Almost all baseline characteristics were tightly balanced. The demographics-adjusted mortality HR for DPO (vs EPO) was 1.06 (95% CI, 1.00-1.13) and was materially unchanged after adjustment for all other baseline characteristics (HR, 1.05; 95% CI, 0.99-1.12). Cardiovascular mortality did not differ between groups (HR, 1.05; 95% CI, 0.94-1.16). Nonfatal outcomes were evaluated among 9,455 patients with fee-for-service Medicare: 4,542 (48.0%) in DPO and 4,913 (52.0%) in EPO facilities. During 10,457 and 10,363 person-years, 248 and 372 events were recorded, respectively, for strokes and MIs. We found no differences in adjusted stroke or MI rates or their composite with cardiovascular death (HR, 1.10; 95% CI, 0.96-1.25)., Limitations: Nonrandom treatment assignment, potential residual confounding., Conclusions: In incident hemodialysis patients, mortality and cardiovascular event rates did not differ between patients treated at facilities predominantly using DPO versus EPO., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary: Microalgae are considered as attractive expression systems for the production of biologics. As photosynthetic unicellular organisms, they do not require costly and complex media for growing and are able to secrete proteins and perform protein glycosylation. Some biologics have been successfully produced in the green microalgae Chlamydomonas reinhardtii. However, post‐translational modifications like glycosylation of these Chlamydomonas‐made biologics have poorly been investigated so far. Therefore, in this study, we report on the first structural investigation of glycans linked to human erythropoietin (hEPO) expressed in a wild‐type C. reinhardtii strain and mutants impaired in key Golgi glycosyltransferases. The glycoproteomic analysis of recombinant hEPO (rhEPO) expressed in the wild‐type strain demonstrated that the three N‐glycosylation sites are 100% glycosylated with mature N‐glycans containing four to five mannose residues and carrying core xylose, core fucose and O‐methyl groups. Moreover, expression in C. reinhardtii insertional mutants defective in xylosyltransferases A and B and fucosyltransferase resulted in drastic decreases of core xylosylation and core fucosylation of glycans N‐linked to the rhEPOs, thus demonstrating that this strategy offers perspectives for humanizing the N‐glycosylation of the Chlamydomonas‐made biologics. [ABSTRACT FROM AUTHOR]

Background: Left ventricular hypertrophy (LVH) is highly prevalent in haemodialysis (HD) patients and is associated with an increased risk of death. Roxadustat and recombinant human erythropoietin (rHuEPO, abbreviated as EPO) are the main treatment strategies for renal anaemia in HD patients, but it has not been clear whether there is a difference in their effect on LVH. Methods: In this multi‐centre, prospective, randomized trial of 12‐month duration, study participants were randomized in a 1:1 ratio to the roxadustat group or the EPO group. The doses of both treatment regimens were adjusted so that the patients had a haemoglobin level of 10.0–12.0 g per dL. The primary study endpoint was the change from baseline to 12 months in the left ventricular mass index (LVMI, g/m2) measured by echocardiography. Results: In total, 114 patients were enrolled. The mean age was 50 years, and the median dialysis duration was 33 months. Sixty‐one patients were men, and 24 were diabetic. LVMI decreased from 116.18 ± 27.84 to 110.70 ± 25.74 g/m2 in the roxadustat group. However, it increased from 109.35 ± 23.41 to 114.99 ± 28.46 g/m2 in the EPO group, with a significant difference in the change in LVMI between the two groups [−5.48 (−11.60 to 0.65) vs. 5.65 (0.74 to 10.55), p < 0.05]. Changes in left ventricular mass, end‐diastolic volume and 6‐min walk test seemed superior in the roxadustat group. There were no significant differences in other cardiac geometry, biochemical parameters and major adverse cardiovascular events between the two groups. Conclusions: Compared to EPO, roxadustat is more helpful in the regression of LVH in HD patients. [ABSTRACT FROM AUTHOR]

Anaemia is one of the most common complications of chronic kidney disease (CKD), having a significant impact on quality of life, and is also associated with a number of adverse clinical outcomes. Its pathogenesis is multifactorial, caused largely by an inadequate production of erythropoietin from the diseased kidneys, with iron deficiency, inflammation, shortened red cell lifespan and enhanced blood loss also being contributory factors. The management of this condition was transformed in the late 1980s by the advent of recombinant human erythropoietin (epoetin), and treatment paradigms have developed over the last three decades, largely focusing on a combination of epoetin or its analogues (erythropoiesis-stimulating agents; ESAs) along with iron supplementation, often administered intravenously due to increased hepcidin levels limiting iron absorption from the gut. Indeed, in patients with early CKD and iron deficiency, iron per se may be sufficient to improve the anaemia, delaying the need for ESA therapy. Other causes of anaemia should be excluded and corrected (if possible) before resorting to treatment with ESAs and iron. More recently, the hypoxia-inducible factor–prolyl hydroxylase inhibitors have entered the therapeutic arena; these are orally active agents that upregulate endogenous erythropoietin production as well as a number of iron-regulatory genes which may also enhance erythropoiesis. The latter drugs are highly efficacious, and may have advantages in inflammatory conditions causing resistance to conventional ESA therapy, but concerns exist regarding their safety, particularly in the longer term. This article reviews the current standards of treatment, as well as recent novel developments in the management of anaemia in CKD. [ABSTRACT FROM AUTHOR]

Background: The bone regeneration potential of erythropoietin (EPO) is not yet fully investigated, but some previous experimental studies demonstrated that its application activated the differentiation of osteoblasts and promoted bone formation. Aim: The aim of the present study was to evaluate the effects of recombinant human erythropoietin (rhEpo) on bone healing in cats with fragmented long bone fractures. Methods: Twelve cats were divided into two groups--control (n = 6) in which physiological saline was applied at the fracture gap site and EPO (n = 6) with the application of 1,000 IU rhEpo. The effects of EPO on blood erythrocyte counts, hemoglobin content, and hematocrit were monitored by serial complete blood cell tests, whereas bone formation was evaluated by clinical and radiographic examinations on post-operative weeks 1, 2, 3, 4, 6, and 8. Results: All tested blood parameters were within the reference range. A faster fracture healing and full limb weight-bearing were observed in the EPO group, with statistically significant differences with respect to the control group. Conclusion: The obtained results confirmed that the local application of rhEpo promoted bone healing in cats with fragmented femoral fractures and increased bone callus strength without having significant systemic effects. [ABSTRACT FROM AUTHOR]

This study is to evaluate the efficacy of erythropoietin in treating sepsis-associated acute respiratory distress syndrome (ARDS). One hundred patients with sepsis-related ARDS were randomized into the placebo group and Erythropoietin (EPO) group. Patients in the placebo group received saline as placebo on the standard therapy, while the EPO group received recombinant human erythropoietin injections on the standard therapy. It was found that the heart rate and mean arterial pressure did not differ significantly between days 7 and 14 after treatment initiation (p > 0.05). The partial pressure of oxygen (PaO2) and oxygenation index levels measured on days 7 and 14 were significantly higher than the placebo group and partial pressure of carbon dioxide (PaCO2) was significantly lower than the placebo group (p < 0.05). Lung capacity and functional residual capacity (and FRC) increased significantly in tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and C-reactive protein (CRP) concentrations (p < 0.05). In the EPO group, the duration of mechanical ventilation was significantly shorter and the mortality rate was significantly reduced (Log-Rank test, χ² = 4.651, p = 0.031). The results confirm that EPO significantly improves lung function and blood gas parameters, reduces serum levels of inflammatory markers, and reduces the risk of death in sepsis-induced ARDS patients, highlighting the potential therapeutic role of EPO in the management of this disease. [ABSTRACT FROM AUTHOR]

In orthopaedics, erythropoietin (EPO) is applied in the preoperative management of anaemic patients, but also as a stimulating factor to assist bone regeneration due to its angiogenic and osteoinductive potential. Since orthopaedists mainly rely on their clinical experience to assess bone healing, additional and more objective methods such as studying the dynamics of bone markers are needed. Therefore, the aim of this study was to investigate the plasma activity of bone-specific alkaline phosphatase (BALP), the N-terminal propeptide of type I collagen (PINP), the C-terminal telopeptide of type I collagen (CTX), and deoxypyridinoline (DPD) during the first 2 months of healing of comminuted fractures in cats, either non-stimulated or locally stimulated with recombinant human erythropoietin (rhEPO). The study included twelve cats of mixed breeds, aged 7.2 ± 4 months, weighing 2.11 ± 1.1 kg, with comminuted diaphyseal fractures of the femur. Surgical treatment with plate osteosynthesis was performed in all animals. The cats were randomly divided into two groups—a control (n = 6) and an EPO group (n = 6). The locally applied EPO leads to the increased activity of bone formation markers (BALP and PINP) during the second week after the osteosynthesis, preceding the peaks in the control group by two weeks. The studied bone resorption markers (DPD, CTX) varied insignificantly during the studied period. In conclusion, erythropoietin could serve as a promoter of bone healing in comminuted fractures in cats. [ABSTRACT FROM AUTHOR]

Recombinant human erythropoietin (EPO) is a biopharmaceutical frequently used in the treatment of anemia. It is a heavily glycosylated protein with a diverse and complex glycome. EPO N-glycosylation influences important pharmacological parameters, prominently serum half-life. Therefore, EPO N-glycosylation analysis is of the utmost importance in terms of controlling critical quality attributes. In this work, we performed an interlaboratory study of glycoanalytical techniques for profiling and in-depth characterization, namely (1) hydrophilic interaction liquid chromatography with fluorescence detection after 2-aminobenzamide labeling (HILIC-FLD(2AB)) and optional weak anion exchange chromatography (WAX) fractionation and exoglycosidase digestion, (2) HILIC-FLD after procainamide labeling (PROC) optionally coupled to electrospray ionization-MS and (3) matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS). All techniques showed good precision and were able to differentiate the unique N-glycosylation profiles of the various EPO preparations. HILIC-FLD showed higher precision, while MALDI-TOF-MS covered the most analytes. However, HILIC-FLD differentiated isomeric N-glycans, i.e., N-acetyllactosamine repeats and O-acetylation regioisomers. For routine profiling, HILIC-FLD methods are more accessible and cover isomerism in major structures, while MALDI-MS covers more minor analytes with an attractively high throughput. For in-depth characterization, MALDI-MS and HILIC-FLD(2AB)/WAX give a similar amount of orthogonal information. HILIC-FLD(PROC)-MS is attractive for covering isomerism of major structures with a significantly less extensive workflow compared to HILIC-FLD(2AB)/WAX. [ABSTRACT FROM AUTHOR]

The polymorphism of the angiotensin-converting enzyme (ACE) gene and interleukin-1 beta (IL-1b) gene could be associated with resistance in the treatment of anemia in dialysis patients with recombinant human erythropoietin (rHuEPO). The aim of the study was to evaluate the association between the polymorphism of the ACE and IL-1b genes and the response to rHuEPO therapy in dialysis patients with anemia. The study investigated 69 patients on dialysis with anemia treated with recombinant human erythropoietin for 12 months. Genotyping of ACE and IL-1b polymorphism was done in all study patients at the initiation of the study. The patient's demographic characteristics, dialysis vintage, and laboratory parameters were also evaluated as factors associated with rHuEPO resistance. The erythropoietin resistance index (ERI) was calculated as the weekly rHuEPO dose per kg of body weight, divided by the hemoglobin (Hb) concentration in g/dl. The Hb ≥ 110 g/l was registered in 37 (53.6%) patients. Patients with Hb ≥ 110 g/l were characterized by significantly higher serum levels of albumin, cholesterol, and iron than those with Hb < 110 g/l. The serum level of the CRP, the weekly dose of rHuEPO, and ERI were significantly higher in patients with Hb < 110 g/l compared to patients with Hb ≥ 110 g/l. The ERI value of ≥ 10 IUkg/weekly/g/dl was present in 27 (39.1%) patients. The serum levels of ferritin and CRP, and weekly dose of rHuEPO were significantly higher in patients with ERI value ≥ 10 IU kg/weekly/g/dl compared with the patients with ERI value < 10 IUkg/weekly/g/dl. There was no significant association between the ERI and polymorphism of the ACE and IL-1b genes in study patients. The polymorphism of the ACE and IL-1b genes was not significantly associated with the response to erythropoietin therapy in dialysis patients with anemia. Iron deficiency, malnutrition, and inflammation were factors associated with anemia and resistance to erythropoietin therapy in dialysis patients. [ABSTRACT FROM AUTHOR]

Introduction: The use of Doxorubicin (DOX) as an antineoplastic drug is limited by its cardiotoxicity. Erythropoietin (EPO) has been suggested to be a powerful cardioprotective agent. Aim of the Work: This study aimed to evaluate the protective role of EPO in DOX-induced cardiotoxicity and its role in mobilization of bone marrow-derived stem cells to the injured tissue. Materials and methods: Thirty-two adult male albino rats (180-200 gm) were divided into equal four groups: control group (Gp I) received physiological saline (1 ml/kg/day), EPO group (Gp II) received recombinant human erythropoietin (rhEPO) (2500 IU/kg, 3 times a week), DOX-treated group (Gp III) received DOX (2.5 mg /kg, 3 times a week), DOX+EPO group (Gp IV) received DOX and rhEPO concomitantly. All medications were given by intraperitoneal injection for two weeks. Specimens from the left ventricles of all rats were prepared and stained for light microscopic study (hematoxylin & eosin, Masson’s trichrome and phospho-tungstic acid hematoxylin in addition to immunohistochemical staining for caspase-3 and CD34) and transmission electron microscopic study. Results: DOX caused marked alteration in the histological features of the cardiac muscle fibers as well as a highly significant increase in area percentage of the collagenous fibers and caspase-3 immunoexpression. Co-administration of EPO with DOX caused improvement in the histological features of the cardiac muscle fibers, a highly significant decrease in area percentage of the collagenous fibers and caspase-3 immunoexpression, and a highly significant increase in CD34 immunoexpression. Conclusion: EPO exerts cardioprotective effects on DOX-induced cardiomyopathy via anti-apoptotic and anti-fibrotic pathways as well as via mobilization of stem cells to the injured tissues. [ABSTRACT FROM AUTHOR]

Background: Recombinant human erythropoietin (rhEPO) lost its role in minimizing red blood cell transfusion in very preterm infants after it had been associated with severe retinopathy of prematurity (ROP). Previous systematic reviews did not stratify ROP by gestation and birth weight (BW). Objectives: The aim of this study was to investigate the effect of early prophylactic rhEPO on ROP in a stratified meta-analysis of randomized controlled trials (RCTs). Methods: The databases EMBASE, MEDLINE, and the Cochrane Central Register of Controlled Trials were searched in January 2022 and complemented by citation searching. RCTs comparing early rhEPO treatment with no treatment or placebo were selected if they were published in a peer-reviewed journal and reported ROP outcomes. Previously unpublished data were requested from the study authors to allow stratified analyses by gestational age (GA) and BW. Data were extracted and analyzed using the standard methods of the Cochrane Neonatal Review Group. Pre-specified outcomes were "ROP stage ≥3" (primary outcome) and "any ROP." Results: Fourteen RCTs, comprising 2,040 infants of <29 weeks of GA, were included for meta-analysis. Data syntheses showed no effects of rhEPO on ROP stage ≥3 or on any ROP, neither in infants of <29 weeks GA, nor in infants of <1,000 g BW, nor in any GA strata. The risk ratio (95% confidence interval) for ROP stage ≥3 in infants of <29 weeks of GA was 1.13 (0.84, 1.53), p = 0.41 (quality of evidence: moderate). Conclusions: The present meta-analysis detected no effects of early rhEPO on ROP in any comparison, but most stratified analyses were limited by low statistical power. [ABSTRACT FROM AUTHOR]

The athlete's desire to achieve victory is why some of them decide to use doping. Blood doping, the common type of doping in endurance sports, increases hemoglobin, oxygen tissue delivery, and improves maximal aerobic capacity. The usual illegal ways for hemoglobin elevation are abuse of substances (recombinant human erythropoietin, hypoxia inducible factors) and/or methods (autologous blood transfusions) in concordance with exact protocols to avoid adverse finding either with approved direct methods or with indirect methods. Since direct methods are not sensitive enough to autologous blood transfusion, novel molecular substances, or to detect variants of recombinant human erythropoietin in different application protocols, indirect anti-doping methods marking the changes in the human body related to doping abuse, are being developed. The successful example of indirect method is the Athlete biological passport, with its hematological module consisting of twelve parameters and two scores that enable longitudinal monitoring of each athlete. Personal reference values at each sampling time are calculated in concordance with the Bayes network. Changes in blood parameters related to doping are marked as adverse analytical finding with 99% certainty, but new application regimens and various athletes' explanations for adverse analytical finding represent a novel challenge. Possibility of doping detection with Athlete biological passport has triggered athletes and their teams to use various explanations for the adverse analytical findings, which is why numerous studies are being conducted to explain influence of various conditions on the hematological biomarkers. Simultaneously, authorities are developing novel direct (e.g., additional substances, metabolites) and indirect (e.g., immature reticulocyte fraction, transcriptomics, etc.) methods to detect anti-doping rules violation. The substances/methods used by athletes to improve maximal aerobic capacity are being developed more rapidly than anti-doping authorities can implement adequate direct detection methods, which is why constant novel studies are needed. [ABSTRACT FROM AUTHOR]

Patients with renal anaemia are usually treated with recombinant human erythropoietin (EPO) because of insufficient renal EPO secretion. The establishment of a good haemoglobin (Hgb) response model is a necessary condition for dose optimization design. The purpose of this paper is to apply physics‐informed neural networks (PINN) to build the Hgb response model under EPO treatment. Neural network training is guided by a physiological model to avoid overfitting problems. During the training process, the parameters of the physiological model can be estimated simultaneously. To handle differential equations with impulse inputs and time delays, we propose approximate model equations for the pharmacokinetic (PK) model and the pharmacodynamic (PD) model, respectively. The modified PK/PD model was incorporated into PINN for training. Tests on simulated data and clinical data show that the proposed method has better performance than data‐driven modelling methods and the traditional physiological modelling based on the least squares method. [ABSTRACT FROM AUTHOR]

Background: Administration of recombinant erythropoietin (EPO), a kidneyproduced hormone with erythropoietic functions, has been shown to have multiple immunoregulatory effects in mice and humans, but whether physiological levels of EPO regulate immune function in vivo has not been previously evaluated. Methods: We generated mice in which we could downregulate EPO production using a doxycycline (DOX)-inducible, EPO-specific silencing RNA (shEPOrtTAPOS), and we crossed them with B6.MRL-Faslpr/J mice that develop spontaneous lupus. We treated these B6.MRL/lpr shEPOrtTAPOS with DOX and serially measured anti-dsDNA antibodies, analyzed immune subsets by flow cytometry, and evaluated clinical signs of disease activity over 6 months of age in B6.MRL/lpr shEPOrtTAPOS and in congenic shEPOrtTANEG controls. Results: In B6.MRL/lpr mice, Epo downregulation augmented anti-dsDNA autoantibody levels and increased disease severity and percentages of germinal center B cells compared with controls. It also increased intracellular levels of IL-6 and MCP-1 in macrophages. Discussion: Our data in a murine model of lupus document that endogenous EPO reduces T- and B-cell activation and autoantibody production, supporting the conclusion that EPO physiologically acts as a counterregulatory mechanism to control immune homeostasis. [ABSTRACT FROM AUTHOR]

Purpose: Recombinant erythropoietin (EPO) administered for traumatic brain injury (TBI) may increase short-term survival, but the long-term effect is unknown. Methods: We conducted a pre-planned long-term follow-up of patients in the multicentre erythropoietin in TBI trial (2010–2015). We invited survivors to follow-up and evaluated survival and functional outcome with the Glasgow Outcome Scale-Extended (GOSE) (categories 5–8 = good outcome), and secondly, with good outcome determined relative to baseline function (sliding scale). We used survival analysis to assess time to death and absolute risk differences (ARD) to assess favorable outcomes. We categorized TBI severity with the International Mission for Prognosis and Analysis of Clinical Trials in TBI model. Heterogeneity of treatment effects were assessed with interaction p-values based on the following a priori defined subgroups, the severity of TBI, and the presence of an intracranial mass lesion and multi-trauma in addition to TBI. Results: Of 603 patients in the original trial, 487 patients had survival data; 356 were included in the follow-up at a median of 6 years from injury. There was no difference between treatment groups for patient survival [EPO vs placebo hazard ratio (HR) (95% confidence interval (CI) 0.73 (0.47–1.14) p = 0.17]. Good outcome rates were 110/175 (63%) in the EPO group vs 100/181 (55%) in the placebo group (ARD 8%, 95% CI - 3 to 18%, p = 0.14). When good outcome was determined relative to baseline risk, the EPO groups had better GOSE (sliding scale ARD 12%, 95% CI 2–22%, p = 0.02). When considering long-term patient survival, there was no evidence for heterogeneity of treatment effect (HTE) according to severity of TBI (p = 0.85), presence of an intracranial mass lesion (p = 0.48), or whether the patient had multi-trauma in addition to TBI (p = 0.08). Similarly, no evidence of treatment heterogeneity was seen for the effect of EPO on functional outcome. Conclusion: EPO neither decreased overall long-term mortality nor improved functional outcome in moderate or severe TBI patients treated in the intensive care unit (ICU). The limited sample size makes it difficult to make final conclusions about the use of EPO in TBI. [ABSTRACT FROM AUTHOR]

Germinal matrix-intraventricular hemorrhage (GM-IVH) is among the devastating neurological complications with mortality and neurodevelopmental disability rates ranging from 14.7% to 44.7% in preterm infants. The medical techniques have improved throughout the years, as the morbidity-free survival rate of very-low-birth-weight infants has increased; however, the neonatal and long-term morbidity rates have not significantly improved. To this date, there is no strong evidence on pharmacological management on GM-IVH, due to the limitation of well-designed randomized controlled studies. However, recombinant human erythropoietin administration in preterm infants seems to be the only effective pharmacological management in limited situations. Hence, further high-quality collaborative research studies are warranted in the future to ensure better outcomes among preterm infants with GM-IVH. [ABSTRACT FROM AUTHOR]

Purpose: We investigated the effects of early postnatal administration of erythropoietin (EPO) on neurodevelopmental outcomes and morbidities in preterm infants with severe grades of intraventricular hemorrhage (IVH). Methods: We retrospectively reviewed the medical records of preterm infants of gestational age 23+0 weeks to 31+6 weeks, who were diagnosed with severe grades of IVH and received EPO over at least 2 weeks. We compared clinical characteristics, major complications, and neurodevelopmental impairment between the two groups. The primary outcome was severe neurodevelopmental impairment at 18 to 26 months of corrected age. Severe neurodevelopmental impairment was defined as a mental developmental index or psychomotor developmental index of <70 on the Bayley Scales of Infant Development II or diagnosis of cerebral palsy. Results: The study included 33 preterm infants (mean gestational age 25.2±1.6 weeks and mean birth weight 775.1±224.5 g). EPO was administered at a dose of 400 or 1,000 IU/kg thrice weekly and was maintained over a mean period of 58.6± 25.9 days beginning from 10.7±6.9 days after birth. We observed no difference in perinatal characteristics between the EPO (n=14) and the control group (n=19). Similarly, severe neurodevelopmental impairment rates did not differ between the EPO (85.7%) and control groups (78.9%). The incidence of neonatal morbidities including bronchopulmonary dysplasia, necrotizing enterocolitis, and retinopathy of prematurity was also similar between the EPO and control groups. Conclusion: Early administration of EPO did not reduce the risk of severe neurodevelopmental impairment in preterm infants with severe IVH. [ABSTRACT FROM AUTHOR]

Neuroprotective drugs to protect the brain against cerebral ischemia and reperfusion (I/R) injury are urgently needed. Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been demonstrated to have excellent neuroprotective functions in preclinical studies, but its neuroprotective properties could not be consistently translated in clinical trials. The clinical failure of rhuEPOM was thought to be mainly due to its erythropoietic activity-associated side effects. To exploit its tissue-protective property, various EPO derivatives with tissue-protective function only have been developed. Among them, asialo-rhuEPO, lacking terminal sialic acid residues, was shown to be neuroprotective but non-erythropoietic. Asialo-rhuEPO can be prepared by enzymatic removal of sialic acid residues from rhuEPOM (asialo-rhuEPOE) or by expressing human EPO gene in glycoengineered transgenic plants (asialo-rhuEPOP). Both types of asialo-rhuEPO, like rhuEPOM, displayed excellent neuroprotective effects by regulating multiple cellular pathways in cerebral I/R animal models. In this review, we describe the structure and properties of EPO and asialo-rhuEPO, summarize the progress on neuroprotective studies of asialo-rhuEPO and rhuEPOM, discuss potential reasons for the clinical failure of rhuEPOM with acute ischemic stroke patients, and advocate future studies needed to develop asialo-rhuEPO as a multimodal neuroprotectant for ischemic stroke treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: The treatment of large bone defects caused by fractures is still a challenge in orthopaedics. In this study, the potential of erythropoietin to promote bone regeneration was investigated in an experimental rat model. Bone healing was monitored through objective radiological, osteodensitometric and histological methods. The assessment suggests that erythropoietin, applied locally on a collagen scaffold alone or in combination with a bone substitute, is capable of inducing bone healing in femoral defects. Critical-size bone defect models are the standard in studies of the osteogenic potential of biomaterials. The present investigation aimed to evaluate the ability of recombinant human erythropoietin (EPO) to induce trabecular bone healing either alone or combined with a xenograft in a rat femoral critical-size defect model. Five-mm bone defects were created in the femoral diaphysis of fifty-six skeletally mature male Wistar albino rats. The animals were divided into six groups: one control group and five experimental groups. The defects in the control group were left empty, whereas an absorbable collagen cone soaked either with saline or erythropoietin (alone or in combination with xenograft) was placed in locally treated groups. The systemic treatment group received EPO subcutaneously. Bone formation was objectively evaluated through radiography, osteodensitometry and histological examination on post-operative days 30 and 90. The results demonstrate that EPO, locally applied on a collagen scaffold, was capable of inducing bone healing, whereas the single systemically administered high EPO dose had only an insignificant effect on bone formation. The combination of EPO with a bone substitute under the form of cancellous granules resulted in more rapid integration between the xenograft and host bone. [ABSTRACT FROM AUTHOR]

Recombinant human erythropoietin (rHuEPO) is one of the most effective drugs for the treatment of anemia in patients with chronic kidney disease. However, EPO-resistance is an important contributor to the increased risk of adverse effects. We previously showed that EPO treatment could induce precursor cell depletion, resulting in EPO-resistance. We further found that the combination of EPO with romiplostim, a thrombopoietin receptor agonist that can stimulate the expansion of hematopoietic stem cells, can treat EPO-resistance. In this study, we performed interspecies pharmacodynamic (PD) scaling of this combination therapy for human dose prediction. The pharmacokinetic parameters of both rHuEPO and romiplostim in humans were obtained from previous studies. The PD parameters obtained in rats were scaled to humans using allometric equations. The relationship between PD parameters of the megakaryocyte lineage from rats, monkeys, and humans was in agreement with those from the literature on allometric scaling. The PD response was translated to humans based on allometric scaling and agreed with the observed data. These parameters were used to simulate hemoglobin and platelet response in humans. RHuEPO 50 IU/kg thrice weekly and romiplostim 1 μg/kg once every 4 weeks from the second week is the recommended combination dosing regimen according to the model prediction. Our work successfully scaled the PD of rHuEPO and romiplostim monotherapy from rats to humans. The predicted dosing regimen of each drug in the combination therapy is less intensive than the approved starting dose of each drug, which supports additional evaluations of the combination therapy in humans. [ABSTRACT FROM AUTHOR]

Purpose: Our study aims to evaluate the effectiveness of intravenous erythropoietin (EPO) in patients with indirect traumatic optic neuropathy (TON), assess the side effects, and compare the visual function results among three groups of patients who had received different treatment options – EPO, steroids, and observation. Methods: Patients with indirect TON presenting to the neuro-ophthalmology clinic from August 2019 to March 2020, were assigned to three groups, with six patients in each group. In group 1, patients were recruited prospectively and received recombinant human erythropoietin, whereas, in groups 2 and 3, patients were recruited retrospectively and received intravenous methylprednisolone followed by oral steroids and multivitamins, respectively. Groups 1 and 2 included patients presenting within 2 weeks of trauma, whereas group 3 included those presenting beyond that. Best-corrected visual acuity, pupillary reaction, color vision, and visual fields following treatment were measured. Results: Initial visual acuity in the EPO group ranged from 20/80 to no perception of light (No PL). The mean initial BCVA (1.82 logMAR, standard deviation [SD] = 0.847) improved to 1.32, SD = 0.93 logMAR after treatment recorded at the third month (P = 0.0375), with no significant adverse effects. The initial BCVA of group 2 ranged from 20/120 to No PL. The mean initial BCVA (1.95, SD = 0.77 logMAR) improved to 1.45 logMAR, SD = 0.97 after treatment (P = 0.0435) but three patients had side effects of steroids. Initial visual acuity in Group 3 ranged from 20/40 to no PL. The mean initial BCVA (1.09 logMAR, SD = 1.10) worsened to 1.19 logMAR, SD = 1.06 after treatment after treatment (P = 0.0193). The improvement in BCVA when compared between the three groups was not significant. Conclusion: Both erythropoietin and steroids are effective in the management of traumatic optic neuropathy. However, erythropoietin shows lesser or no side effects when compared to steroids. [ABSTRACT FROM AUTHOR]

Background & Objective: Critically sick patients often develop moderate to severe anemia, due to various factors. We tested the hypothesis that administering prophylactic recombinant human erythropoietin (rHuEPO) together with early identification and correction of iron deficiency anemia in traumatic brain injury patients would have a positive effect on the incidence of RBCs transfusions. Methodology: One hundred and seventeen head-trauma patients were enrolled. They received 40,000 units of prophylactic rHuEPO, starting on day three after admission and continuing at weekly intervals for 6 weeks, with follow-up of iron biomarkers. Any patient developing iron deficiency anemia was prescribed oral or intravenous iron supplement. The incidence of RBCs transfusion was recorded. Results: The number of RBCs transfusion was significantly less in the prospective group (27.35%) than the retrospective group (42.86%). Hemoglobin and hematocrit levels were significantly less in the first and second weeks after admission and then began to rise. Serum iron, transferrin saturation, and ferritin were at their lowest levels in the fourth week. Meanwhile, total iron binding capacity was higher in the fourth week and lower in the sixth. Conclusion: The administration of prophylactic rHuEPO with early discovery and correction of iron deficiency anemia resulted in a significant reduction in the incidence of RBCs transfusion in the head-trauma patients. [ABSTRACT FROM AUTHOR]

Background: β-thalassemia is the most common hereditary disease in Iran, and more than 2 million carriers of β-thalassemia live in Iran. On the other hand, our country is located in the thalassemia belt, and no comprehensive study has been conducted regarding the effect of erythropoietin on blood parameters in thalassemia intermedia patients in our region. Therefore this study aimed to investigate the effect of erythropoietin on blood parameters of thalassemia intermedia patients. Materials and Methods: This prospective cross-sectional study was conducted on all patients suspected of thalassemia intermedia in Shahid Sadoughi hospital from March 2021 to M 2022. In the case of diagnosis of microcytic anemia, an electrophoresis test was performed, and people diagnosed with thalassemia intermedia entered the study. Then patients were divided into two groups (the intervention and control groups). The erythropoietin dose was 50-100 units/kilogram (body weight) three times a week for six months. The measurement of hematocrit and hemoglobin were done using CBC cell counter (Sysmex KX21). Other data were extracted from medical records. ResultL: In the current study, the mean age of patients in the intervention and control groups was 9.15±1.53 and 8.35± 6.90 years old, respectively (p=0.9). The mean hematocrit level in the intervention and control groups was 28.05± 4.06 and 23.45± 3.22 %, respectively (P<0.001). The mean hemoglobin level in the two groups was 9.15± 1.53 and 7.65± 1.23 g/dL respectively (p=0.002). The mean hematocrit level before and after the intervention was 25±3.71 and 28.05±4.06 %, respectively. The mean hemoglobin levels before and after therapy were 7.9±1.52 and 9.15±1.53 g/dL, respectively. Conclusion: According to the findings, hemoglobin and hematocrit increased in thalassemia intermedia patients taking erythropoietin. Therefore it seems that recombinant erythropoietin can be helpful in these patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Erythropoietin (ЕPО) is a glycoprotein hormone which, apart from its physiological function in the regulation of erythropoiesis, has additional functions in the animal and human body. In orthopaedic patients EPO treatment induces regeneration of bone defects, proliferation of osteoblasts and new blood vessel formation, improvement of early endochondral ossification and bone mechanical strength, thus resulting in enhanced bone healing. The purpose of this study was to compare the effects of local and systemic application of recombinant human erythropoietin (rhEPO) on the healing of rat calvarial defects. The systemic effect of EPO treatment was monitored by haematological analyses on days 0, 30 and 90. Bone healing was monitored via radiography and computed tomography on the same time intervals. The results demonstrated that intraperitoneally-injected rhEpo did not result in bone formation. Simultaneously, it affects the erythropoiesis by increasing the erythrocyte counts, haemoglobin and haematocrit. At the same time, single local dose rhEpo applied on collagen carrier could be used for enhancing bone healing without systemic effect. The purpose of this study was to compare the effects of local and systemic application of recombinant human erythropoietin (rhEPO) on the healing of rat calvarial defects. Twenty-four male skeletally-mature Wistar rats were used. Two bone 5 mm critical size defects were created in calvarial bones of each rat. In rats from experimental group I (n = 12), EPO was applied locally on a collagen cone in left defects, whereas a collagen cone soaked with physiological saline was placed in right defects. The rats from experimental group II were injected once intraperitoneally with 4900 IU/kg EPO; a collagen cone was only placed in left defects, whereas the right defects were left empty. The systemic effect of EPO treatment was monitored by haematological analyses on days 0, 30 and 90. Bone healing was monitored via radiography and computed tomography on the same time intervals. The results demonstrated that local EPO application had no significant effect on haemopoiesis, unlike the systemic application. At the same time, it resulted in new bone formation and therefore, could be successfully used as a means of promoting bone regeneration. [ABSTRACT FROM AUTHOR]

Purpose. We performed a genome-wide analysis of long noncoding RNA (lncRNA) expression to identify novel targets for the further study of recombinant human erythropoietin (rhEPO) treatment of acute spinal cord injury (SCI) in rats. Methods. Nine rats were randomly divided into 3 groups. No operation was performed in group 1. In groups 2 and 3, a laminectomy was performed at the 10th thoracic vertebra, and a contusion injury was induced by extradural application of an aneurysm clip. Group 1 rats did not receive any treatment, group 2 rats received a single intraperitoneal injection of normal saline, and group 3 rats received rhEPO. Three days after injury, spinal cord tissues were collected for RNA-Seq, microarray, differentially expressed genes (DEGs), Gene Ontology (GO) function enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction (PPI) analyses. Results. Compared with group 1, 4,446 genes were found to be differentially expressed in group 2. Furthermore, 99 lncRNAs were found to be changed in the injury group. The data indicate that 2,471 mRNAs were upregulated, and 1,975 mRNAs were downregulated in group 2 as compared with group 1. In addition, 45 of the lncRNAs were upregulated, and the other 44 lncRNAs were downregulated. The top 5 upregulated and top 5 downregulated lncRNAs that were different between group 2 and group 1 are shown. The top 5 downregulated and the top 5 upregulated lncRNAs that were different between group 3 and group 2 are shown. Conclusion. RhEPO treatment alters the expression profiles of the differentially expressed lncRNAs and genes beneficial to the development of new treatments. [ABSTRACT FROM AUTHOR]

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In vitro cell experiments have suggested that recombinant human erythropoietin (rhEPO) and peroxisome proliferator activated receptor γ (PPARγ) activation exert protective effects on neurons. This study observed the learning and memory ability, antioxidant capacity and the ratio of apoptotic cells after rhEPO intervention and investigated the relationship among rhEPO, PI3K/Akt and PPARγ in the anti‐neural oxidative stress injury process in vivo. The results showed that rhEPO significantly improved the learning and memory abilities of rats subjected to oxidative stress, enhanced the antioxidant capacity of cells, and reduced neuronal apoptosis. Then, the PI3K/Akt and PPARγ pathways were inhibited, and TUNEL staining were used to observe the changes in the effect of rhEPO. After the PI3K/Akt and PPARγ pathways were inhibited, the effect of rhEPO on rats subjected to oxidative stress was significantly weakened, suggesting that both the PI3K/Akt and PPARγ pathways are involved in the process by which rhEPO protects neurons. Finally, Western blotting and immunofluorescence staining were used to observe the changes in PI3K/Akt and PPARγ signalling proteins in the neurons after the rhEPO intervention and to explore the relationship among the three. The results showed that rhEPO significantly increased the levels of the p‐Akt and PPARγ proteins and the level of the PPARγ protein in the nucleus, indicating that the PI3K/Akt pathway was located upstream of and regulates PPARγ. In conclusion, this study suggested that rhEPO activates the PI3K/Akt to upregulate PPARγ, enhance the cellular antioxidant capacity, and protect neurons in rats subjected to oxidative stress. rhEPO reduces apoptosis and protects oxidative stress injury neurons by enhancing the antioxidant capacity of cells. PI3K/Akt and PPARγ pathways are involved in rhEPO‐protected neurons of rats subjected to oxidative stress. In this process, PI3K/Akt is located upstream of PPARγ pathway regulation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Pure red cell aplasia (PRCA) is an uncommon cause of anemia in end-stage kidney disease (ESKD). It is attributed to recombinant human erythropoietin (rHuEPO) administration. Although immunosuppression is the mainstay therapy, its effectiveness varies from 30% to 70%. PRCA in ESKD has been reported to improve following kidney transplantation. CASE REPORT: A 46-year-old woman with ESKD secondary to lupus nephritis was treated for uremia at our center. She developed severe anemia. Bone marrow aspiration and biopsy revealed a reduction of erythroid precursors, consistent with PRCA. Because she had no sibling’s blood group matched with her, ABO-incompatible kidney transplantation was an option for treatment. She underwent a desensitization protocol consisting of rituximab 375 mg/m², tacrolimus, mycophenolate mofetil, and prednisolone 4 weeks before surgery, in addition to 3 sessions of double-filtration plasmapheresis (DFPP) every other day followed by intravenous immunoglobulin (IVIG) and 1 session of specific immunoadsorption (Glycosorb® B column) at pre-transplant day -1. She also received low-dose rabbit anti-thymocyte globulin (rATG) (Thymoglobulin®) (total 2.0 mg/kg). Maintenance therapy included tacrolimus, mycophenolate mofetil, and prednisolone. Allograft function normalized a few days after transplantation and her Hb gradually increased. CONCLUSIONS: We report a rare case of PRCA in a patient with ESKD undergoing ABO-incompatible kidney transplantation. The outcome was satisfactory, with complete correction of anemia and kidney function. [ABSTRACT FROM AUTHOR]

Successful uterus transplantation, a potential treatment method for women suffering from absolute uterine infertility, is negatively affected by ischemia–reperfusion injury (IRI). The aim of this study is to investigate the protective effect of relaxin (RLX) or/and erythropoietin (EPO) on experimental uterus IRI. Eighty rats, randomly assigned into eight groups (n = 10/group), were pretreated with either saline, 5 μg/kg human relaxin-2, 4000 IU/kg recombinant human erythropoietin or their combination. Ischemia was achieved by clamping the aorta and ovarian arteries for 60 min, following 120 min of reperfusion and tissue sampling. For sham animals, clamping was omitted during surgery. There were no differences in tissue histological score, malondialdehyde (MDA) and superoxide dismutase (SOD) levels, myeloperoxidase (MPO) and TUNEL-positive cell count between all sham-operated rats. Pretreatment with RLX preserved normal tissue morphology, reduced MDA levels, MPO and TUNEL-positive cell count, preserved SOD activity and upregulated NICD and HES1 gene expression when compared to the control group. Pretreatment with EPO reduced MDA levels. In conclusion, pretreatment with RLX, EPO or a combination of both EPO and RLX significantly alleviates uterine tissue damage caused by IRI. [ABSTRACT FROM AUTHOR]

A number of drugs based on recombinant erythropoietin contain human serum albumin as an auxiliary component. The presence of this protein hinders the proper control of the drug quality in accordance with the requirements of regulating agencies. We propose the novel method for separation of recombinant erythropoietin (epoetin beta) and human serum albumin. It is based on the subsequent use of hydrophobic sorbent and anion exchange resin placed in gravity flow columns (without the use of spin-columns). The proposed approach makes it possible to concentrate and purify the preparations containing the epoetin beta both at high and at minimal concentrations (the ratio of the amount of albumin and erythropoietin in the used preparations can reach 125:1). The average yield of epoetin beta after the use of hydrophobic sorbent and anion exchange resin was 75 % and 97 %, respectively. It was shown that the determined conditions of sample preparation had no affect on the content of the epoetin beta in the product. [Display omitted] • Epoetin beta and HSA well separated with hydrophobic and anion-exchange sorbents. • Epoetin beta preparations with high content of HSA had been concentrated and purified. • Sample preparation conditions have not an effect on the content of related impurities. [ABSTRACT FROM AUTHOR]

Recombinant human erythropoietin (rhEPO) has been shown to exert anti-apoptotic and anti-inflammatory effects after cerebral ischemia. Inflammatory cytokines interleukin-1β and -18 (IL-1β and IL-18) are crucial mediators of apoptosis and are maturated by multiprotein complexes termed inflammasomes. Microglia are the first responders to post-ischemic brain damage and are a main source of inflammasomes. However, the impact of rhEPO on microglial activation and the subsequent induction of inflammasomes after ischemia remains elusive. To address this, we subjected human microglial clone 3 (HMC-3) cells to various durations of oxygen-glucose-deprivation/reperfusion (OGD/R) to assess the impact of rhEPO on cell viability, metabolic activity, oxidative stress, phagocytosis, migration, as well as on the regulation and activation of the NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes. Administration of rhEPO mitigated OGD/R-induced oxidative stress and cell death. Additionally, it enhanced metabolic activity, migration and phagocytosis of HMC-3. Moreover, rhEPO attenuated post-ischemic activation and regulation of the NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes as well as their downstream effectors CASPASE1 and IL-1β. Pharmacological inhibition of NLRP3 via MCC950 had no effect on the activation of CASPASE1 and maturation of IL-1β after OGD/R, but increased protein levels of NLRP1, NLRC4, and AIM2, suggesting compensatory activities among inflammasomes. We provide evidence that EPO-conveyed anti-inflammatory actions might be mediated via the regulation of the inflammasomes. [ABSTRACT FROM AUTHOR]

Purpose: Treatment with recombinant human erythropoietin (rHuEPO) may correct anemia in patients with chronic kidney disease. However, up to 10% of these patients exhibit EPO resistance or hyporesponsiveness, which may be caused by the depletion of erythroid progenitor cells. Thrombopoietin (TPO) stimulates the self-renewal of stem cells and promotes the growth of early erythroid progenitor cells. The objective of this study was to determine whether the combination of recombinant human TPO (rHuTPO) and rHuEPO could correct the depletion of erythroid precursor cells to treat EPO-resistant anemia. Methods: To test our hypothesis, pharmacokinetic (PK) and pharmacodynamic (PD) studies of rHuEPO and rHuTPO were performed in healthy rats. Rats received rHuEPO or rHuTPO alone or in combination. Plasma concentrations of rHuTPO and rHuEPO were measured. PD responses, including erythropoietic and thrombopoietic responses, were assessed in peripheral blood. Results: On one hand, the results demonstrated the synergistic effect of the combination of rHuEPO and rHuTPO on erythropoiesis. Compared with rHuEPO monotherapy, the combination therapies further stimulated the production of red blood cells and hemoglobin. On the other hand, rHuEPO inhibited the platelet production induced by rHuTPO and mitigate the risk of blood clots. Furthermore, we successfully developed a mechanism-based PD model to simultaneously characterize the responses of the two molecules. Conclusions: Overall, our study indicated that a combination therapy of rHuTPO and rHuEPO could be used to treat EPO-resistant anemia and provided a quantitative basis for further optimizing the combination therapy for clinical use. [ABSTRACT FROM AUTHOR]

Background: Erythroferrone (ERFE) is a hormone identified recently as a master regulator connecting iron homeostasis and erythropoiesis. Serum ERFE concentrations significantly increase in animals and humans with normal or impaired kidney function after receiving exogenous erythropoiesis-stimulating agents (ESAs), which suggests it might be a predictive factor for erythropoiesis. To evaluate whether ERFE is an early, sensitive biomarker for long-term erythropoietic effects of ESAs, we investigated the relationship between ERFE dynamics and time courses of major erythropoietic responses to ESA treatment. Methods: Healthy rats received single dose and multiple doses (thrice a week for 2 weeks) of recombinant human erythropoietin (rHuEPO) at three dose levels (100, 450, and 1350 IU/kg) intravenously. The rHuEPO and ERFE concentrations in plasma were determined at a series of time points after dosing. Erythropoietic effects including red blood cell count and hemoglobin concentrations were continuously monitored for 24 days (single dose) or 60 days (multiple doses). The expansion of erythroblasts in bone marrow was quantified by flow cytometry analysis. Results: ERFE significantly increased within a few hours and return to baseline at 24 h after rHuEPO treatment. The ERFE response was enhanced after repeated treatment, which was consistent with the observed expansion of erythroblasts in the bone marrow. In addition, the dynamics of ERFE showed double peaks at approximately 2 and 10 h after rHuEPO stimulation, and the ERFE baseline displayed a significant circadian rhythm. There was a strong positive correlation between peak values of short-term ERFE responses and the long-term hemoglobin responses. Conclusion: The stimulated release of ERFE is a rapid process within 24 h. The second peak in the ERFE response to rHuEPO suggests the presence of a feedback mechanism counterregulating the ESA stimulation. The early increase of ERFE at 2 h appears to be a predictor of the hemoglobin response at 14 days after single dose of rHuEPO. Under multiple-dose regimen, the enhanced ERFE responses still correlate with the peak hemoglobin responses. The ERFE baseline also exhibits a circadian rhythm. [ABSTRACT FROM AUTHOR]

Background: Anti-erythropoietin (EPO) antibodies against a recombinant form of Erythropoietin were associated with pure red cell aplasia. These antibodies probably cross-react with endogenous EPO causing severe anemia. Objective: This study was done to estimate the prevalence of anti-EPO antibody in prevalent hemodialysis patients and its relation to anemia and EPO resistance. Patients and Methods: This is cross-sectional study included 90 End-Stage Renal Disease (ESRD) patients who were on recombinant human erythropoietin for > 6 months with hemoglobin< 11 g/dL. Serum anti-EPO antibody was measured for all Patients that were divided into 2 groups: anti-EPO antibody positive and anti-EPO antibody negative. Results: The prevalence of anti-EPO antibodies was 45.6% (41 patients). 58.5% (24 patients) received EPO treatment by S.C route. Anti-EPO antibody-positive patients Showed lower hemoglobin level, serum iron, and Transferrin saturation with mean ±SD values 8.60±0.99 g/dl, 52.91±16.30 mcg/dl, 22.62±8.27 % respectively also lower MCHC with median (IQR)29.7 (2.50) g/dl. On comparing it with antibody-negative patients (N=49) there were significant differences with p values (< 0.0001), (0.038), (0.034), (0.002) respectively. These patients received higher EPO doses with high erythropoietin resistance index with median (IQR) values 8000 (4000) IU/week, 10.79 (8.495) respectively. The anti-EPO antibody was negatively correlated with hemoglobin {r-0.661, p value 0.0001} and positively correlated with EPO doses, iron doses, and erythropoietin resistance index {r 0.309, p value 0.011, r 0.266, p value 0.003, r 0.417, p value 0.0001} respectively. Conclusions: It could be concluded that anti-EPO antibodies are commonly present in prevalent hemodialysis patients and were associated with resistant anemia and higher doses of EPO therapy. [ABSTRACT FROM AUTHOR]

Abstract Objectives To evaluate the effect of recombinant erythropoietin on hospitalised COVID-19 patients. Trial design Concealed, randomized, single-blinded, phase 2 controlled clinical trial with two arm parallel-group design of 20 patients allocated with 1:1 ratio and using the placebo in the control group. Participants This study will be performed at Shahid Mohammadi Hospital in Bandar Abbas, Hormozgan in Iran. All positive (PCR confirmed) COVID-19 patients ≤65 years old who have Hb≤9 and at least one of the severe COVID-19 symptoms (tachypnea (breathing rate> 30 beats per minute), hypoxemia (O2 ≤93 saturation, the partial pressure ratio of arterial oxygen 50% of lung field within 24 to 48 hours), progressive lymphopenia, LDH>245 U/I, CRP>100) and are willing to cooperate in this project will be included in the study. Patients with a history of coronary heart disease, thrombosis, deep vein thrombosis, chronic lung disease, diabetes mellitus, weakened immune system, end-stage renal disease, liver disease, and patients with a history of taking oral contraceptive pills, systolic blood pressure more than 160 mm Hg, diastolic blood pressure more than 90 mm Hg and age over 65 and erythropoietin above 500 are excluded. Intervention and comparator Patients will receive the standard of care (SOC) based on the treatment protocols of the Iranian National Committee of COVID-19 and recombinant erythropoietin (EPREX Manufactured by Johnson and Johnson Pharmaceutical Company) 300 units / Kg or 4000IU as subcutaneous (SQ) injection three times a day for 5 days and simultaneously Enoxaparin 1 mg/kg SQ daily is also taken to prevent thrombosis in the intervention group. Patients' blood pressure, along with other vital signs, are checked regularly and at regular intervals. In the control group, patients received SOC and the placebo (distilled water) is given as a subcutaneous injection three times a day for 5 days. We use sterile water for injection (EXIRpharmaceutical company) as the placebo. To the same appearance of the placebo and the recombinant erythropoietin, they are taken in a separate room in the same size syringes and cover with labels before injection. Main outcomes The main outcome for this study is a composite endpoint for Patient clinical symptoms (Respiratory rate, Oxygen saturation state and arterial oxygen partial pressure ratio, Lung infiltration status, blood pressure), Laboratory tests (LDH, CRP, Lymphocyte count, Endogenous erythropoietin, and Haemoglobin level). All of these will be assessed at the beginning of the study (before the intervention) and day 5 after the intervention. The study will also evaluate side effects and how to manage them. Randomisation Eligible participants (20) will be randomized in two arms in the ratio of 1: 1 (10 per arm) by permuted block randomization method using online web-based tools. Blinding (masking) Patients participating in the study will not be aware of the assignment to the intervention or control group. The principal investigator, health care personnel, data collectors, and those evaluating the outcome are aware of patient grouping. Numbers to be randomised (sample size) A total of 20 patients will participate in this study, who are randomly allocated to the 2 arms with a 1:1 ratio; 10 patients in the intervention group will receive SOC and recombinant erythropoietin, and 10 patients in the control group will receive SOC and placebo. Trial Status The protocol version is 3.0, approved by the Deputy of Research and Technology and the ethics committee of Hormozgan University of Medical Sciences on 6th June 2020, with the local grant number of 990108. The expected recruitment end date was on 21th December 2020 but since we had a wide and careful exclusion criteria because of the adverse reactions of the medication, the recruitment (for both cases and controls) was not so easy and did not finish on the expected date and we are still recruiting now. Recruitment began on 17th August 2020 and the updated expected recruitment end date is 1st August 2021. Trial registration The protocol was registered before starting subject recruitment under the title: Evaluation of the effect of recombinant erythropoietin on the improvement of COVID-19 patients, IRCT20200509047364N1, at Iranian Registry of clinical trials ( https://en.irct.ir/trial/49282 ) on 2020/08/09. Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).

Background: Treatment with erythropoietin is well established for anemia in chronic kidney disease patients but not well studied in acute kidney injury. Methods: This is a multicenter, randomized, pragmatic controlled clinical trial. It included 134 hospitalized patients with anemia defined as hemoglobin < 11 g/dL and acute kidney injury defined as an increase of serum creatinine of ≥ 0.3 mg/dL within 48 h or 1.5 times baseline. One arm received recombinant human erythropoietin 4000 UI subcutaneously every other day (intervention; n = 67) and the second received standard of care (control; n = 67) during the hospitalization until discharge or death. The primary outcome was the need for transfusion; secondary outcomes were death, renal recovery, need for dialysis. Results: There was no statistically significant difference in transfusion need (RR = 1.05, 95%CI 0.65,1.68; p = 0.855), in renal recovery full or partial (RR = 0.96, 95%CI 0.81,1.15; p = 0.671), in need for dialysis (RR = 11.00, 95%CI 0.62, 195.08; p = 0.102) or in death (RR = 1.43, 95%CI 0.58,3.53; p = 0.440) between the erythropoietin and the control group. Conclusions: Erythropoietin treatment had no impact on transfusions, renal recovery or mortality in acute kidney injury patients with anemia. The trial was registered on ClinicalTrials.gov (NCT03401710, 17/01/2018). [ABSTRACT FROM AUTHOR]

Anemia is a common complication of chronic kidney disease (CKD). Recombinant human erythropoietin (rHu-EPO) is used extensively in patients with CKD. However, anti-erythropoietin (anti-EPO) antibody has been reported during rHu-EPO treatment, which causes pure red cell aplasia (PRCA). We presented a case of 75-year-old man, who underwent hemodialysis for 2 years. He developed PRCA during rHu-EPO treatment. The rHu-EPO was immediately discontinued, and the patient was given roxadustat treatment. After 6 months of roxadustat treatment, the anti-EPO antibody was disappeared, and hemoglobin recovered normal range. The results suggest that roxadustat can be used to treat patients with anti-EPO antibody-mediated PRCA without immunosuppressive therapy. [ABSTRACT FROM AUTHOR]

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients. The in‐vivo activity of EPO is carbohydrate‐dependent with the number of sialic acid residues regulating its circulatory half‐life. EPO carries three N‐glycans and thus obtaining pure glycoforms provides a major challenge. We have developed a robust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids. EPO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments. The glycopeptides were obtained by pseudoproline‐assisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1 complexes with recombinant EPO receptor. [ABSTRACT FROM AUTHOR]

Switching to an alternative recombinant erythropoietin agent in patients with myelodysplastic syndromes: a second honeymoon? Median time from diagnosis to first rEPO was 3-4 months (0-87), median endogenous (e)EPO was 59 U/l (3-257) and 12 patients were transfusion dependent (median 4 units/8 weeks, range 2-7). Keywords: erythropoietin; myelodysplastic syndromes; transfusion EN erythropoietin myelodysplastic syndromes transfusion e147 e150 4 11/18/21 20211115 NES 211115 Duration of response to recombinant (r)EPO in myelodysplastic syndromes (MDS) is limited and further options are scanty, such that most subjects finally become transfusion dependent. [Extracted from the article]

Anemia is a prevalent complication endured by patients with chronic renal disease. Renal anemia also leads to the development of cardio-vascular complications. Epoetin alpha and beta are recombinant human erythropoietin prioritized for managing anemia in hemodialysis patients. The current study aimed to compare the therapeutic efficacy of both erythropoietin alpha and erythropoietin beta in treating renal anemia. This prospective observational study was conducted in a Renal Dialysis Centre at a tertiary care Hospital of Karachi, Pakistan for a period of 3 months. The two erythropoietin products used were human recombinant erythropoietin alpha (Tropin®) and erythropoietin beta (Recormon®). Both groups were age-matched, BMI, eGFR, gender, and comorbidities like diabetes and hypertension were indifferent. The comparative analysis was performed after the completion of 3 months. A total of 94 participants were included in the analysis, 54 in group A and 40 in group B. Mean albumin, urea, creatinine, ferritin, iron, and transferrin saturation at inclusion were statistically insignificant, TIBC was higher in group A (p = 0.005) and CRP levels were slightly higher in group B (p = 0.050). There was significant improvement in Hb level (p = 0.025), PCV (p = 0.001), and RBC count (p = 0.007) in group B. While in group A, there was significantly increased MCV (p = 0.005) and MCHC (p = 0.002). In intention to treat analysis, 22.2% of subjects in group A and 40.0% in group B reached desired Hb levels of ≥11 g/l after 3 months. In our assessment of hemodialysis patients, erythropoietin beta was found more effective than erythropoietin alpha. [ABSTRACT FROM AUTHOR]

Background: Since fall 2019, SARS-CoV-2 spread world-wide, causing a major pandemic with estimated ~ 220 million subjects affected as of September 2021. Severe COVID-19 is associated with multiple organ failure, particularly of lung and kidney, but also grave neuropsychiatric manifestations. Overall mortality reaches > 2%. Vaccine development has thrived in thus far unreached dimensions and will be one prerequisite to terminate the pandemic. Despite intensive research, however, few treatment options for modifying COVID-19 course/outcome have emerged since the pandemic outbreak. Additionally, the substantial threat of serious downstream sequelae, called 'long COVID' and 'neuroCOVID', becomes increasingly evident. Among candidates that were suggested but did not yet receive appropriate funding for clinical trials is recombinant human erythropoietin. Based on accumulating experimental and clinical evidence, erythropoietin is expected to (1) improve respiration/organ function, (2) counteract overshooting inflammation, (3) act sustainably neuroprotective/neuroregenerative. Recent counterintuitive findings of decreased serum erythropoietin levels in severe COVID-19 not only support a relative deficiency of erythropoietin in this condition, which can be therapeutically addressed, but also made us coin the term 'hypoxia paradox'. As we review here, this paradox is likely due to uncoupling of physiological hypoxia signaling circuits, mediated by detrimental gene products of SARS-CoV-2 or unfavorable host responses, including microRNAs or dysfunctional mitochondria. Substitution of erythropoietin might overcome this 'hypoxia paradox' caused by deranged signaling and improve survival/functional status of COVID-19 patients and their long-term outcome. As supporting hints, embedded in this review, we present 4 male patients with severe COVID-19 and unfavorable prognosis, including predicted high lethality, who all profoundly improved upon treatment which included erythropoietin analogues. Short conclusion: Substitution of EPO may—among other beneficial EPO effects in severe COVID-19—circumvent downstream consequences of the 'hypoxia paradox'. A double-blind, placebo-controlled, randomized clinical trial for proof-of-concept is warranted. [ABSTRACT FROM AUTHOR]