Erythropoietin Formulations Research Articles

A synthetic approach to 'click' neoglycoprotein analogues of EPO employing one-pot native chemical ligation and CuAAC chemistry.

The clinical significance of batch-wise variability on the pharmacokinetics and potency of commercial erythropoietin (EPO), prepared recombinantly as a heterogeneous mixture of glycoforms, necessitates the development of synthetic strategies to afford homogenous EPO formulations. Herein we present a previously unexplored and divergent route towards 'click' neoglycoprotein analogues of EPO, employing one-pot native chemical ligation (NCL) of alkynylated peptides and copper-catalysed azide-alkyne cycloaddition (CuAAC) with azido monosaccharides. By design, our synthetic platform permits glycosylation at virtually any stage, providing flexibility for the synthesis of various glycoforms for biological analysis. Insights obtained from attempted folding of our 'click' neoglycoprotein EPO analogue, bearing four different neutral sugar moieties, highlight the important role played by the charged oligosaccharides present in native EPO glycoproteins.

Semi-mechanistic Pharmacokinetic/Pharmacodynamic model of three pegylated rHuEPO and ior®EPOCIM in New Zealand rabbits

Marketed formulations of erythropoietin (EPO) ior®EPOCIM, MIRCERA® and two newly developed pegylated-EPO analogues (PEG-EPO 32 and 40 kDa) formulations were intravenously administered to New Zealand rabbits. A semi-mechanistic Pharmacokinetic/Pharmacodynamic (PK/PD) model describing in a simultaneous and integrated form the time course of reticulocytes, red blood cells and hemoglobin was built to account for the time course of hematopoiesis stimulation after erythropoietin administration. Data analysis was performed based on the population approach with the software NONMEM version 7.3. Erythropoietin disposition of each of the administered formulations was best described with a two compartment model and linear elimination. Different formulations show different clearance and apparent volume of distribution of the central compartment but share estimates of inter-compartmental clearance and apparent peripheral volume of distribution. A semi-mechanistic model including cell proliferation, maturation, and homeostatic regulation provided a good description of the data regardless the type of erythropoietin formulation administered. The system-, and drug-related parameters showed consistency and differed across formulations, respectively. A single IV administration of PEG-EPO 32 and 40 kDa formulations in New Zealand rabbits achieves a median change of 27% and 22% on RET levels, and of 47% and 63% on RBC and HGB levels, respectively compared to MIRCERA®. The administration of new branched PEG-chains formulations improves PK and PD properties of EPO, in terms of increasing elimination half-lives and pharmacological activity on RET, RBC and HGB compared to commercially available formulations (ior®EPOCIM and MIRCERA®).

Revisiting the role of erythropoietin for treatment of ocular disorders.

Erythropoietin (EPO) is a glycoprotein hormone conventionally thought to be responsible only in producing red blood cells in our body. However, with the discovery of the presence of EPO and EPO receptors in the retinal layers, the EPO seems to have physiological roles in the eye. In this review, we revisit the role of EPO in the eye. We look into the biological role of EPO in the development of the eye and the physiologic roles that it has. Apart from that, we seek to understand the mechanisms and pathways of EPO that contributes to the therapeutic and pathological conditions of the various ocular disorders such as diabetic retinopathy, retinopathy of prematurity, glaucoma, age-related macular degeneration, optic neuritis, and retinal detachment. With these understandings, we discuss the clinical applications of EPO for treatment of ocular disorders, modes of administration, EPO formulations, current clinical trials, and its future directions.

Optimization of amino acid-stabilized erythropoietin parenteral formulation: In vitro and in vivo assessment.

The aim of this study was to optimize the formulation of erythropoietin (EPO) using amino acids instead of human serum albumin (HSA) and to evaluate its in vivo stability in order to avoid the risk of viral contamination and antigenicity. Different EPO formulations were developed in such a way as to allow studying the effects of amino acids and surfactants on the EPO stability profile. The main techniques applied for EPO analysis were ELISA, Bradford method, and SDS gel electrophoresis. The in vivo stability was evaluated in a Balb-c mouse animal model. The results showed that the presence of surfactant was very useful in preventing the initial adsorption of EPO on the walls of vials and in minimizing protein aggregation. Amino acid combinations, glycine with glutamic acid, provided maximum stability. Formulation F4 (containing glycine, glutamic acid and Tween 20) showed minimum aggregation and degradation and in vivo activity equivalent to commercially available HSA-stabilized EPO (Eprex®).

Chemical Modifications of Therapeutic Proteins Induced by Residual Ethylene Oxide

Ethylene oxide (EtO) is widely used in sterilization of drug product primary containers and medical devices. The impact of residual EtO on protein therapeutics is of significant interest in the biopharmaceutical industry. The potential for EtO to modify individual amino acids in proteins has been previously reported. However, specific identification of EtO adducts in proteins and the effect of residual EtO on the stability of therapeutic proteins has not been reported to date. This paper describes studies of residual EtO with two therapeutic proteins, a PEGylated form of the recombinant human granulocyte colony-stimulating factor (Peg-GCSF) and recombinant human erythropoietin (EPO) formulated with human serum albumin (HSA). Peg-GCSF was filled in an EtO sterilized delivery device and incubated at accelerated stress conditions. Glu-C peptide mapping and LC-MS analyses revealed residual EtO reacted with Peg-GCSF and resulted in EtO modifications at two methionine residues (Met-127 and Met-138). In addition, tryptic peptide mapping and LC-MS analyses revealed residual EtO in plastic vials reacted with HSA in EPO formulation at Met-328 and Cys-34. This paper details the work conducted to understand the effects of residual EtO on the chemical stability of protein therapeutics.

Physicochemical and Biological Evaluation of Bioadhesive Polymers for the Development of Intranasal Low Sialic Acid Erythropoietin Formulations

Physicochemical and Biological Evaluation of Bioadhesive Polymers for the Development of Intranasal Low Sialic Acid Erythropoietin Formulations

Comparison of the Therapeutic Effects of Two Recombinant Erythropoietin Beta, Biosimilar and Reference Formulations in Patients with Chronic Kidney Disease under Hemodialysis

Anemia is common in patients with chronic kidney diseases under hemodialysis and it’s managed with recombinant erythropoietin formulation. The objective of the study is to compare the safety and efficacy of two recombinant erythropoietin formulations in patients undergoing hemodialysis with chronic kidney diseases. In this randomized, controlled, prospective, parallel open study 70 patients were treated for 24 weeks with either reference β recombinant erythropoietin (100 IU/kg) or biosimilar β recombinant erythropoietin (100 IU/kg). The primary efficacy endpoint was the hemoglobin and hematocrit levels from baseline to 24 week of treatment. The secondary endpoints were safety, weekly doses of both erythropoietins required to maintain hemoglobin levels and immunogenicity. There was no significant difference between the two preparations in terms of hemoglobin and hematocrit levels achieved. The weekly doses of both erythropoietins required to maintain hemoglobin levels were the same in both groups. The frequency of adverse events was similar in the two groups of treatment. Two patients of the reference erythropoietin group developed anti-erythropoietin antibodies. The biosimilar erythropoietin was comparable since the safety and efficacy point of view with the innovator erythropoietin in hemodialysis patients based on hemoglobin changes. The biosimilar erythropoietin when administered subcutaneously will be equally efficacious and may be interchangeable as therapy.

Intranasal formulation of erythropoietin (EPO) showed potent protective activity against amyloid toxicity in the Aβ25-35 non-transgenic mouse model of Alzheimer’s disease

Erythropoietin (EPO) promotes neurogenesis and neuroprotection. We here compared the protection induced by two EPO formulations in a rodent model of Alzheimer's disease (AD): rHu-EPO and a low sialic form, Neuro-EPO. We used the intracerebroventricular administration of aggregated Aβ₂₅₋₃₅ peptide, a non-transgenic AD model. rHu-EPO was tested at 125-500 µg/kg intraperitoneally and Neuro-EPO at 62-250 µg/kg intranasally (IN). Behavioural procedures included spontaneous alternation, passive avoidance, water-maze and object recognition, to address spatial and non-spatial, short- and long-term memories. Biochemical markers of Aβ₂₅₋₃₅ toxicity in the mouse hippocampus were examined and cell loss in the CA1 layer was determined. rHu-EPO and Neuro-EPO led to a significant prevention of Aβ₂₅₋₃₅-induced learning deficits. Both EPO formulations prevented the induction of lipid peroxidation in the hippocampus, showing an antioxidant activity. rHu-EPO (250 µg/kg) or Neuro-EPO (125 µg/kg) prevented the Aβ₂₅₋₃₅-induced increase in Bax level, TNFα and IL-1β production and decrease in Akt activation. A significant prevention of the Aβ₂₅₋₃₅-induced cell loss in CA1 was also observed. EPO is neuroprotective in the Aβ₂₅₋₃₅ AD model, confirming its potential as an endogenous neuroprotection system that could be boosted for therapeutic efficacy. We here identified a new IN formulation of EPO showing high neuroprotective activity. Considering its efficacy, ease and safety, IN Neuro-EPO is a new promising therapeutic agent in AD.

Erythropoietin neuroprotection is enhanced by direct cortical application following subdural blood evacuation in a rat model of acute subdural hematoma

Recombinant human erythropoietin (EPO) has been successfully tested as neuroprotectant in brain injury models. The first large clinical trial with stroke patients, however, revealed negative results. Reasons are manifold and may include side-effects such as thrombotic complications or interactions with other medication, EPO concentration, penetration of the blood–brain-barrier and/or route of application. The latter is restricted to systemic application. Here we hypothesize that EPO is neuroprotective in a rat model of acute subdural hemorrhage (ASDH) and that direct cortical application is a feasible route of application in this injury type. The subdural hematoma was surgically evacuated and EPO was applied directly onto the surface of the brain.We injected NaCl, 200, 2000 or 20,000IU EPO per rat i.v. at 15min post-ASDH (400μl autologous venous blood) or NaCl, 0.02, 0.2 or 2IU per rat onto the cortical surface after removal of the subdurally infused blood t at 70min post-ASDH. Arterial blood pressure (MAP), blood chemistry, intracranial pressure (ICP), cerebral blood flow (CBF) and brain tissue oxygen (ptiO2) were assessed during the first hour and lesion volume at 2days after ASDH.EPO 20,000IU/rat (i.v.) elevated ICP significantly. EPO at 200 and 2000IU reduced lesion volume from 38.2±0.6mm3 (NaCl-treated group) to 28.5±0.9 and 22.2±1.3mm3 (all p<0.05 vs. NaCl). Cortical application of 0.02IU EPO after ASDH evacuation reduced injury from 36.0±5.2 to 11.2±2.1mm3 (p=0.007), whereas 0.2IU had no effect (38.0±9.0mm3). The highest dose of both application routes (i.v. 20,000IU; cortical 2IU) enlarged the ASDH-induced damage significantly to 46.5±1.7 and 67.9±10.4mm3 (all p<0.05 vs. NaCl). In order to test whether Tween-20, a solvent of EPO formulation ‘NeoRecomon®’ was responsible for adverse effects two groups were treated with NaCl or Tween-20 after the evacuation of ASDH, but no difference in lesion volume was detected.In conclusion, EPO is neuroprotective in a model of ASDH in rats and was most efficacious at a very low dose in combination with subdural blood removal. High systemic and topically applied concentrations caused adverse effects on lesion size which were partially due to increased ICP. Thus, patients with traumatic ASDH could be treated with cortically applied EPO but with caution concerning concentration.

Aluminum in Erythropoietin Formulations: Lyophilized versus Liquid Forms

Background: Erythropoietin (EPO) formulations may comprise aluminum (Al) as a contaminant. Due to the toxicity of Al in chronic kidney disease patients, possible sources of Al were investigated. Since EPO formulations are stored in container-closure systems made of glass and rubber, and both contain Al, formulation ingredients may enable its leaching into the solution during shelf-life. Methods: Individual solutions of formulation ingredients were stored in new glass vials and in contact with the rubber stopper and kept at 4 ± 2°C. For 12 months, aliquots of each solution were collected for analysis. Fifteen commercial samples of EPO were analyzed for their Al content. Aluminum was determined by atomic absorption spectrometry. Results: Glass and rubber are sources of Al for EPO formulations. Storage assay showed that citrate and phosphate (used as buffers) extracted high amounts of Al from the container/closure parts. The most important difference, however, was found when comparing liquid and lyophilized samples. While in liquid forms the Al level reached 943 μg/L, in lyophilized forms the level did not exceed 20 μg/L. The container system was also confirmed as a source of Al in reconstituted lyophilized samples. Al in reconstituted samples stored in their own vials increased 19-fold in 12 months. Lyophilized powders stored for 2 years in glass vials contained less Al than in 1 month after dissolution. Conclusion: The difference in the Al measured in liquid forms of EPO and in lyophilized powders suggests that the latter would be the best pharmaceutical form for CKD patients.

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

BackgroundApart from its hematopoietic effect, erythropoietin(EPO) is a molecule with high neuroprotective potential.However, its prolonged application may cause seriousadverse effects due to the erythropoiesis stimulation.Therefore, an EPO derivative with neuroprotective prop-erties but low hematopoietic activity, designated as neu-ropoietin (rhNEPO), was developed in our lab using analternative purification process of the recombinanthuman erythropoietic counterpart (rhEPO) produced inCHO cells [1]. The in vitro cytoprotective activity ofrhNEPO on neural phenotype cells and its brain uptakefrom blood are herein analyzed.ResultsIn vitro citoprotective activity of rhNEPO was analyzedon rat pheochromocytoma cells (PC-12) differentiated toneural phenotype with neural growth factor (NGF).Apoptosis was triggered by NGF and serum withdrawalfrom cell cultures. Thus, nuclear DNA fragmentationwas analyzed by colorimetric TUNEL detection. One-way analysis of variance was carried out followed byDunnett´s multiple comparison test. Probabilities lowerthan 0.05 were considered significant (p 0.05), confirming their prop-erties to protect PC-12 cells from apoptosis. Therefore,this novel combination of erythropoietin glycoforms(rhNEPO) with lower sialic acid content and antennaritythan rhEPO [1] preserved its binding receptor capacityexerting an in vitro neuroprotective activity even betterthan the mentioned counterpart. Also, rhAEPO showedan in vitro activity that is similar to that of rhNEPO,having both derivatives the lowest content of carbohy-drates. It is well known that the affinity of EPO analo-gues for EPO receptor is inversely related to thesialylation of their attached carbohydrate [2] and thatremoval of sialic acid turns it into a molecule with avery short half-life with almost no erythropoietic activ-ity. This is the case of rhAEPO that explains its rapidhepatic clearance from blood [3]. For that reason,rhNEPO emerges as a neuroprotective candidate dis-playing higher in vitro activity than rhEPO.Taking into account the cytoprotective activity ofrhNEPO on neural phenotype cells, cerebrospinal fluid(CSF) and blood pharmacokinetics of rhEPO andrhNEPO were evaluated in rats following intravenousadministration of a single dose of each protein, aimingto evaluate their CSF uptake from plasma.The distribution and the elimination half-lives ofrhNEPO in blood were significantly shorter than thecorresponding ones for rhEPO. Differences in the sialic

Evidence for aluminum-binding erythropoietin by size-exclusion chromatography coupled to electrothermal absorption atomic spectrometry

Erythropoietin (EPO) is a glycoprotein that stimulates erythropoiesis and is clinically used for treating anemia during chronic renal failure and for anemia in preterm infants. EPO formulations usually have elevated rates of contamination due to aluminum (Al), which is toxic to both types of patients. Size-exclusion chromatography (SEC) coupled with graphite furnace atomic absorption spectrometry (GF AAS) was employed to separate proteins and to quantify the amount of aluminum present in the elution volume corresponding to EPO and, therefore, to evaluate possible binding. Because EPO formulations contain human serum albumin (HSA), a chromatographic method was optimized for the separation of these proteins. Subsequent to the chromatographic separation, 1-mL fractions of the column effluent were collected, and the Al content in these aliquots was measured by GF AAS. EPO and HSA samples were incubated with Al for 4 h at 4 °C and 37 °C as well as for 16 h at 4 °C and 37 °C. Afterwards, they were injected into the chromatographic system. These samples were also submitted to ultrafiltration (10 and 50 kDa membranes), and Al was measured in the ultrafiltrates. The results showed that Al was present in the eluent volume corresponding to the EPO peak but not in the HSA peak in the chromatograms. Temperature strengthened the interaction because the Al present in the EPO fraction was 3 times higher at 37 °C compared to 4 °C. Thirty-eight percent of the Al present in a 2.4 μg/mL EPO standard solution, and approximately 50% of the Al in formulation samples containing approximately 11 μg/mL EPO and either citrate or phosphate, were non-ultrafiltrable, which suggests that EPO is an effective Al acceptor in vitro.

Pharmacokinetic and pharmacodynamic comparison of two recombinant human erythropoietin formulations after single subcutaneous administration: An open-label, sequence-randomized, two-treatment crossover study in healthy Korean male volunteers

Background: Recombinant human erythropoietin is indicated for the treatment of anemia resulting from chronic renal failure or chemotherapy. It is also used for patients at high risk for transfusions because of significant blood loss during surgery. A new recombinant human erythropoietin (epoetin alfa) that excludes fetal bovine serum and human serum albumin from among its ingredients was developed in Korea. This study was planned as part of a product development project at the request of the Korean regulatory agency. Objective: The aim of this study was to compare the pharmacokinetic and pharmacodynamic characteristics of a new recombinant human erythropoietin (test) formulation with an existing branded (reference) for- mulation after a single subcutaneous administration. Methods: An open-label, sequence-randomized, 2-period, 2-sequence, 2-treatment crossover study was conducted. Healthy male subjects were randomly assigned with a random number table into 1 of 2 sequence groups, and each subject was given recombinant human erythropoietin 4000 IU SC in the upper arm as the test formulation in one period and the reference formulation in the other period, according to the sequence group. Each period was separated by a 4-week washout period. Serial blood samples were taken up to 120 hours after drug administration for the pharmacokinetic assessments and up to 240 hours for reticulocyte counts as the pharmacodynamic end point. Pharmacokinetic analysis was performed without baseline correction. Adverse events (AEs) were collected by spontaneous reporting of the subjects or solicited by asking general health-related questions. Results: Twenty healthy men (mean [range] age, 25.6 [21–36] years; height, 175 [167–187] cm; weight, 70 [57.6–85.5] kg) were enrolled in and completed the study. The mean (SD) baseline erythropoietin plasma concentrations were 10.4 (2.4) mIU/mL for the test formulation and 10.8 (3.5) mIU/mL for the reference formulation. After the injection of 4000 IU SC per subject, the erythropoietin plasma concentrations reached a maximum at a median T max of 10 hours for both formulations (range: test formulation, 7.00–95.95 hours; reference formulation, 6.98–24.13 hours). The mean (SD) C max values for the test and reference formulations were 74.34 (30.63) and 80.46 (30.56) mIU/mL, respectively; the mean AUC 0–last values were 3664 (731.5) and 3553 (723.2) mIU·h/mL. The ratios of the geometric mean (test/reference) for C max and AUC 0–last were 0.92 (90% CI, 0.81–1.05) and 1.03 (90% CI, 0.98–1.09). The mean baseline hemoglobin, hematocrit, and reticulocyte counts were 15.4 g/dL, 45.5%, and 49.6 · 10 3/μL, respectively, for the test formulation and 15.5 g/dL, 45.3%, and 47.5 · 10 3/μL for the reference formulation. The mean reticulocyte counts slowly reached T max for both formulations at a median of 120 hours after administration (test formulation, 120.0 hours [range, 95.5–240.8 hours]; reference formulation, 120.1 hours [range, 72.0–240.5 hours]). The mean (SD) maximum reticulocyte counts for the test and reference formulations were 77.7 (12.2) · 10 3/μL and 80.7 (15.2) · 10 3/μL, respectively; values for area under the effect curve to the last observation (AUEC 0–last) were 14,781.5 (2439.2) · 10 3/μL · h and 14,783.8 (2415.4) · 10 3/μL · h. The 2 agents did not exhibit any significant differences in maximum reticulocyte counts or AUEC 0–last. During the study, a total of 6 AEs were reported, which were mild in severity. After the administration of test formulation, 1 case each of rhinorrhea, epigastric discomfort, and joint sprain (left ankle) were reported. After the administration of reference formulation, 2 cases of rhinorrhea and 1 case of cough were reported. Conclusion: In this small, selected group of healthy male volunteers, there were no significant differences in pharmacokinetic parameters or effects on reticulocytes between a test formulation and a reference formulation of recombinant human erythropoietin.

Determination of metallic contaminants in human recombinant erythropoietin (EPO) formulations by adsorptive cathodic stripping voltammetry and UV photo-decomposition of samples

The contamination of renal failure patients by metals can be associated to the presence of toxic metals in the medication, since they can be absorbed by the organism. Among the possible contaminants, there are nephrotoxic metals such as aluminum, chromium and nickel. In the present work, the presence of Al, Cr and Ni as contaminants in erythropoietin formulations (EPO) was investigated by optimizing the adsorptive cathodic stripping voltammetric (AdCSV) method and the photo-decomposition of samples by UV irradiation. The UV irradiation of acidified EPO samples from 2 to 3 h in the presence of H2O2 allowed the efficient sample decomposition and the determination of the contaminants by AdCSV without matrix interferences. The method was applied for the determination of the metals in commercial EPO formulations used routinely in the treatment of anemia in patients undergoing chronic renal failure. The contamination levels determined in the samples ranged from 420.2 to 840.5 ng mL-1 (RSD 2-5%) for Al, 12.0 to 40.0 ng mL-1 (RSD 2-6%) for Cr and 4.1 to 8.2 ng mL-1 (RSD 3-8%). The results obtained by AdCSV were in good agreement with those obtained by the comparative GFAAS measurements. The contamination of EPO formulations with Al, Cr and Ni was shown to be a serious contamination source for anemic patients undergoing chronic renal failure.

Comparison of two recombinant erythropoietin formulations in patients with anemia due to end-stage renal disease on hemodialysis: A parallel, randomized, double blind study

BackgroundRecombinant human erythropoietin (EPO) is used for the treatment of last stage renal anemia. A new EPO preparation was obtained in Cuba in order to make this treatment fully nationally available. The aim of this study was to compare the pharmacokinetic, pharmacodynamic and safety properties of two recombinant EPO formulations in patients with anemia due to end-stage renal disease on hemodialysis.MethodsA parallel, randomized, double blind study was performed. A single 100 IU/Kg EPO dose was administered subcutaneously. Heberitro (Heber Biotec, Havana, formulation A), a newly developed product and Eprex (CILAG AG, Switzerland, formulation B), as reference treatment were compared. Thirty-four patients with anemia due to end-stage renal disease on hemodialysis were included. Patients had not received EPO previously. Serum EPO level was measured by enzyme immunoassay (EIA) during 120 hours after administration. Clinical and laboratory variables were determined as pharmacodynamic and safety criteria until 216 hours.ResultsBoth groups of patients were similar regarding all demographic and baseline characteristics. EPO kinetics profiles were similar for both formulations; the pharmacokinetic parameters were very close (i.e., AUC: 4667 vs. 4918 mIU.h/mL; Cmax: 119.1 vs. 119.7 mIU/mL; Tmax: 13.9 vs. 18.1 h; half-life, 20.0 vs. 22.5 h for formulations A and B, respectively). The 90% confidence intervals for the ratio between both products regarding these metrics were close to the 0.8 – 1.25 range, considered necessary for bioequivalence. Differences did not reach 20% in any case and were not determined by a formulation effect, but probably by a patients' variability effect. Concerning pharmacodynamic features, a high similitude in reticulocyte counts increments until 216 hours and the percentage decrease in serum iron until 120 hours was observed. There were no differences between formulations regarding the adverse events and their intensity. The more frequent events were pain at injection site (35.3%) and hypertension (29%). Additionally, further treatment of the patients with the study product yielded satisfactory increases in hemoglobin and hematocrit values.ConclusionThe formulations are comparable. The newly developed product should be acceptable for long-term application.