The human body generates 2.5 million new red blood cells (RBCs) per second from the bone marrow to replenish the continuous elimination of erythrocytes effect. The production of red blood cells (erythropoiesis) is controlled by a complex interaction between various humoral factors and cytokines. A specific cytokine, a sialoglycoprotein known as erythropoietin, which acts directly on certain precursors and precursors of red blood cells in the bone marrow, controls the proliferation, differentiation and maturation of red blood cells. Erythropoietin expression is significantly increased in the kidneys during the hypoxic state, a condition mediated by transcription factor HIF-1. The ultimate effect is to increase erythropoiesis in order to maintain the supply of oxygen to vital organs. This article provides an overview of erythropoietin from a historical and scientific perspective, followed by a discussion of its current and potential uses in clinical medicine. Darbepoetin alfa is a second-generation ESA that is a supersialized analogue of EPO and has two additional nitrogen-bound glycosylation chains. This property confers greater metabolic stability and a lower clearance rate in vivo, and the elimination half-life of this compound in humans after single intravenous administration is three times greater than that of epoetin alfa (25.3 hours versus 8.5 hours). Therefore, this agent can generally be administered less frequently than standard epoetins, with dosing intervals of once a week and once a week alternately.27 Unlike epoetins, the dosage requirements for darbepoetin alfa are the same for correcting anemia and maintaining Hb concentration in patients with CKD when administered intravenously and subcutaneously. The conversion factor for switching from epoetin alfa or beta to darbepoetin alfa is typically 200:1, but there can be significant differences depending on patient population, dose, and route of administration.25 Patients in the intensive care unit require regular blood transfusions. Anemic patients have unreasonably low endogenous serum erythropoietin.86 A recent prospective, multicenter, randomized trial showed that RHuEPO treatment (started with 300 U/kg subcutaneously from day 3 for five consecutive days, followed by alternating daily administration until the volume of packaged cells reaches 38%) reduced the need for blood transfusions by 50%.
In addition, there were no significant differences in mortality and frequency of adverse events compared to controls.87 Erythropoietin (EPO) is a protein hormone produced by the kidneys that plays a critical role in the production and maturation of red blood cells (RBCs), which carry oxygen from the lungs to the rest of the body. EPO contains 165 amino acids, which contributes to the relative molecular weight of about 30,600 daltons. The post-translational modification of this protein results in the addition of 4 carbohydrate chains: 3N bound glycosylation and 1 O1,2,3 bound glycosylation, after which the molecular weight of erythropoietin is increased by about 40% compared to its initial mass. Recombinant human erythropoietin (rHuEPO) is given to patients with lower haemoglobin levels because they are unable to produce enough endogenous erythropoietin. These include patients with chronic kidney disease (CKD), dialysis patients, HIV infection and malignancy. rHuEPO has also been used to accelerate erythropoiesis in surgery, after chemotherapy and after transplantation4. In fact, treating anemia with rHuEPO has been shown to improve the quality of life (QoL) of these patients5,6. The human erythropoietin gene is encoded on chromosome 7q11-22. Susantad, T., Fuangthong, M., Tharakaraman, K. et al.
Recombinant modified human erythropoietin with potentially reduced immunogenicity. Sci Rep 11, 1491 (2021). doi.org/10.1038/s41598-020-80402-1 This study was approved by the Human Research Ethics Committee of the Chulabhorn Research Institute, Thailand. All methods with human participants were carried out in accordance with the guidelines and regulations of the Institutional Ethics Committee. All volunteers gave their written consent. Blood was drawn from healthy volunteers in vacuum tubes treated with EDTA (Becton Dickinson). The HLA-DRB1 type was determined using sequence-specific oligonucleotide primer PCR (PCR-SSO) using Luminex technology at Chulalongkorn University School of Medicine, Thailand. Common infectious diseases such as hepatitis B, hepatitis C, syphilis and HIV were also studied. PBMCs were isolated using the IsoPrep solution (Robbins Scientific Corporation). Before the availability of recombinant human erythropoietin (RHuEPO), the only treatment for patients with anaemia or chronic renal failure was a blood transfusion. Unfortunately, blood transfusions had to be administered regularly to maintain hemoglobin levels. In addition, various transfusion-related problems, especially iron overload, have significantly affected the treatment and outcomes of patients with kidney disease.
Due to the promising results in the animal model, erythropoietin was considered the main candidate as a replacement therapy. Once RHuEPO was made available for human trials, a number of clinical trials were immediately conducted to evaluate its effectiveness in correcting chronic renal anemia. Early results showed that RHuEPO was able to restore the volume of packed cells, eliminate the need for regular blood transfusions in dialysis patients, and improve overall well-being.2–4 The results of these studies were so impressive that RHuEPO was approved as a therapeutic agent for patients with anemia or chronic kidney disease in 1988. only three years after its discovery. To test this hypothesis, the immunogenicity of the EPO protein was evaluated with PBMC from negative and positive HLA-DRB1*09 volunteers. Screening for HLA-DRB1*09 in volunteers living in Bangkok, Thailand, revealed that 22% of volunteers have HLA-DRB1*09, confirming the high incidence of this particular HLA in Thailand. Therefore, since the types of MHC molecules expressed in experimental animals differ from those in humans, we used an ex vivo human primary model instead of an animal model. Based on the small study group, in addition to the t-test, we also confirmed statistically significant differences in T-cell response between EPO mutants between HLA-DRB1*09-negative and positive groups with the nonparametric MannâWhitney U test. Statistical differences in pala¤â0.01 were found for all EPO mutants compared to EPO-WT (data not shown).
Statistical differences in pala¤â0.01 were found for all EPO mutants compared to EPO-WT (data not shown). The level of immunogenicity observed in the 5 EPO mutants was significantly reduced in HLA-DRB1*09-positive subjects compared to HLA-DRB1*09-negative subjects. These altered proteins with reduced immunogenicity may be associated with reduced HLA-DRB1*09 binding affinity.