• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제37권1호
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• pp.9-14
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• 2011

Recently, the population of patients who refuse transfusion has increased for both religious and non-religious reasons, even in life threatening emergency situations. Their refusal has highlighted the need to develop nonblood transfusion surgery techniques to decrease the risk from blood transfusions. A 57-year woman with an ulcerative lesion on the gingiva of the right upper molar area visited the department of oral and maxillofacial surgery in Dankook University Dental Hospital. After a preliminary evaluation, the patient was diagnosed with squamous cell carcinoma. As she refused blood transfusion during surgery for religious reasons, surgery was planned using recombinant human erythropoietin (rHuEPO) without a blood transfusion. The patient underwent a partial maxillectomy, supraomohyoid neck dissection, free radial forearm flap and split thickness skin graft under general anesthesia. rHuEPO and iron were used before and after surgery. The hemoglobin/hematocrit (Hb/Hct) level, iron (Fe) and total iron-binding capacity (TIBC) were assessed. The patient recovered completely without any blood transfusions. rHuEPO is a viable alternative for patients with religious objections to receiving blood transfusions.

• Biomolecules & Therapeutics
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• 제6권3호
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• pp.317-327
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• 1998

The subchronic toxicity study of rHuEPO, a newly developed recombinant erythropoietin, was investigated for 13 weeks in Beagle dogs intravenously treated with doses of 100, 500 and 2,500 lU/kg/day. There were no significant changes in body weight, food intake, physical and opthalmic examination, urine analysis, etc. Any toxic response was not observed except for enlarged spleen and extramedullary hematopoiesis. These results indicate that the no-observed adverse effect level (NOAEL) of rHuEPO is 100 lU/kg in Beagle dogs.

• BMB Reports
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• 제30권5호
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• pp.315-319
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• 1997

It was discovered that monoclonal anti-erythropoietin (EPO) antibody CFC-6 can neutralize EPO-induced cell activation. To know the binding site of CFC-6, recombinant human erythropoietin (rhEPO) was digested with Glu-C, followed by a separation using high performance liquid chromato graphy (HPLC). Each HPLC fraction was blotted on the nitrocellulose membrane and the membrane was treated with anti-EPO antibody CFC-6 and anti-mouse antibody which is modified with peroxidase. Only one spot showed the color and the fraction was sequenced by Edman degradation. The results suggest that CFC-6 recognizes amino acid sequence V63-W-Q-G-L-A-L-L-S-E72 which is a part of helix II of the EPO molecule. Binding of CFC-6 to EPO may inhibit EPO binding to its receptor, which implies that the antibody binding site and the receptor binding site are close or overlapping.

• BMB Reports
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• 제29권3호
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• pp.266-271
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• 1996

A recombinant human erythropoietin (EPO), expressed in Chinese hamster ovary (CHO) cells, is glycosylated at Asn 24, Asn 38, Asn 83, and Ser 126. After release of the N-linked carbohydrate chains by $peptide-N^{4}-(N-acetyl-{\beta}-glucosaminyl)$ asparagine amidase F, the oligosaccharides were analyzed by FACE (Fluorophore-Assisted Carbohydrate Electrophoresis). The O-linked carbohydrate chain was separated by hydrazine, and analyzed by FACE. The monosacccharide composition of recombinant EPO showed man nose, fucose, galactose, N-acetylglucosamine, N-acetylneuraminic acid, and a trace of N-acetylgalactosamine, which are typical monosaccharides in the glycoproteins from the CHO cell. Sequences of N-linked and O-linked oligosaccharides were determined. The structure and composition of oligosaccharides attached to recombinant human EPO, expressed in the CHO cell, are identical to the reported oligosaccharide structure in human EPO isolated from urine.

• Reproductive and Developmental Biology
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• 제34권2호
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• pp.111-115
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• 2010

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. In this study, we analyzed by immunohistochemical methods adaptive mechanisms to excessive erythrocytosis in transgenic (tg) mice expressing dimeric human erythropoietin (dHuEPO) gene. Splenomegaly was observed over 11~21 times in the tg mice. The 2,672 candidate spleen-derived genes were identified through the microarray analysis method, and decreased genes were higher than increased genes in the spleen. The specific proteins in the increased and decreased genes were analyzed by immunohistochemical methods. Our results demonstrate that problems of abnormal splenomegaly would solve in tg mice overexpressing dHuEPO gene.

• Toxicological Research
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• 제14권3호
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• pp.393-400
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• 1998

As a series of safety studies on DA-3585, a recombinant human erythropoietin, its local irritancy was examined in rabbits after the following treatments; application into the conjunctival sac of the eye(single), subcutaneous injection (single and -day repeated)and intravenous injection (7-day repeated.)In addition, perivascular irritation of DA-3585 was investigated in mice. In the result of ocular irritation test, 10,000IU/ml solution of DA-3585 could be considered as a non-irritating material. The local irritation of DA-3585 by a single and 7-day repeated subcutaneous injection was negligible and not so much different from that of saline. In the vascular irritancy test, macro-and microscopic observations revealed that local irritation of DA-3585 was comparable to that of saline when injected into retroauricular vein of rabbits for 7 consecutive days. Furthermore the perivascular administration of DA-3585 upto the concentration of 10,000 IU/ml did not induce any morphological abnormalities at injection sites. The results obtained from the present study suggest that the local irritancy of DA-3585 is not different from that of saline when injected through intravenous or subcutaneous route for clinical practice.

• Biomolecules & Therapeutics
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• 제4권1호
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• pp.78-83
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• 1996

The pharmacokinetics and tissue distribution of DA-3285 (recombinant human erythropoietin, recently manufactured by Research Laboratories of Dong-A Pharmaceutical Company) were studied in the laboratory animals. The plasma, urine, and tissue concentration of DA-3285 were measured by a double-antibody sandwich enzyme immunoassay. After intravenous administration of DA-3285, 20, 100, 500 and 2500 units/kg to rats, the plasma concentrations declined polyexponentially with the terminal half-lives of 2.15, 2.10, 2.31, and 2.35 hr, respectively. Total body clearance (20.7∼26.6 mι/hr/kg) and apparent volume of distribution at steady state (57.2∼70.1 mι/kg) were independent of the dose and AUC increased proportionally with the dose. The renal clearance was much lower than total body clearance, suggesting that extrarenal clearance, presumably metabolism , plays a significant role in elimination of DA-3285. In all rat tissues, the tissue to plasma ratios were smaller than unity, indicating less affinity of DA-3285 to rat tissues and was proved by considerably less value of Vdss. After 3 times a week for consecutive 3 weeks i.v. administration of DA-3285, 100 units/kg to rats, the plasma concentrations and pharmacokinetic parameters of DA-3285 were not significantly different from those in a single administration. After s.c. administration to the rat, plasma concentrations of DA-3285 peaked at 6 hr and the extent of bioavailability was 26.7%. In mice, rabbits and dogs, at DA-3285 dose of 100 units/kg, the mean terminal haw-lives were 2.78, 3.05, and 4.01 hr, respectively. Compared with reported data in the literatures, DA-3285 has similar properties to rh-EPO manufactured by other companies in view of pharmacokinetics.

• Biomolecules & Therapeutics
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• 제4권1호
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• pp.68-77
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• 1996

Efficacy and general pharmacology of recombinant human erythropoietin (rHu-EPO), were investigated. Efficacy studies were conducted in normal, cisplatin- and acute hemorrhage-induced anemic rats. Normal and anemic animals were treated intravenously with rHu-EPO for 5 days and the changes in the numbers of red blood cells (RBC), hemoglobin (Hb), hematocrit (Hct) and reticulocytes (Ret) were examined. In normal rats, rHu-EPO significantly increased RBC, Hb, Hct and Ret at doses of 50∼ 1250 IU/kg/day. Cisplatin-induced anemic rats showed significant increase of RBC, Hb, Hct and Ret in a dose-dependent manner after administration of rHu-EPO (50∼200 lU/kg/day). And in acute hemorrhage-induced anemic rats, rHu-EPO(4∼100 Iu/kg/day) accelerated recovery of anemia with significant increase in Ret. These changes disappeared gradually after cessation of the treatment. The general pharmacological effects of rHu-EPO were investigated in mice, rats, guinea-pigs, rabbits and cats. rHu-EPO had no influences on central nervous, cardiovascular, gastrointestinal and blood coagulation system. It also had no influence on the contraction of phrenic nerve-diaphragm preparation. rHu-EPO produced significant increase of urine volume at a dose of 7000 IU/kg. These results suggest that rHu-EPO might be useful for the therapy of anemia without serious side effect.

• Clinical and Experimental Pediatrics
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• 제59권2호
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• pp.100-103
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• 2016

Patients with hemolytic-uremic syndrome (HUS) can rapidly develop profound anemia as the disease progresses, as a consequence of red blood cell (RBC) hemolysis and inadequate erythropoietin synthesis. Therefore, RBC transfusion should be considered in HUS patients with severe anemia to avoid cardiac or pulmonary complications. Most patients who are Jehovah's Witnesses refuse blood transfusion, even in the face of life-threatening medical conditions due to their religious convictions. These patients require management alternatives to blood transfusions. Erythropoietin is a glycopeptide that enhances endogenous erythropoiesis in the bone marrow. With the availability of recombinant human erythropoietin (rHuEPO), several authors have reported its successful use in patients refusing blood transfusion. However, the optimal dose and duration of treatment with rHuEPO are not established. We report a case of a 2-year-old boy with diarrhea-associated HUS whose family members are Jehovah's Witnesses. He had severe anemia with acute kidney injury. His lowest hemoglobin level was 3.6 g/dL, but his parents refused treatment with packed RBC transfusion due to their religious beliefs. Therefore, we treated him with high-dose rHuEPO (300 IU/kg/day) as well as folic acid, vitamin B12, and intravenous iron. The hemoglobin level increased steadily to 7.4 g/dL after 10 days of treatment and his renal function improved without any complications. To our knowledge, this is the first case of successful rHuEPO treatment in a Jehovah's Witness child with severe anemia due to HUS.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.77-83
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• 2009

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. We constructed three EPO mutants ($\Delta$69, $\Delta$105 and $\Delta$69,105), containing an additional oligosaccharide chains. EPOWT and EPO$\Delta$69 were effectively expressed in transient and stably transfected CHO-K1 cell lines. But, it wasn't detected any protein in the culture medium of EPO$\Delta$105 and EPO$\Delta$69,105 mutants. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure the cytokine dependency and in vitro bioactivity of rec-hEPO. MTT assay values were increased by survival of F36E cells at 24h. To analysis biological activity in vivo, two groups of ICR-mice (7 weeks old) were injected subcutaneously with 10 IU per mice of rec-hEPO molecules on days 0 and 2. Red blood cell and hematocrit values were measured on 6 days after the first injection. The hematocrit values were remarkably increased in all treatment groups. The pharmacokinetic analysis was also affected in the mice injected with rec-hEPO molecules 2.5 IU by tail intravenous. Protein samples were detected by Western blotting. An EPO$\Delta$69 protein migrated as a broad band with an average apparent molecular and detected slightly high band. Enzymatic N-deglycosylation resulted in narrow band and was the same molecular size. The biological activity of EPO$\Delta$69 was enhanced to compare with wt-hEPO. The half-life was longer than wt-hEPO. The results suggest that hyperglycosyalted recombinant human erythropoietin (EPO$\Delta$69) may have important biological and therapeutic good points.