• Biomolecules & Therapeutics
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• 제4권4호
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• pp.323-329
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• 1996

A recombinant human erythropoietin (rHu-EPO), was administered intravenously to beagle dogs at doses of 100, 500 and 2, 500IU/kg/day for 30 days. There were no significant clinical signs such as body weight, food intake, physical and opthalmic examination, urine analysis, etc. Any toxic response was not observed except for enlarged spleen and extramedullary hematopoiesis. No observed adverse effect level (NOAEL) of rHu-EPO for 30 days was considered to be 100IU/kg/day in beagle dogs.

• KSBB Journal
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• 제30권1호
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• pp.44-51
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• 2015

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for the commercial production of recombinant proteins. However, they show relatively low yields of recombinant proteins in comparison with microbial cells. Various strategies have been tried to overcome this drawback. The acetyl moieties are attached to the N-terminus of histone by histone acetyltransferase (HAT) while histone deacetylase (HDAC) removes histone-bound acetyl groups. HDAC inhibitor (HDACi), such as sodium butyrate, sodium propionate and valproic acid, can enhance specific productivity of CHO cells. Human albumin-erythropoietin (Alb-EPO) is a novel 105 kDa protein comprising recombinant human EPO fused to human albumin. In this study, we examined the effects of HDACi on the production of Alb-EPO in CHO cells with various concentrations in the range of 0-1 mM. The results showed that sodium butyrate was found to be the best HDACi for enhancing productivity. It enhanced not only the production of Alb-EPO but also the apoptosis of recombinant CHO cells.

• Biomolecules & Therapeutics
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• 제6권2호
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• pp.182-190
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• 1998

DA-3585 is a recombinant human erythropoietin produced by Dong-A pharmaceutical Co. Ltd. using recombinant DNA technique. Recently, recombinant human erythropoietin (rHu-EPO) has been used to treat various types of anemia. In this study, we examined acute and subacute toxicity of DA-3585 in rats. DA-3585 was intravenously administered to rats at dose levels of 0, 6,250, 12,500 and 25,000 lU/kg for single dose toxicity study and at dose levels of 0,100,500 and 2,500IU/kg daily for 4 week-repeated dose toxicity study. In the single dose toxicity study, there were no death, clinical signs and changes in body weight gain related to the treatment. Necropsy revealed no evidence of toxicity related to DA-3585, In the repeated dose toxicity study, all the rats survived throughout the study. There were no treatment-related changes in clinical signs, food and water intake, and body weight. Hematological examination showed increases in the number of erythrocytes, hemoglobin concentration, hematocrit value and mean corpuscular volume, and decrease in the number of platelet in 500 and 2,500 lU/kg dosed groups. Extramedullary hematopoiesis in the spleen and erythroid hyperplasia in the bone marrow were noted as treatment-related histological changes. Toxicologically significant changes were not observed in blood biochemistry, urinalysis, organ weights and in any other examinations. The treatment-related changes observed in this study were hematological or histological changes associated with pharmacological effects of DA-3585. On the basis of the results of this study, LD5n value of DA-3585 was above 25,000 lU/kg and the no-observed-adverse-effect-level was estimated to be 100 lU/kg.

• 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.

• Toxicological Research
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• 제11권1호
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• pp.77-80
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• 1995

Antigenic potential of genetically-engineered human erythropoietin (EPO) was assessed in guinea pigs (active systemic anaphylaxis [ASA] ; passive cutaneous anaphylaxis [PCA]) and in vitro (hemagglutination test [PHA]). In ASA, EPO at 70~700 U/kg elicited a weak anaphylactic response tvhereas the positive control ovalbumin (OVA) did cause intensive responses leading to death in 40% animals. However, the extract of CHO cells, to which EPO gene was introduced, did not cause any symptom. In PCA and PHA tests, neither EPO nor CHO cell extract induced positive responses. OVA, in contrast, produced high titers in both PCA and PHA tests. It was concluded that, in light of the fact that EPO was slightly antigenic only in ASA but not in PCA or PHA and also that human EPO is a foreign protein to guinea pigs, the present EPO may not be antigenic in humans.

• 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.

• KSBB Journal
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• 제13권3호
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• pp.295-302
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• 1998

Human Erythropoietin (EPO) gene is cloned in quail fibrosarcoma cell, QT35. Because molecular weight of EPO is similar to that of serum albumin, cell culture with serum containing medium makes purification of EPO very difficult. Using fractional factorial study, we have developed serum free medium for the recombinant QT35 cell lines, QT N4D4 and QT SY-IMP, which have cloned EPO with glutamine synthetase (GS) gene amplification system and with puromycin selective marker, respectively. Among the seven frequently used medium components, fibronectin, BSA, and EGF were the most important for EPO production. However, sufficient fibronectin supplement to the medium did not make any good attachment of QT35 to culture plate over 3 days. Therefore, to maximize EPO production, we attempted a medium-shift at confluence from serum containing medium to serum free medium(QT SFM6). Using the medium-shift protocol with QT SFM6, nearly the same productivity of EPO was achieved comparing with that without medium-shift. This result was true in both QT35 cell lines in three types of culture, i.e. T flask, microcarrier and roller bottle cultures.

• Biomolecules & Therapeutics
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• 제8권2호
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• pp.184-193
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• 2000

To evaluate GC-rhEPO, human erythropoietin produced by recombinant DNA technique, its general pharmacological properties were investigated in experimental animals administering intravenously and in vitro test system. GC-rhEPO at doses of 70,700 and 7,000 IU/kg body weight had no influence on general behavior, spontaneous motor activity, thiopental-inducted sleeping time, writhing syndrome induced by acetic acid, strychnine-induced convulsions, charchoal meal propulsion in mice, and body temperature, gastric juice secretion, urine and electrolyte excretion in rats. In anesthetized rabbits, GC-rhEPO (70, 700 and 7,000 lU/kg, i.v.) did not alter respiratory rate, blood pressure, heat rate. In in vitro experiments, GC-rhEPO did not affect the contractions of the isolated ileum of guinea pigs and the muscle twitchs of isolated neuromuscular junction of the rats. In addition, GC-rhEPO did not affect the blood coagulation time and ADP-induced platelet aggregation in plasma of rabbits. Taken together, these results indicate that GC-rhEPO does not induce any adverse effects in the experimental animals.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1087-1092
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• 2001

The effect of Sodium Butyrate (NaBu) on the N-linked oligosaccharide structure of Erythropoietin (EPO) was investigated. Recombinant human EPO was produced by CHO cells grown in an $MEM{\alpha}$ medium with or without 5 mM NaBu, and purified from the culture supernatants using a heparin-sepharose affinity column and immunoaffinity column. The N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were then labeled with a fluorescent dye, 2-aminobenzamide, and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of a GU (glucose unit) vague. A glycan analysis by HPLC showed that the most significant characteristic effect of NaBu was a reduction in the proportion of glycans with Sri-and tetrasialylated oligodaccharides from $21.30\%$ (tri-) and $14.86\%$ (tetra-) in the control cultures (without NaBu) to $8.72\%$ (tri-) and $1.25\%$ (tetra-) in the NaBu-treated cultures, respectively. It was also found that the proportion of asialo-glycan increased from $12.54\%\;to\;23.6\%$ when treated with NaBu.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.338.1-338.1
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• 2002

The efficient Erythropoietin(EPO)-expression system in mammalian cells is required for massive production for therapeutic use. Ammonium ion is a major problem in the production of useful proteins by cultured animal cells and therefore it is of importance to devise a system by which a high productivity of human therapeutic recombinant protein can be maintained or enhanced under low ammonium concentration. (omitted)