The pharmacodynamics and pharmacokinetic properties of beneficial glycoproteins can be altered using glycosylation.Glycosylation is a process in which glycosyl groups are added to a protein resulting in the formation of glycoprotein. There is an increase in solubility of protein and thermodynamic stability due to glycosylation and it is also a common post translational modification(2).N and O- linked are the two types of glycosylation. N linked glycosylation attacks the Asn site of the consensus sequence of Asn-X-Ser whereas O-linked glycosylation attacks the Ser site(3).There are many examples of glycoproteins such as erythropoietin. Kidney is the main unit which is responsible for the synthesis of erythropoietin. Erythropoietin is a protein which is heavily glycosylated. Erythropoietin results in activation of erythropoiesis by its action on erythroid progenitor cell(5).Structurally 40% carbohydrate is present in erythropoietin. These carbohydrates are necessary for various properties like pharmacokinetics, stability ,antigenicity (4).Activated human erythropoietin is composed of 3 N-glycosylation sites at Asn 24, Asn 38,Asn 83 and one O-glycosylation sites at Ser 126 in the single polypeptide chain of 165 amino acids(4).For the biological activity of erythropoietin glycosylation is necessary. The pharmacodynamics and the speed of catabolism depends upon the number of sialic acid residues and the arrangement of N-linked oligosaccharides.
HOSTS REQUIRED FOR THE PRODUCTION OF ERYTHROPOIETIN:
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For the production of recombinant erythropoietin the common hosts are Chinese hamster ovary(CHO) and baby hamster kidney(BHK) cells. Three types of recombinant human erythropoietin are available, they are epoetin alpha, epoetin beta and epoetin omega. CHO cells are used for the synthesis of epoetin alpha and beta. BHK cells are used for the synthesis of epoetin omega(4). The three major advantages of CHO cells are:
Increase in the yield of erythropoietin.
Its capability to produce oligosaccharide chain structures.
These cells die by necrosis and are not apoptotic.
For the production of erythropoietin it is favorable to choose non apoptotic cells. In recombinant CHO cells there is an increase in erythropoietin production and longer life span of the culture when the optimum pH is maintained. The optimum pH is adjusted by the addition of sodium lactate in the medium which results in decrease in the production of lactate(6). Addition of 40Mm sodium lactate increases the life span of the culture by 187h and there was a 2.7 fold increases in the concentration of erythropoietin(6). In mammalian cells lactate is a waste compound formed by glycolysis. Aggregation of lactate causes increase in the acidity of the medium which leads to lower pH and hence arrests cell growth. Hence to reduce the toxicity of lactate sodium lactate is added in the medium which results in the activation of lactate dehydrogenase resulting in the conversion of lactate to pyruvate.(6)
BHK cells are cultured at increased quantity on microcarriers captured by calcium alginate gel particles for the production of recombinant human erythropoietin. The microcarriers and the available spaces in the alginate gel particles are used up for the proliferation of the BHK cells. Immobilization of the alginate gel and microcarriers are advantageous for high density cultivation of these cells and increased productivity of erythropoietin.
GLYCOSYLATION ANALOGS OF ERYTHROPOIETIN :
1)Carbamylated erythropoietin: There are eight lysine residues in the mature erythropoietin molecule. Nine primary amino groups are provided by the N-terminal alanine which is required for carbamylation which results in the formation of a molecule called as carbamylated erythropoietin(CEPO). The carbamylation process begins with the parent recombinant human erythropoietin molecule. This chemical reaction is controlled in the existence of potassium cyanate. This reaction results in the conversion of lysine residues to homoitrulline. Hence carbamylation process is used to modify eight lysine residues and N-terminal amino acid for the formation of CEPO(13). It is used in the treatment of acute ischemic stroke.
2)Darbepoetin alpha: It constitutes of two supplementary N-linked carbohydrates, three fold increase in the half life of the serum and an higher in vivo activity. It is a hyper glycosylated erythropoietin. It is used to treat anemia caused by cancer during chemotherapy treatment and disorders related to kidney.(14)
1) ERYTHROPOIETIN IN INHERITED RETINAL DEGENERATION .
A group of inherited retinal degeneration is retinitis pigmentosa which is induced by mutation in over 190 genes. Preipherin/rds is one of the factor causing retinitis pigmentosa and it encodes a structural protein placed at the end of the photoreceptor outer segment discs(7). Inherited retinal degeneration has a common degenerative mechanism in which apoptosis of the photoreceptor results in loss of vision. Erythropoietin and its receptor were repoted to be present in some non hematopoietic tissues which also includes the eye. Apoptosis is blocked in numerous models of neuronal cell death. When erythropoietin is administered directly into the eye, it arrests the death of retinal ganglion cells, it safeguards the photoreceptors from photo oxidative stress evoked cell death. All forms of erythropoietin such as hyperglycosylated erythropoietin and deglycosylated erythropoietin protects the neurons of the photoreceptors in the retinal degeneration slow mouse(7). Erythropoietin prevents cell death of the neurons related with acute or chronic injuries. Systemically administered erythropoietin which is able to cross the blood retina boundary protects the retina from genetic degeneration and light damage, injury caused due to obstruction in the blood vessels.(8)
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2) ERYTHROPOIETIN IN MANAGEMENT OF ANEMIA.
Erythropoietin is used in treating anemia in patients with chronic renal failure. This has considerably changed clinical practice in the science that deals with disorders related to kidney. The necessity of blood transfusion has decreased due to extensive use of recombinant erythropoietin. In the bone marrow erythropoietin binds to its receptor. There is an induction of intracellular signaling cascade on binding of the receptor which results in transcription of anti-apoptotic genes.(9) Hence there is synthesis of red blood cells which results in improvement of anemia. It has also improved the quality of life in dialysis patients and it has also improved left ventricular hypertrophy. Epoetin alpha a type of recombinant erythropoietin treats anemia by depicting the action of the erythropoietin hormone. Erythropoietin therapy is beneficial to patients suffering from chronic kidney disease, anemia caused by AIDS, numerous hematologic disorders.(10)
ERYTHROPOIETIN MOLECULES TO TREAT ACUTE ISCHEMIC STROKE.
Ischemic stroke is a significant cause of disability and death. Several neuroprotective drugs have failed in preclinical trials. For the treatment of various acute and chronic neurological disorders erythropoietin has been reported to be an effective drug. Erythropoietin molecules have been reported to possess neuroprotective properties which are used in treatment of acute ischemic stroke(11). Erythropoietin treatment results in increase in the level of circulating erythropoietin and it also improves the function of the neurons and the clinical consequence in patients after acute ischemic stroke(12). An EPO analog CEPO is used for improving the safety and pharmacokinetic properties of the clinical trial for the treatment of acute ischemic stroke.
Glycosylation in erythropoietin is important for treatment of various acute and chronic disorders of kidney. It also results in structural alteration of the erythropoietin resulting in the formation of various analogs recombinant erythropoietin that are used for many therapeutic purposes.