KR20030045416A - Conjugates of erythropoietin and polyethylene glycol derivatives - Google Patents

Abstract

The present invention provides a novel form of methoxypolyethylene glycol-propionaldehyde in the alpha-amino group of the amino-terminus (N-terminus) of the erythropoietin (EPO). ) Conjugates in which the derivatives are bound to the conjugates, wherein the combinations improve the pharmacokinetic profile by increasing the remaining time in the body while reducing the immunogenicity of erythropoietin and maximally reducing the degradation of physiological activity. ) And pharmacological properties, it can be useful in clinical treatments related to erythrocyte formation and hematopoiesis in anemia caused by renal anemia in chronic renal failure and diseases such as cancer and AIDS.

Description

Compound of erythropoietin and polyethylene glycol derivatives {CONJUGATES OF ERYTHROPOIETIN AND POLYETHYLENE GLYCOL DERIVATIVES}

The present invention relates to a polyethylene glycol-erythropoietin conjugate (conjugate) in which a novel type of methoxy polyethylene glycol-propionaldehyde derivative is bound to the amino terminus (amino-terminus, N-terminus) of erythropoietin. will be.

Erythropoietin (EPO) is a glycoprotein that promotes the production of red blood cells. Human EPO consists of 165 amino acids and contains three N-glycosyl moieties and one O-glycosyl moiety, and the entire sugar chain is about the molecular weight of this glycoprotein. 40% (Lai et al., J. Biological Chemistry 261 (1986), 3116). Sugar chains added in EPO have been found to play an important role in in vivo activity but are independent of activity in vitro (Dordal et al., Endocrinology 116 (1985), 2293), and play an important role in the structural stability of EPO. (Linda et al., J. Biological Chemistry 266 (1991), 23022-23026), and thereafter, studies related to sugar chains of EPO continue (Amgen, U.S. Patent No. 5,856,298, European Patent No. EP0640619, and Ergrie et al. , British J. Cancer 84 (2001), 3-10).

However, the EPO derivatives obtained from such sugar chain studies have a potential risk of causing antigenicity in the body by changing the amino acid sequence of the original EPO to add more sugar chain branches. Accordingly, polyethylene glycol derivatives have been developed as one method for giving various functions to proteins while reducing antigenicity, which is a potential threat (Yoh Kodera et al., Prog. Polym. Sci. 23 (1998), 1233).

Polyethylene glycol (PEG) is a high-molecular compound having a structure of HO-(-CH ₂ CH ₂ O-) _n -H. Because of its high hydrophilicity, polyethylene glycol can increase its solubility. Proper binding also increases the molecular weight of the bound protein while maintaining key biological functions such as enzymatic activity and receptor binding, thereby reducing kidney filtration, protecting the protein from cells and antibodies that recognize foreign antigens, Can also reduce the degradation of proteins. As such, the molecular weight range of PEG that can be bound to proteins is approximately 1,000 to 100,000, and when the molecular weight of PEG is 1,000 or more, it is known that the toxicity is quite low. Its molecular weight ranges from 1,000 to 6,000 and is distributed throughout the body and metabolized through the kidneys. In particular, branched PEGs with a molecular weight of 40,000 are known to be distributed in organs including blood and liver, and metabolism in the liver.

Medically and pharmacologically useful proteins administered through the parenteral route have antigenicity, and are generally poor in water solubility and have a short remaining period in the body. In US Patent No. 4,179,337 to Frank F. Davis et al., The use of PEG-binding proteins and enzymes as therapeutic agents has the advantages of reducing antigenicity, increasing water solubility, and increasing the length of stay in the body, which are advantages of PEG. It is disclosed that can be obtained. Since this patent, attempts have been made to overcome the drawbacks by combining bioactive proteins with PEG, for example, Veronese et al. , Applied Biochem. And Biotech . 11: 141-152, 1985. ) Combines ribonuclease and superoxide dismutase with PEG. In addition, Carter et al. , U.S. Patent Nos. 4,766,106 and 4,917,888 discloses the binding of a polymer containing PEG to a protein to increase the water solubility of the protein. Nitecki et al. , In US Pat. No. 4,902,502, bind PEG or other polymers to recombinant proteins to reduce antigenicity and increase the duration of life in the body.

In addition to these advantages, there are drawbacks to PEG and protein binding. That is, PEG usually binds covalently to one or more free lysine (lys) residues of the protein to be bound, where a site directly related to protein activity on the surface of the protein is bound to PEG. In that case, the site will no longer be able to perform biological functions, resulting in reduced protein activity. In addition, the binding of PEG and lysine residues usually occurs randomly, so that many kinds of PEG-protein conjugates exist as a mixture depending on the binding position, and thus, the process of pure separation of the desired combination is complicated and difficult. You lose. For example, in the case of interferon-alpha 2b, there are eight free lysine residues on the surface of the protein, which must be PEG-linked in a position-selective manner to amino acid sites that do not affect the biological activity of interferon. Useful formulations can be obtained.

As an example of binding a PEG derivative to EPO, f. Korean Patent Publication No. 2001-0049676 (F. Hoffmann-La Roche AG) (European EP 1 064 951 A2) discloses α-lower alkoxy X acids (X = propionic acid, butyric acid, etc.) of PEG. Succinimidyl esters (US Pat. No. 5,672,662; RO (CH ₂ CH ₂ O) _m (CH ₂ ) _x COO (C ₄ H ₄ O ₂ N) using the free amino group on the surface of EPO (ε- of lysine amino acid) Amino group or amino-terminal amino group) to a PEG having a molecular weight of about 20 to about 40 kDa, wherein the ratio of bound PEG to EPO was 1: 1 or 2: 1, with the 1: 1 form being a chemical bond. It is a major product, and its function is described as better than 2: 1 binding: the effect of binding is increased half-life, extended plasma residence time, reduced clearance, and increased in vivo clinical activity compared to EPO without PEG. It is supposed to be.

Also f. In another patent WO 01/02017 A2 of Hoffman-La Roche-Age, PEG was used as a linker to bind to EPO, with a binding ratio of PEG: EPO of 1: 1 to 3: 1 The structure is as follows:

EPO-N _EPO H-CO-XSY- (OCH ₂ CH ₂ ) _m -OR

Here, R is an alkyl group, X is-(CH ₂ ) _k -or -CH ₂ (OCH ₂ CH ₂ ) _k- , Y is a substance as shown in the following formula (2).

On the other hand, Ortho-McNeil Pharmaceutical, Inc. is a methoxyPEG-hydrazine carboxylate, methoxyPEG-high in Korean Patent No. 0257643 (related US Patent No. 6,077,939) MethoxyPEG-hydrazide, methoxyPEG-semicarbazide, and the like, are bonded to the N-terminal α carbon atom (C _α ) of the EPO by hydrozone (NH-N = C _α ) and reduced. It is bound using a reduced hydrozone (NH-NH-C _α ) or an oxime bond (ON = C _α ) and a reduced oxime bond (O-NH-C _α ). At this time, the bonding ratio of bound methoxy PEG and EPO was 1: 1.

PEG polymers that can selectively bind to the amino terminal alpha-amino groups in the protein structure include methoxy PEG-aldehyde, methoxy PEG-acetaldehyde, methoxy PEG-propionaldehyde And the like have been used since the aldehyde group selectively reacts at the amino terminus. Also, since methoxyPEG-acetaldehyde is sensitive to dimerization by aldol condensation, propionaldehyde forms are known to be easier to synthesize and use than acetaldehyde. The coupling reaction involves the formation of a stable secondary amine bond between the alpha-amino group and the aldehyde group via the Schiff base to form a combination of PEG and protein. Examples of using methoxyPEG-propionaldehyde include the amino terminus (Kinstler et al., Pharm Res., 13 (7) : 996-1002, 1996) of recombinant human granulocyte-colony stimulating factor (G-CSF) . A PEG polymer is bound to the amino terminus of Edward necrosis factor (TNF) receptor type 1 (Edwards et al., Ann. Rheum. Dis., 58 (S1) : I73-I81, 1999).

Considering the conventional PEG-protein binding, the site does not participate in receptor binding in EPO, which is useful for erythrocyte formation and clinical treatment related to hematopoiesis in anemia caused by various diseases such as renal anemia. It would be very useful to provide a novel form of PEG derivative that exhibits selective reactivity to specific amino acid specific residues of, and to provide a combination of this novel PEG derivative with EPO.

In the present invention, by selectively binding a novel form of methoxyPEG-propionaldehyde derivative to the amino-terminus of the wild-type or recombinant EPO, the immunogenicity of EPO is reduced and physiological Modified EPO with improved pharmacokinetic profile and pharmacological properties to reduce the dose or frequency required for the therapeutic effects of wild-type or recombinant EPO proteins by increasing the retention time in the body while minimizing degradation of activity. It is an object to provide a combination of and PEG.

In order to achieve the above object, in the present invention, ethoxypolyethylene glycol-propionaldehyde (methoxypolyethylene) in the alpha-amino group of the amino terminal (amino-terminus, N-terminus) of erythropoietin (EPO) Provided is a conjugate to which a glycol-propionaldehyde) derivative is bound.

Wherein the erythropoietin may be wild type or recombinant erythropoietin.

In addition, the methoxy polyethylene glycol (PEG)-propionaldehyde derivative is a novel form of methoxyPEG-propionaldehyde derivative that shows a selective reactivity to the alpha-amino group of the amino terminal of the erythropoietin, Linear methoxyPEG-amide-propionaldehyde derivatives and methoxyPEG-urethane-propionaldehyde derivatives, and pendant PEG-amide-propionaldehyde derivatives and PEG-urethanes At least one of propionaldehyde derivatives.

Here, it is preferable that the methoxy polyethylene glycol propionaldehyde derivative has a molecular weight range of 1,000 to 1,000,000. Specifically, the linear methoxyPEG-propionaldehyde derivative preferably has a molecular weight range of 1,000 to 100,000, more preferably 1,000 to 40,000. Moreover, it is preferable that the molecular weight range of PEG skeleton of a pendant PEG-propionaldehyde derivative is 5,000-1,000,000, and it is more preferable that it is 5,000-100,000. The number of amide-propionaldehyde or urethane-propionaldehyde as pendant groups is preferably 1 to 20.

In the present invention, by binding a novel form of methoxyPEG-propionaldehyde derivative showing selective reactivity to the alpha-amino group of the amino terminus of erythropoietin, it limits the type of the combination formed thereby and reduces protein activity. Is suppressed as much as possible. That is, PEG derivatives that are reactive with epsilon-amino groups in the side chain of lysine residues on the secondary and tertiary structures of the protein react with a number of epsilon-amino groups exposed on the surface of the protein. Although the site is inhibited to decrease the activity, the methoxyPEG-propionaldehyde derivative according to the present invention exhibits selective reactivity to the alpha-amino group of the amino terminus so that only one PEG derivative can be bound to the protein of interest. In addition, as long as this amino terminal is a site which does not participate in binding with a receptor, the reduction of the activity of a wild type by formation of a compound with a PEG derivative can be suppressed as much as possible.

According to the invention, the amount of the methoxyPEG-propionaldehyde derivative used in the formation of the combination with erythropoietin must be at least the same equivalent as the erythropoietin and the alpha-amino group at the amino terminus. In order to induce a complete reaction of the methoxyPEG-propionaldehyde derivative is preferably added in excess (mole ratio of PEG derivatives per mole of erythropoietin protein in the range of 1 to 10 times). In addition, the mixing reaction of erythropoietin and methoxyPEG-propionaldehyde takes about 5 to 20 hours under room temperature conditions in the pH range of 6.0 to 7.0.

As described above, the combination of erythropoietin and PEG derivatives obtained by binding a methoxyPEG-propionaldehyde derivative to the alpha-amino group of the amino terminal of erythropoietin reduces the immunogenicity of erythropoietin and reduces physiology. As the degradation of activity is suppressed, the remaining time in the body increases, resulting in improved pharmacokinetic profiles and pharmacological properties.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only examples showing some experimental methods and compositions of the present invention, but the scope of the present invention is not limited thereto.

The PEG derivatives used in the following examples are those using products synthesized by Sun Bio Co., Ltd.

Example 1 Preparation of MethoxyPEG-amide-Propionaldehyde

Methoxy PEG (mPEG-OH) (MW. 20,000) and potassium t-butoxide (potassium t-butoxide) was added to t- butyl alcohol (t-buthyl alcohol) and stirred under the conditions of 60 ℃. Ethyl bromoacetate was slowly added to the mixture and stirred for 15 hours under the conditions of 80 to 85 ° C. After the reaction mixture was filtered, the filtrate was distilled under reduced pressure to remove the organic solvent and dissolved by adding distilled water. Washed once with diethyl ether and extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate (magnesium sulfate) and distilled under reduced pressure to remove the organic solvent. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum reduced pressure to obtain a mPEG-ethylacetate compound in the form of a white powder. The above reaction process is shown in the following scheme 1.

In the following reaction scheme, n = 22 to 2273, and 22 to 909 are preferable. This applies to schemes 1-5.

The mPEG-ethyl acetate was dissolved in 1 N aqueous sodium hydroxide solution and stirred at room temperature for 15 hours. The pH of the reaction solution was acidified to 2 with 1 N aqueous hydrochloric acid and extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate, and the organic solvent was removed by distillation under reduced pressure. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum reduced pressure to obtain an mPEG-acetic acid compound in the form of a white powder. The above reaction process is shown in the following scheme 2.

The mPEG-acetic acid was dissolved in dichloromethane and stirred under 0-5 ° C. conditions. N-hydroxysuccinimide was added to this mixture, and then dicyclohexylcarbodiimide was dissolved in dichloromethane and slowly added under 0 to 5 ° C. The reaction mixture was stirred at room temperature for about 15 hours. The reaction mixture was filtered under reduced pressure to remove byproduct dicyclohexylurea and distilled under reduced pressure to remove the organic solvent. The concentrated reaction mixture was recrystallized from ethyl acetate. The recrystallized compound was washed twice with diethyl ether after filtration under reduced pressure, and dried under vacuum reduced pressure for 12 hours to obtain mPEG-succinimidyl acetate compound in the form of a white powder. The above reaction process is shown in the following scheme 3.

The mPEG-succinimidyl acetate was dissolved in dichloromethane and stirred at room temperature. To this mixture was added 1-amino-3,3-diethoxypropane. The reaction mixture was stirred at room temperature for 2 hours. Precipitation was induced by adding diethyl ether to the reaction mixture, and the precipitated mixture was filtered under reduced pressure and recrystallized with ethyl acetate. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain mPEG-propionaldehyde diethylacetal (mPEG-propionaldehydediethylacetal) compound in the form of a white powder. The above reaction process is shown in the following scheme 4.

The mPEG-propionaldehyde diethyl acetal was dissolved in an aqueous solution of phosphoric acid (phosphoric acid, pH 1) and stirred for 2 hours under 40 to 50 ° C. The reaction mixture was cooled to room temperature, adjusted to pH 6 with 5% sodium bicarbonate aqueous solution, and brine was added thereto, and then extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate, and the organic solvent was distilled off under reduced pressure. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum reduced pressure to obtain a methoxyPEG-amide-propionaldehyde compound in the form of a white powder. The above reaction process is shown in the following scheme 5.

Example 2 Preparation of MethoxyPEG-Urethane-Propionaldehyde

MethoxyPEG (mPEG-OH) (MW. 20,000) was added to dichloromethane and stirred at room temperature. To this mixture, triphosgene dissolved in dichloromethane was slowly added and stirred at room temperature for 15 hours. The reaction mixture was distilled under reduced pressure to remove the organic solvent and remaining phosgene. The mixture distilled under reduced pressure was dissolved in dichloromethane and stirred. N-hydroxysuccinimide was added to the reaction mixture, triethylamine was added thereto, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and the filtrate was distilled under reduced pressure to remove the organic solvent, which was dissolved in warm (50 ° C.) ethyl acetate. The filtrate of the reaction mixture was filtered to induce precipitation at low temperature, and the precipitated compound was filtered under reduced pressure and dried under vacuum pressure to obtain mPEG-succinimidylcarbonate compound as a white powder. The above reaction process is shown in the following Scheme 6.

In the following reaction scheme, n = 22 to 2273, and 22 to 909 are preferable. This applies to Schemes 6-8.

The mPEG-succinimidyl carbonate was dissolved in dichloromethane and stirred at room temperature. To this mixture was added 1-amino-3,3-diethoxypropane. The reaction mixture was stirred at room temperature for 2 hours. Precipitation was induced by adding diethyl ether to the reaction mixture, and the precipitated mixture was filtered under reduced pressure and recrystallized with ethyl acetate. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain mPEG-propionaldehyde diethylacetal compound in the form of a white powder. The above reaction process is shown in the following scheme 7.

The mPEG-propionaldehyde diethyl acetal was dissolved in an aqueous solution of phosphoric acid (pH 1) and stirred for 2 hours under 40 to 50 ° C. After the reaction mixture was cooled to room temperature, the pH was adjusted to 6 with 5% aqueous sodium bicarbonate solution and brine was added thereto, and then extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate, and the organic solvent was distilled off under reduced pressure. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum reduced pressure to obtain a methoxyPEG-urethane-propionaldehyde compound in the form of a white powder. The above reaction process is shown in the following scheme 8.

Example 3 Preparation of Pendant PEG-amide-Propionaldehyde

Nonane was added to a reaction vessel containing methoxy PEG (MW 20,000) or PEG (MW 20,000), and the mixture was stirred while rising to a temperature of 140 to 145 ° C. Acrylic acid (acrylic acid) and the reaction initiator t-butyl peroxybenzoate were slowly added to the reaction mixture for 1.5 hours, respectively. After the addition of the reaction, the mixture was stirred under the conditions of 140 to 145 ° C for about 1 hour. The reaction mixture was distilled under reduced pressure to remove nonane, and methanol was added thereto, followed by heating to a uniform liquid phase, followed by stirring. The mixture was filtered through a filter paper, and a mixed solution of methanol and distilled water (9: 1) was added thereto, and the mixture was purified by a Pall Filtron Ultrafiltration system. The purified mixture was distilled under reduced pressure to remove the solvent, and then a mixed solution of acetone and isopropyl alcohol (1: 1) was added thereto and heated to obtain a uniform solution. The mixed solution was left at 0 ° C. for 12 hours to induce precipitation. The precipitated compound was filtered under reduced pressure, washed three times with a mixed solution of acetone and isopropyl alcohol (1: 1) and once with diethyl ether, and dried under vacuum reduced pressure to obtain a pendant-PEG-propionic acid in the form of a white powder. PEG-propionic acid) compound was obtained. The above reaction process is shown in the following scheme 9.

In the following reaction scheme, n = 113 to 22,728 and 113 to 2273 are preferable. m = 1-20. This applies to schemes 9-12.

The pendant-PEG-propionic acid was dissolved in dichloromethane and stirred under 0-5 ° C. conditions. N-hydroxysuccinimide was added to the mixture, followed by dicyclohexylcarbodiimide (DCC) and 4- (dimethylamino) pyridine (DMAP) in dichloromethane. It was added slowly under ˜5 ° C. conditions. The reaction mixture was stirred at room temperature for about 15 hours. The reaction mixture was filtered under reduced pressure to remove byproduct dicyclohexylurea and distilled under reduced pressure to remove the organic solvent. The concentrated reaction mixture was recrystallized from ethyl acetate. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain a pendant PEG-succinimidyl propionate compound in the form of a white powder. The above reaction process is shown in the following Scheme 10.

The pendant-PEG-succinimidyl propionate was dissolved in dichloromethane and stirred at room temperature. To this mixture was added 1-amino-3,3-diethoxypropane. The reaction mixture was stirred at room temperature for 2 hours. Precipitation was induced by adding diethyl ether to the reaction mixture, and the precipitated mixture was filtered under reduced pressure and recrystallized with ethyl acetate. The recrystallized compound was filtered under reduced pressure, washed twice with diethyl ether, and dried under vacuum reduced pressure for 12 hours to obtain a pendant-PEG-propionaldehyde diethylacetal compound in the form of a white powder. The above reaction process is shown in Scheme 11 below.

The pendant-PEG-propionaldehyde diethylacetal was dissolved in an aqueous solution of phosphoric acid (pH 1) and stirred for 2 hours under 40 to 50 ° C. After the reaction mixture was cooled to room temperature, the pH was adjusted to 6 with 5% aqueous sodium bicarbonate solution and brine was added thereto, and then extracted twice with dichloromethane. The extracted organic layer was dried over magnesium sulfate, and the organic solvent was distilled off under reduced pressure. Diethyl ether was added to the concentrated reaction mixture to induce precipitation, and the precipitated compound was filtered under reduced pressure and dried under vacuum pressure to obtain a pendant PEG-amide propionaldehyde compound in the form of a white powder. . The above reaction process is shown in Scheme 12 below.

Example 4

Conjugation of EPO with Linear Methoxy PEG-amide-Propionaldehyde

The linear methoxy PEG-amide-propionaldehyde and EPO protein having a molecular weight of 1,000 to 40,000 prepared in Example 1 were combined, and the EPO: PEG was prepared to have a molar ratio of 1: 1 to 1: 5. The reaction was carried out in NaH ₂ PO ₄ , pH 6.0 buffer containing sodium cyanoborohydride to form a PEG-EPO combination. The reaction was carried out at room temperature for 5-20 hours, and the completion of the mixture was confirmed by SDS-polyacrylamide electrophoresis.

Example 5

Combination of EPO and Linear MethoxyPEG-Urethane-Propionaldehyde

Formulation of EPO protein with linear methoxyPEG-urethane-propionaldehyde having a molecular weight of 1,000 to 40,000 prepared in Example 2 above, prepared so that the molar ratio of EPO: PEG is 1: 1 to 1: 5 and sodium cyanoboro The reaction was performed in NaH ₂ PO ₄ , pH 6.0 buffer containing hydride to form a PEG-EPO combination. The reaction was carried out at room temperature for 5-20 hours and the completion of the formulation was confirmed by SDS-polyacrylamide electrophoresis.

Example 6

Combination of EPO and pendant PEG-amide-propionaldehyde

A PEG PEG-amide-propionaldehyde having 1 to 20 amide-propionaldehyde groups and an EPO protein were mixed in a PEG skeleton having a molecular weight of 5,000 to 1,000,000 prepared in Example 3, wherein the molar ratio of EPO: PEG was 1: 1. It was prepared to ˜1: 5 and reacted in NaH ₂ PO ₄ , pH 6.0 buffer containing sodium cyanoborohydride to form a PEG-EPO blend. The reaction was carried out at room temperature for 5-20 hours and the completion of the formulation was confirmed by SDS-polyacrylamide electrophoresis.

Example 7

Combination of EPO and pendant PEG-urethane-propionaldehyde

A pendant PEG-urethane-propionaldehyde having 1 to 20 urethane-propionaldehyde groups and an EPO protein are mixed with a PEG skeleton having a molecular weight of 5,000 to 1,000,000, and the molar ratio of EPO: PEG is prepared to be 1: 1 to 1: 5. And reacted in NaH ₂ PO ₄ , pH 6.0 buffer containing sodium cyanoborohydride to form a PEG-EPO blend. The reaction was carried out at room temperature for 5-20 hours and the completion of the formulation was confirmed by SDS-polyacrylamide electrophoresis.

The combination in which the methoxyPEG-propionaldehyde derivative is bound to the alpha-amino group of the amino terminus of erythropoietin prepared according to the present invention, remains in the body while reducing the antigen-induced activity of erythropoietin and reducing physiological activity as much as possible. Increased time and improved pharmacokinetic profile and pharmacological properties make it useful for clinical treatments related to erythropoiesis and hematopoiesis in anemia caused by renal anemia in chronic renal failure and diseases such as cancer and AIDS. Can be.

Claims (5)

A compound in which a methoxypolyethylene glycol-propionaldehyde derivative is bound to an alpha-amino group of an amino-terminus (N-terminus) of erythropoietin (EPO) (conjugate). 2. The combination of claim 1, wherein said erythropoietin is wild type or recombinant erythropoietin. The method of claim 1, wherein the methoxy polyethylene glycol propionaldehyde derivative is a linear methoxy polyethylene glycol-amide- propionaldehyde derivative and methoxy polyethylene glycol urethane propionaldehyde derivative, And at least one of a pendant polyethylene glycol-amide-propionaldehyde derivative and a polyethylene glycol urethane-propionaldehyde derivative. 4. The combination according to claim 3, wherein the methoxy polyethylene glycol propionaldehyde derivative has a molecular weight range of 1,000 to 1,000,000. According to claim 3, wherein the linear methoxy polyethylene glycol propionaldehyde derivative has a molecular weight range of 1,000 to 100,000, the pendant polyethylene glycol propionaldehyde derivative has a molecular weight range of 5,000 to 1,000,000 polyethylene glycol skeleton, pendant group A compound wherein the number of phosphorus amide-propionaldehyde or urethane-propionaldehyde is 1 to 20.