HK1136838B - Pegylated erythropoietin conjugate and preparation method and uses thereof - Google Patents

Abstract

A PEGylated erythropoietin conjugate with the general formula rhuEPO-NH-CH2-X-S-Y-mPEG is provided. The preparation method of the conjugate is also provided, which includes a reductive amination reaction between erythropoietin and aldehydes containing protected thiol groups to form activated erythropoietin with a - NH-CH2- bond, and then coupling reaction with methoxypolyethylene glycol derivatives to obtain the conjugate. The conjugate or pharmaceutical composition containing it can be used to treat diseases lacking erythropoietin or red blood cell deficiency or deficiency.

Description

Polyethylene glycol erythropoietin conjugate and preparation method and application thereof

Technical Field

The invention relates to a polyethylene glycol erythropoietin conjugate, which has the biological activity of improving the hemoglobin content and reticulocyte number in vivo, and the application field of the invention relates to biochemistry, medicinal chemistry and treatment of human diseases.

Technical Field

Erythropoietin (EPO) is a glycoprotein hormone with a molecular weight of about 34 kD. Erythropoietin present in plasma consists of 165 amino acids, is glycosylated to a high degree, and the glycosyl component is predominantly sialic acid. Naturally occurring erythropoietin is classified into two types, the alpha type containing 34% carbohydrate and the beta type containing 26% carbohydrate, depending on the carbohydrate content. The two types are identical in biological characteristics, antigenicity and clinical application effect. The human erythropoietin gene is located in chromosome 7, region 22. In 1985, cDNA was successfully cloned, and large-scale production of recombinant human erythropoietin (rHuEPO) was started by gene recombination technology, and widely used in clinic. Erythropoietin (Egrie, JC, Strickland, Tw, Lane, J et al (986) immunobiology (lmmunobi0l) 72: 213-224), which is the product of a cloned human erythropoietin gene inserted into and expressed in ovarian tissue cells (CHO cells) of Chinese hamster, has been biosynthesized using recombinant DNA technology. Naturally occurring human erythropoietin is first translated into a polypeptide chain containing 166 amino acids with arginine at position 166. Arginine at position 166 is cleaved by hydroxypeptidase in post-translational modifications. The molecular weight of the multi-skin chain of human EPO without a sugar group is 18236da in the intact erythropoietin molecule, the sugar group represents approximately 40% of the total molecular weight (j.biol.chem.262: 12059).

Erythropoietin is the first cytokine to be used clinically and is the hemoglobin-increasing preparation which has the single action and is safe and reliable to date. Has certain curative effect on renal anemia, aplastic anemia, multiple myeloma, paroxysmal nocturnal hematuria and the like; in addition, the application of EPO can reduce the blood transfusion amount in operation and can correct anemia caused by malignant tumor, chemotherapy and rheumatoid arthritis to a certain extent. Since erythropoietin is mainly produced by tubular endothelial cells, anemia caused by renal disorders is the first indication for erythropoietin; EPO has almost 100% efficacy in correcting renal anemia, but does not improve renal function. The treatment of the erythropoietin is safe and effective, is suitable for long-term treatment and can also avoid the blood source tension. In the world biotech drug market in 2006, recombinant drugs of the erythropoietin class account for $ 119 billion, with enormous market capacity.

As early as 1989, the U.S. FDA approved recombinant human erythropoietin (EPOGEN) for the treatment of renal anemia, but it was not marketed in China until 1992. The annual incidence rate of chronic nephritis in China is about 0.25%, a considerable number of patients can be finally converted into renal failure, and about 50-60 ten thousand of patients with annual renal anemia are treated. According to the conservative drug consumption estimation, if the drug is taken by other patients with 30-40 yuan per patient according to the current price and cancer-related anemia and the like, the domestic market volume is about 12-16 million yuan or more (the average weight of the patients is calculated by 50 Kg). Since the later 90 s in the 20 th century, EPO entered the popular drug line in hospitals in key cities in China, and in 2003, the amount of medication in sample hospitals in key cities in China was 6213 ten thousand yuan, and the rank was 56 th. In 2004, the medicine purchase amount of sample hospitals in key cities in China is increased to 8049 ten thousand yuan, which is increased by 30% on year-on-year basis.

Erythropoietin is an endocrine hormone which acts on bone marrow hematopoietic cells to promote the proliferation and differentiation of erythroid progenitor cells and finally matures. Plays an important role in regulating and controlling the oxygen supply condition of organisms. EPO is produced in the early embryonic stage by the liver and then gradually transferred to the kidney, and is secreted mainly by tubular interstitial cells after birth.

During the process of erythropoietin-induced erythroid differentiation, globins are induced, which enable the cells to take up more iron to synthesize functional hemoglobin, which can bind with the oxygen in mature red blood cells, and thus, red blood cells and hemoglobin play an extremely important role in providing body oxygen. This process is caused by the interaction between erythropoietin and surface receptors of the erythroid cells.

When a person is in a healthy state, the tissue can absorb enough oxygen from the existing red blood cells, where the body's concentration of erythropoietin is low, such a normal low erythropoietin concentration is completely stimulating to promote normal loss of red blood cells due to age problems.

When the level of oxygen transport by red blood cells in the circulatory system is reduced and hypoxia occurs, the amount of erythropoietin in the body will increase and the hypoxic state of the body can be caused by: excessive radiation, decreased oxygen intake due to high altitude or long-term coma, various types of anemia, and the like. In response to the tissue being subjected to hypoxic pressure, an increase in erythropoietin levels stimulates the differentiation of the red blood cells to the point of increasing erythropoiesis. When the number of red blood cells in the body is greater than that required by normal tissue, the levels of erythropoietin in the circulatory system are reduced. Because erythropoietin plays a crucial role in erythropoiesis, this class of hormones holds great promise for the treatment and diagnosis of hematological disorders characterized by poor and defective erythropoiesis. Recent studies have provided the basis for the speculation of the utility of erythropoietin therapy in a variety of diseases, disorders and hematological abnormalities, including: the use of erythropoietin in the treatment of anemia in patients with Chronic Renal Failure (CRF) and EPO in the treatment of anemia in AIDS and cancer patients undergoing chemotherapy (Danna, RP, Rudnick, SA, Abels, RI, in: MB, Garnic, eds., erythropoietin in Clinical Applications-International patent application. Marcel Dekker; 1990: p 301-324).

However, currently available unmodified erythropoietins have short plasma half-lives, are subject to protease degradation, and are poorly available, which prevents them from achieving maximum clinical efficacy. Therefore, obtaining long-acting erythropoietin has become a hot spot of competitive research and development of various research institutions and pharmaceutical enterprises, for example, the marketed long-acting erythropoietin product (aransp) of amgen company increases the number of glycosylation sites by means of genetic engineering, further improves the glycosylation degree, achieves injection once every two weeks, improves the half-life period of erythropoietin in vivo, but the product still cannot avoid the enzymolysis influence of protease in vivo, so the degree of prolonging the half-life period in vivo is limited, and the production cost is higher.

Disclosure of Invention

The invention aims to provide a polyethylene glycol modified erythropoietin conjugate with better biological activity and higher bioavailability and a preparation method of the conjugate.

It is also an object of the present invention to provide a pharmaceutical composition comprising the modified erythropoietin conjugate for the treatment of diseases characterized by a deficiency of erythropoietin or a deficiency or defect in the red blood cell population.

The invention discloses a new erythropoietin conjugate modified by polyethylene glycol, which has a structural general formula

P-NH-CH₂-X-S-Y-(OCH₂CH₂)_m1-OCH₃

The conjugate is formed by methoxy polyethylene glycol group through-CH₂-CH in-X-S-Y-₂-the group forms-NH-CH with the amino group of erythropoietin₂Linked by a linkage, wherein P is recombinant human erythropoietin and X is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_k-k is selected from 2 to 10, m1 is selected from integers between 100 and 2000, Y is selected from:

wherein the numbers of m and n are respectively and independently selected from 2-10.

Among them, the recombinant human erythropoietin is preferably recombinant human erythropoietin alpha or beta, especially alpha. Erythropoietin is produced by techniques disclosed in the art.

In the scheme, m and n are preferably integers of 2.

In this embodiment, where X is- (CH)₂)_k-, k is selected from 2 to 10.

In the scheme, the number of k is preferably 2-4, and more preferably 2.

In the scheme, the average molecular weight of the methoxypolyethylene glycol group is preferably 5,000-40,000 daltons, and more preferably 20,000 daltons.

In this embodiment, the conjugate according to a preferred embodiment has the formula:

wherein P is recombinant human erythropoietin and X is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_kThe number of k is 2 to 10, the number of m and n is 2 to 10, m₁Is selected from an integer of 100 to 2000.

Wherein X is preferably- (CH)₂)_kThe number of k may further preferably be 2 to 4, and most preferably 2.

Further preferably, an embodiment of the present invention corresponds to the structural formula:

m₁an integer selected from 450 to 600, wherein P is recombinant human erythropoietin, more preferably recombinant human erythropoietin alpha or beta, most preferably alpha.

The invention also provides a conjugate of the following structural formula:

wherein P is recombinant human erythropoietin glycoprotein and X is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_kThe number of k is 2 to 10, the number of m is 2 to 10, m₁Is selected from an integer of 100 to 2000.

Further preferably, P in the structural formula refers to recombinant human erythropoietin α; x is- (CH)₂)_k-, where k is 2; the number of m is selected from 2; m is₁An integer selected from 450 to 600.

The invention also provides a conjugate of the following structural formula:

wherein P is recombinant human erythropoietin and X is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_kThe number of k is 2 to 10, the number of m is 2 to 10, m₁Is selected from an integer of 100 to 2000.

Further preferably, in the structural formula, P refers to recombinant human erythropoietin alpha; x is- (CH)₂)_k-, where k is 2; the number of m is selected from 2; m is₁An integer selected from 450 to 600.

The invention also provides a conjugate of the following structural formula:

wherein P is recombinant human erythropoietin and X is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_kThe number of k is selected from 2 to 10, m₁Is selected from an integer of 100 to 2000.

Further preferably, P in the structural formula refers to recombinant human erythropoietin α; x is- (CH)₂)_k-, where k is 2; m is₁An integer selected from 450 to 600.

The invention also provides a conjugate of the following structural formula:

wherein P is recombinant human erythropoietin and X is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_kThe number of k is selected from 2 to 10, m₁Is selected from an integer of 100 to 2000.

Further preferably, P in the structural formula refers to recombinant human erythropoietin alpha; x is- (CH)₂)_k-, where k is 2; m is₁An integer selected from 450 to 600.

The invention also provides a conjugate of the following structural formula:

wherein P is recombinant human erythropoietin and X is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_kThe number of k is 2 to 10, the number of m and n is 2 to 10, m₁Is selected from an integer of 100 to 2000.

Further preferably, P in the structural formula refers to recombinant human erythropoietin α; x is- (CH)₂)_k-, where k is 2; the number of m and n is selected from 2; m is₁An integer selected from 450 to 600.

The conjugate disclosed by the invention is erythropoietin through a connecting body (-CH)₂-X-S-Y-) is covalently linked to a methoxypolyethylene glycol group and is modified, the key being that erythropoietin forms-NH with the linker₂-CH₂The conjugate provided by the invention can improve the retention time and the circulation half-life of plasma, reduce the clearance rate, has higher biological activity in vivo and has the same application as erythropoietin compared with the unmodified erythropoietin. Compared with polyethylene glycol erythropoietin conjugate in the form of-NH-CO-bond, the conjugate has the advantages of more single reaction site and controllable quality. The polyethylene glycol erythropoietin conjugates provided by the invention can be used to treat diseases characterized by a deficiency of erythropoietin or a deficiency or defect in the red blood cell population.

The invention also discloses a preparation method of the conjugate, which comprises the following steps: firstly, erythropoietin and aldehyde substance containing protected sulfhydryl group are subjected to reductive amination reaction to form alpha-NH-CH₂-linked activated erythropoietin, which is then deprotected, coupled with an activated methoxypolyethylene glycol derivative, and purified by further isolation.

The invention also discloses a pharmaceutical composition, comprising: (1) a therapeutic amount of the pegylated erythropoietin conjugate described above; (2) a pharmaceutically acceptable pharmaceutical carrier.

The invention comprises any of the above therapeutic amounts of a pegylated erythropoietin conjugate for treating a condition characterized by a deficiency in erythropoietin or a deficiency or defect in a population of red blood cells. In particular for the treatment of the following diseases: end-stage renal failure or dialysis; AIDS-related anemia, autoimmune diseases, or malignancies; cystic fibrosis; early stage prematurity anemia; anemia associated with chronic inflammatory disease; spinal cord injury; acute blood loss; aging and neoplastic diseases accompanied by abnormal red blood cell production.

Preparation of erythropoietin

Erythropoietin is produced by expression of a protein through activation of an endogenous gene, and is well known in the art. Their preparation and therapeutic use are described in detail in US patents US5547933, US5621080 and US5955422, EP-B0209539 and EP-B0148605 and EP-B0209539, and Huang, S.L., Proc. Natl.Acad.Sci.USA (1984) 2708-.

Purification of recombinant erythropoietin is generally carried out using purification methods known in the art such as hydroxyapatite gel, ion exchange chromatography, gel chromatography and the like, and is described in detail in U.S. Pat. Nos. 5547933, 5621080 and 5955422; nobuo, I.et al, biochemical impurities (J.biochem)107(1990)352-359 also describe methods for the purification of recombinant EPO.

Preparation of polyethylene glycol erythropoietin conjugate

The invention provides a preparation method of a polyethylene glycol erythropoietin conjugate, which comprises the following steps:

(1) the small molecule aldehyde compounds of the general structural formula I are prepared by techniques well known to those skilled in the art, such as the michael addition of thioacetic acid to enal compounds:

wherein the number of k is 2-10, preferably 2;

(2) reacting the micromolecular aldehyde compound with the structural general formula I and erythropoietin in a buffer solution, and adding a reducing agent to obtain activated erythropoietin with the structural general formula II;

wherein P is recombinant human erythropoietin alpha or beta, preferably recombinant human erythropoietin alpha, the pH value of the buffer solution is 4.0-6.0, preferably 6.0, and the reducing agent can be sodium borocyanide or sodium cyanoborocyanide, preferably sodium cyanoborocyanide;

(3) a deprotection agent is added to a buffer containing activated erythropoietin by a technique known in the art to remove the acetyl protecting group of the activated erythropoietin of the general structural formula (II), and then activated methoxypolyethylene glycol of the general structural formula III is added to carry out a pegylation reaction.

Wherein the deprotecting agent added is preferably hydroxylamine; the pH value of the buffer system is selected from 5.0-7.0, and preferably 6.2; AG is selected from:

wherein the number of m and n is 2-10, preferably 2;

(4) purification of the polyethylene glycol erythropoietin conjugate is carried out by techniques known to those skilled in the art, such as ion exchange chromatography, gel chromatography.

Testing of biological Activity

The biological activity of erythropoietin or the polyethylene glycol erythropoietin conjugates provided by the present invention can be determined by a variety of assays well known in the art. In vivo activity test by subcutaneous injection of erythropoietin and polyethylene glycol erythropoietin conjugate provided by the invention to mice for three consecutive days, then the mice are sacrificed, whole blood is taken for peripheral blood cell and reticulocyte counting, and the blood cell counting is carried out by adopting a full-automatic blood cell counter. The pharmacodynamics study is carried out on the intravenous injection of macaques, the single administration dose is 1.35mg/kg, the administration dose of erythropoietin used as a comparative medicine is 240 mu/kg, the medicine is taken three times per week for six weeks, and blood samples are collected for carrying out the analysis of related hematology indexes.

The test data of the polyethylene glycol erythropoietin conjugate provided by the invention show that: the polyethylene glycol erythropoietin conjugate provided by the invention can obviously stimulate the increase of the count of mouse peripheral blood network red blood cells, so that the conjugate can stimulate the generation of red blood cells, and meanwhile, the half-life period of the conjugate in vivo can be greatly prolonged. The polyethylene glycol erythropoietin conjugate has no obvious influence on mature red blood cells, hematocrit and hemoglobin content and has no obvious influence on peripheral blood leukocyte counting liquid.

Preparation of pharmaceutical compositions

Pharmaceutical compositions suitable for injection can be formulated using pharmaceutically acceptable carriers or excipients by methods well known in the art. Preferred pharmaceutically acceptable carriers for formulating the products of the invention are human serum albumin, human plasma proteins and the like. The compounds of the invention can be formulated in a 10mM sodium/potassium phosphate buffer pH7 containing 132mM sodium chloride. Optionally, the pharmaceutical composition may contain a preservative. The pharmaceutical compositions may contain varying amounts of erythropoietin, preferably 10-1000 micrograms/ml.

Drawings

FIG. 1: effect of pegylated erythropoietin conjugate (HH-EPO-014A) on macaque hematocrit.

FIG. 2: effect of pegylated erythropoietin conjugate (HH-EPO-014A) on cynomolgus hemoglobin content.

Detailed Description

Example 1: preparation of acetylmercaptopropionaldehyde

11.2g (20mmol) of acrolein and 100ml of dry THF were charged into a reaction flask, cooled to 0 ℃ and then a mixed solution of 1.52g (0.2mol) of thioacetic acid/20 ml of THF was slowly added dropwise. After the dropwise addition, the reaction was carried out for 2 hours under heat preservation. Excess acrolein was removed by concentration at 35 ℃ under reduced pressure. Then the mixture is quickly loaded on a column (pure hexane → n-hexane/ethyl acetate 50/1) and product points are collected and concentrated under reduced pressure to be dry to obtain 0.6g of oily liquid.

Example 2: preparation of mPEG-MAL-01(20kD)

Putting 20g (1mmol) mPEG-OH (20kD) into a 200ml single-neck bottle, adding 100ml toluene, refluxing, and carrying out water separation reaction for 2.5 hr; the toluene was then distilled off, cooled to room temperature, 100ml of DCM were added, followed by 1.18g (4mmol) of triphosgene (triphosgene) and the reaction was stirred overnight at room temperature under sealed conditions; the next day of treatment: the reaction solution was eluted into 200ml of anhydrous ether in a fume hood, filtered and dried under vacuum to obtain 15g of a white solid. Putting 15g of the above white solid into a 200ml single-neck flask, adding 100ml of Toluene/DCM (2: 1) solution, adding 0.25g of HOSu, then adding 0.3g of triethylamine, and reacting for 4hr (or overnight) under sealed stirring at room temperature; after the reaction is finished, filtering the reaction solution, directly eluting the filtrate into 100ml of anhydrous ether, filtering, and drying in vacuum to obtain 14g of white solid, namely SC-mPEG (20 kD);

dissolving 1.4g anhydrous ethylenediamine in 50ml DCM in a 200ml reaction flask, dissolving 14gSC-mPEG (20kD) in 100ml DCM, adding into the ethylenediamine solution, and reacting overnight; stopping reaction and filtering the reaction the next day, adding 500ml of saturated saline solution into the filtrate for washing, separating an organic layer, extracting a water layer with DCM for three times (200ml multiplied by 3), combining the organic layers, drying the organic layers by anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to 100ml, precipitating the filtrate in 500ml of anhydrous ether to separate out a solid, filtering, and drying in vacuum to obtain 13g of a white solid, namely mPEG-NHCH₂CH₂NH₂(20kD)；

1.9g of MAL-ONP was dissolved in 50mL DCM, 0.04g triethylamine was added, and 13g of mPEG-NHCH was added₂CH₂NH₂(20kD) was dissolved in 100ml of freshly opened DCM and added to the above solution of MAL-ONP in DCM and reacted overnight at room temperature; the next day, DCM is concentrated under reduced pressure, the residue is added into 200ml of anhydrous ether, solid is precipitated by sedimentation, and white solid 12.5g is obtained by vacuum drying, namely mPEG-MAL-01(20 kD).

Example 3: preparation of mPEG-MAL-02(20kD)

2.0g of MAL-ONP was dissolved in 50mL DCM, 0.05g triethylamine was added, and 15g of mPEG-NH was added₂(20kD) was dissolved in 100ml of freshly opened DCM and added to the above solution of MAL-ONP in DCM and reacted overnight at room temperature; the next day, DCM is concentrated under reduced pressure, the residue is added into 200ml of anhydrous ether, solid is precipitated by sedimentation, and white solid 13g, namely mPEG-MAL-02(20kD), is obtained by vacuum drying.

Example 4: preparation of mPEG-OPPS-01(20kD)

0.5g MPPS was dissolved in 50ml DCM, followed by the addition of 0.05g triethylamine and 20g mPEG-NH₂(20kD), stirring at room temperature for reaction overnight, concentrating DCM under reduced pressure the next day, adding 200ml of anhydrous ether into the residue, separating out a solid, filtering, and drying in vacuum to obtain 19g of a white solid, namely mPEG-OPPS-01(20 kD).

Example 5: preparation of mPEG-OPPS-02(20kD)

0.5g MPPS was dissolved in 50ml DCM and 0.05g triethylamine and 20g mPEG-NHCH were added₂CH₂NH₂(20kD), stirring at room temperature for reaction overnight, concentrating DCM under reduced pressure the next day, adding 200ml of anhydrous ether into the residue, separating out a solid, filtering, and drying in vacuum to obtain 18.5g of a white solid, namely mPEG-OPPS-02(20 kD).

Example 6: preparation of activated erythropoietin

60 mg of erythropoietin stock solution is taken, the protein concentration is 1.5 mg/ml, the total volume is 40 ml, the system is 0.1M sodium phosphate buffer solution, and the pH value is 6.0; dissolving 2.5 mg of acetylmercaptopropionaldehyde in 80 microliters of acetonitrile, and adding the protein solution; then 50 mg of sodium cyanoborohydride is weighed and added into the reaction solution, and the mixture is stirred slowly for reaction, and the reaction temperature is controlled at 10 ℃ in ice bath for 24 hours; then the reaction solution was transferred to a dialysis bag (molecular weight cut-off 3500), and dialyzed against 0.1M sodium phosphate buffer solution containing 2mM EDTA, pH6.25, to remove excess small molecular aldehydes, and then hydroxylamine was added to remove acetyl groups, to obtain activated erythropoietin.

Example 7: preparation of HH-EPO-014A

10mg of activated erythropoietin (1.4 mg/ml, 0.1M sodium phosphate buffer, 2mM EDTA, pH6.25) obtained in example 6 was added with 100 mg of mPEG-MAL-01(20 kD). Stirring and reacting for 60 minutes; adding N-methylmaleimide to the concentration of 5mM, and reacting at room temperature for 30 minutes to remove the residual sulfydryl on the protein; the reaction was then dialyzed against a 20mM acetate-acetate buffer system.

Purification of the reaction mixture was carried out by ion exchange chromatography (SP Sepharose H.P) and gel chromatography (Superdex200) in this order to obtain about 5mg of pegylated erythropoietin (HH-EPO-014A).

Example 8: preparation of HH-EPO-014B

10mg of activated erythropoietin (1.4 mg/ml, 0.1M sodium phosphate buffer, 2mM EDTA, pH6.25) having free thiol groups obtained in example 6 was added with 100 mg of mPEG-MAL-02(20 kD). The reaction was stirred for 60 minutes (25 ℃); adding N-methylmaleimide to the concentration of 5mM, and reacting at room temperature for 30 minutes to remove the residual sulfydryl on the protein; the reaction was then dialyzed against a 20mM acetate-acetate buffer system.

Purification of the reaction mixture was carried out by ion exchange chromatography (SP Sepharose H.P) and gel chromatography (Superdex200) in this order to obtain pegylated erythropoietin (HH-EPO-014B) in an amount of about 5.5 mg.

Example 9: preparation of HH-EPO-014C

To a solution of 10mg of activated erythropoietin (1.4 mg/ml, 0.1M sodium phosphate buffer, containing 2mM EDTA, pH6.25) obtained in example 6, 100 mg of mPEG-MAL (20KD) was added, and the reaction was stirred for 60 minutes (25 ℃ C.), N-methylmaleimide was added to a concentration of 5mM, and the reaction was carried out at room temperature for 30 minutes to react off the remaining thiol groups on the protein; the reaction was then dialyzed against a 20mM acetate-acetate buffer system.

Purification of the reaction mixture was carried out by ion exchange chromatography (SP Sepharose H.P) and gel chromatography (Superdex200) in this order to obtain pegylated erythropoietin (HH-EPO-014C) in an amount of about 6.2 mg.

Example 10: preparation of HH-EPO-014D

To a solution of 10mg of activated erythropoietin (1.4 mg/ml, 0.1M sodium phosphate buffer, containing 2mM EDTA, pH6.25) obtained in example 6, 100 mg of mPEG-OPPS (20KD) was added, and the reaction was stirred for 60 minutes (25 ℃ C.), N-methylmaleimide was added to a concentration of 5mM, and the reaction was carried out at room temperature for 30 minutes to react off the remaining thiol groups on the protein; the reaction was then dialyzed against a 20mM acetate-acetate buffer system.

Purification of the reaction mixture was carried out by ion exchange chromatography (SP Sepharose H.P) and gel chromatography (Superdex200) in this order to obtain pegylated erythropoietin (HH-EPO-014D) in an amount of about 6.0 mg.

Example 11: preparation of HH-EPO-014E

To a solution of 10mg of activated erythropoietin (1.4 mg/ml, 0.1M sodium phosphate buffer, containing 2mM EDTA, pH6.25) obtained in example 6, 100 mg of mPEG-OPPS-01(20KD) was added, and the reaction was stirred for 60 minutes (25 ℃ C.), and N-methylmaleimide was added to a concentration of 5mM and reacted at room temperature for 30 minutes to react the remaining thiol groups on the protein; the reaction was then dialyzed against a 20mM acetate-acetate buffer system.

Purification of the reaction mixture was carried out by ion exchange chromatography (SP Sepharose H.P) and gel chromatography (Superdex200) in this order to obtain pegylated erythropoietin (HH-EPO-014E) in an amount of about 5.6 mg.

Example 12: preparation of HH-EPO-014F

To a solution of 10mg of activated erythropoietin (1.4 mg/ml, 0.1M sodium phosphate buffer, containing 2mM EDTA, pH6.25) obtained in example 6, 100 mg of mPEG-OPPS-02(20KD) was added, and the reaction was stirred for 60 minutes (25 ℃ C.), and N-methylmaleimide was added to a concentration of 5mM and reacted at room temperature for 30 minutes to react the remaining thiol groups on the protein; the reaction was then dialyzed against a 20mM acetate-acetate buffer system.

Purification of the reaction mixture was carried out by ion exchange chromatography (SP Sepharose H.P) and gel chromatography (Superdex200) in this order to obtain about 5.6mg of pegylated erythropoietin (HH-EPO-014F).

Examples of the experiments

Experimental example 1: effect of polyethylene glycol erythropoietin conjugate on mice

Purpose of the experiment:

the effect of polyethylene glycol erythropoietin conjugate and erythropoietin protein on mouse erythropoiesis was evaluated and compared.

The material and the method are as follows:

the polyethylene glycol erythropoietin conjugate HH-EPO-014A, HH-EPO-014B, HH-EPO-014C, HH-EPO-014D, HH-EPO-014E, HH-EPO-014F is provided by Jiangsu Haison pharmaceutical industry GmbH; erythropoietin (positive control): purchased from shenyang sansheng pharmaceutical llc; kunming mouse, purchased from Shanghai laboratory animal center of Chinese academy of sciences, with weight of 25-30 g, female parent and animal number of each group: 10 pieces of the Chinese herbal medicine.

Injecting polyethylene glycol erythropoietin conjugate and erythropoietin subcutaneously into mice for three consecutive days, then killing the mice, taking whole blood to count peripheral blood cells and reticulocytes, and counting the blood cells by using a full-automatic blood cell counter.

Results and discussion:

according to the current dosing regimen, both the polyethylene glycol erythropoietin conjugate and erythropoietin significantly stimulated the increase in mouse peripheral blood reticulocyte counts, indicating that they stimulated erythropoiesis (see Table I). The polyethylene glycol erythropoietin conjugate has no obvious influence on mature red blood cells, hematocrit and hemoglobin content (see table two) and has no obvious influence on peripheral blood leukocyte counting solution (see table three).

TABLE I Effect of polyethylene glycol erythropoietin conjugates on mouse reticulocyte production

TABLE II Effect of polyethylene glycol erythropoietin conjugate on mouse erythropoiesis, hematocrits and hemoglobin content

Epimeris, Effect of polyethylene glycol erythropoietin conjugate on mouse thrombopoiesis and leukopoiesis

Experimental example 2: effect of polyethylene glycol erythropoietin conjugate on macaques

Purpose of the experiment:

evaluation of the Effect of polyethylene glycol erythropoietin conjugates on macaque erythropoiesis

The material and the method are as follows:

polyethylene glycol erythropoietin conjugate HH-EPO-014A, provided by Jiangsu Haison pharmaceutical Co., Ltd; erythropoietin (positive control): purchased from Shenyang Sansheng pharmaceuticals, Inc. Before use, the cells were diluted with 0.1% BSA in physiological saline.

The macaque is 5.5-8.5 kg in weight, is unlimited in male and female, and is purchased from the experimental animal center of the Zhongshan Zhongke Suzhou province. The macaques are grouped according to the basic hemoglobin, and each group comprises three macaques. HH-EPO-014A, 1.35mg/kg, administered intravenously; EPO240 mu/kg, three times per week, continuous administration for 6 weeks, 1-2 hematology indexes measured every week

Results and discussion:

a single intravenous injection of HH-EPO-014A resulted in an increase in the peripheral hemoglobin content and an increase in hematocrit in macaques, indicating that HH-EPO-014A stimulated hemoglobin production, which peaked 35 days after administration and then slowly declined, with a hemoglobin-stimulating effect of approximately 33%. The positive control erythropoietin also increases the peripheral blood hemoglobin content of the macaque and increases the hematocrit, and the effect of the positive control erythropoietin is slowly weakened after stopping the medicine application. According to the current administration scheme, a single intravenous injection of HH-EPO-014A and multiple successive intravenous injections of erythropoietin have comparable stimulation effects on the production of hemoglobin in macaques (see figures 1 and 2).

Claims (29)

1. A polyethylene glycol erythropoietin conjugate with a general structural formula

P-NH-CH₂-X-S-Y-(OCH₂CH₂)_m1-OCH₃

The conjugate is formed by methoxy polyethylene glycol group through-CH₂-CH in-X-S-Y-₂-the group forms-NH-CH with the amino group of erythropoietin₂Linked by a linkage, wherein P is recombinant human erythropoietin and X is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_k-k is an integer from 2 to 10, m1 is an integer from 100 to 2000, Y is an integer from:

wherein the numbers of m and n are respectively and independently selected from integers of 2-10;

the average molecular weight of the methoxy polyethylene glycol group is 20,000-40,000 daltons.

2. The conjugate of claim 1, wherein said recombinant human erythropoietin is recombinant human erythropoietin α or β.

3. The conjugate of claim 2, characterized in that the recombinant human erythropoietin is recombinant human erythropoietin α.

4. The conjugate of claim 1, wherein m-2 and n-2 in Y.

5. The conjugate of claim 1, wherein X is- (CH)₂)_k-, k is an integer of 2 to 10.

6. The conjugate of claim 5, wherein k is an integer selected from 2 to 4.

7. The conjugate of claim 6, wherein k is 2.

8. The conjugate of claim 1, wherein the structural formula of the conjugate is:

wherein P is recombinant human erythropoietin;

x is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_k-；

The number of k is an integer of 2-10;

m and n are integers of 2-10;

m₁is selected from an integer of 100 to 2000.

9. The conjugate of claim 8, wherein X is- (CH)₂)_kThe number of k is an integer of 2 to 4.

10. The conjugate of claim 9, wherein X is- (CH)₂)_kThe number of k is 2.

11. The conjugate of claim 1, wherein the structural formula of the conjugate

Wherein m is₁An integer selected from 450 to 600.

12. The conjugate of claim 8, wherein said recombinant human erythropoietin is recombinant human erythropoietin α or β.

13. The conjugate of claim 12, wherein said recombinant human erythropoietin is recombinant human erythropoietin α.

14. The conjugate of claim 1, having the formula:

wherein P is recombinant human erythropoietin;

x is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_k-；

The number of k is an integer of 2-10;

m is an integer of 2-10;

m₁is selected from an integer of 100 to 2000.

15. The conjugate of claim 14, wherein P is recombinant human erythropoietin α, k is 2, m is 2, and m1 is an integer selected from 450 to 600.

16. The conjugate of claim 1, having the formula:

wherein P is recombinant human erythropoietin;

x is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_k-；

k is an integer of 2-10;

m is selected from 2-10;

m₁is selected from an integer of 100 to 2000.

17. The conjugate of claim 16, wherein P is recombinant human erythropoietin α and X is- (CH)₂)₂-, m is 2, and m1 is an integer selected from 450 to 600.

18. The conjugate of claim 1, wherein the structural formula of the conjugate is:

wherein P is recombinant human erythropoietin;

x is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_k-；

k is an integer of 2-10;

m₁is selected from an integer of 100 to 2000.

19. The conjugate of claim 18, wherein P is recombinant human erythropoietin α and X is- (CH)₂)₂-，m₁An integer selected from 450 to 600.

20. The conjugate of claim 1, wherein the structural formula of the conjugate is:

wherein P is recombinant human erythropoietin;

x is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_k-；

The number of k is an integer of 2-10;

m is an integer of 2-10;

m₁is selected from an integer of 100 to 2000.

21. The conjugate of claim 20, wherein P is recombinant human erythropoietin α and X is- (CH)₂)₂-, m is₂，m₁An integer selected from 450 to 600.

22. The conjugate of claim 1, having the formula:

wherein P is recombinant human erythropoietin;

x is- (CH)₂)_k-or-CH₂(OCH₂CH₂)_k-；

The number of k is an integer of 2-10;

m and n are integers of 2-10;

m₁is selected from an integer of 100 to 2000.

23. The conjugate of claim 22, wherein P is recombinant human erythropoietin α and X is- (CH)₂)₂-, m and n are 2, m₁An integer selected from 450 to 600.

24. A process for preparing a conjugate according to any one of claims 1 to 23, comprising the steps of:

(1) the erythropoietin and aldehyde substances containing protected sulfhydryl groups are subjected to reductive amination reaction to form activated erythropoietin connected by-NH-CH 2-bonds;

(2) deprotection of the activated erythropoietin, coupling with an active methoxypolyethylene glycol derivative.

25. A pharmaceutical composition comprising:

(1) a therapeutic amount of a pegylated erythropoietin conjugate according to any one of claims 1 to 23, and

(2) a pharmaceutically acceptable pharmaceutical carrier.

26. Use of a conjugate according to any one of claims 1 to 23 in the manufacture of a medicament for the treatment of a disorder characterised by a deficiency in erythropoietin or a deficiency or defect in the red blood cell population.

27. Use of a pharmaceutical composition according to claim 25 in the manufacture of a medicament for the treatment of a disorder characterized by a deficiency in erythropoietin or a deficiency or defect in the red blood cell population.

28. Use according to claim 26 or 27, characterized in that the disease characterized by a deficiency of erythropoietin or a deficiency or defect in the red blood cell population is end-stage renal failure or dialysis; AIDS-related anemia, autoimmune diseases, or malignancies; cystic fibrosis; early stage prematurity anemia; anemia associated with chronic inflammatory disease; spinal cord injury; acute blood loss and aging.

29. The use according to claim 26 or 27, wherein the disorder characterized by a deficiency of erythropoietin or a deficiency or defect in the red blood cell population is a neoplastic disorder accompanied by abnormal red blood cell production.