TWI430811B - Pegylated erythropoietin conjugates, preparing method and use thereof - Google Patents

Description

PEGylated erythropoietin conjugate and preparation method and use thereof

The present invention relates to a PEGylated erythropoietin conjugate having biological activity for increasing hemoglobin content and number of reticulocytes in vivo, and the field of application of the present invention relates to biochemistry, medicinal chemistry and human diseases. treatment.

Erythropoietin (EPO) is a glycoprotein hormone with a molecular weight of approximately 34kD. The erythropoietin present in plasma is composed of 165 amino acids, with a high degree of glycosylation, and the glycosyl component is mainly sialic acid. Depending on the carbohydrate content, naturally occurring erythropoietin is divided into two types, alpha containing 34% carbohydrate and beta containing 26% carbohydrate. Both types are identical in biological properties, antigenicity, and clinical application. The human erythropoietin gene is located in the long 22 region of chromosome 7. In 1985, its cDNA was successfully cloned, and genetic recombinant technology was used to start mass production of recombinant human erythropoietin (rHuEPO), which is widely used in clinical practice. Erythropoietin has been biosynthesized using recombinant DNA technology (Egrie, JC, Strickland, TW, Lane, J et al. (986) Immunobiology (lmmunobiol 72: 213-224), which is inserted into the ovarian tissue of Chinese hamsters. The product of the cloned human erythropoietin gene expressed in cells (CHO cells). The naturally occurring human erythropoietin is first translated into a polypeptide chain containing 166 amino acids and position 166 is arginine. The arginine at position 166 was cleaved with hydroxypeptidase in post-translational modification. More people without glycosyl EPO The molecular weight of the skin chain is 18236 Da. In the intact erythropoietin molecule, the glycosyl group accounts for about 40% of the entire molecular weight (J. Biol. Chem. 262: 12059).

The erythropoietin is the earliest clinically applied cytokine and is the single most effective and reliable hemoglobin preparation known to date. For renal anemia, aplastic anemia, multiple myeloma and paroxysmal nocturnal hematuria, etc.; in addition, the use of EPO can also reduce the amount of blood transfusion during surgery, and can be corrected to some extent by malignant tumors, chemotherapy And anemia caused by rheumatoid arthritis. Since erythropoietin is mainly produced by renal tubular endothelial cells, anemia caused by renal diseases is the first choice for erythropoietin; EPO corrects renal anemia by almost 100%, but does not improve renal function. The treatment of erythropoietin is safe and effective, suitable for long-term treatment, and can also avoid blood shortage. In the global biotech market in 2006, recombinant drugs that promote erythropoietin accounted for $11.9 billion, with huge market capacity.

As early as 1989, the US FDA officially approved the recombinant human erythropoietin (EPOGEN) for the treatment of renal anemia, but it was not listed in mainland China until 1992. The annual incidence of chronic nephritis in mainland China is about 0.25%, and a considerable number of patients will eventually become renal failure. The annual incidence of renal anemia is about 500,000-600,000. According to the conservative drug dosage, if the current price is 30-40 yuan / support (the currency value in this article refers to the RMB), plus other patients such as cancer-related anemia, the mainland market capacity is about 1.2-1.6 billion yuan. Larger (patient average weight is calculated at 50Kg). Since the late 1990s, EPO has entered the ranks of best-selling drugs in key cities in mainland China, 2003 In the sample hospitals in key cities in mainland China, the amount of medicine used was 62.13 million yuan, ranking 56th. In 2004, the amount of medicine purchased by sample hospitals in key cities in the mainland increased to 80.49 million yuan, a year-on-year increase of 30%.

As a kind of endocrine hormone that acts on bone marrow hematopoietic cells and promotes proliferation, differentiation and final maturation of erythroid progenitor cells. It plays an important regulatory role in the oxygen supply of organisms. In the early stages of the embryo, EPO is produced by the liver and then gradually metastasized to the kidney, which is mainly secreted by the tubulointerstitial cells after birth.

In the process of erythropoietin-induced red cell differentiation, globulin is induced, which enables cells to absorb more iron and synthesize functional hemoglobin, which can bind to oxygen in mature red blood cells. Therefore, red blood cells and hemoglobin play an extremely important role in providing organic oxygen. This process is caused by the interaction between erythropoietin and surface receptors of red blood cells.

When the human body is in a healthy state, the tissue can absorb enough oxygen from the existing red blood cells, and the concentration of erythropoietin in the body is very low. This normal lower erythropoietin concentration can stimulate the promotion. Red blood cells that are normally lost due to age problems.

When the level of oxygen transport in the circulatory system by red blood cells is reduced and hypoxia occurs, the amount of erythropoietin in the body will increase, and the hypoxia state of the organism can be caused by the following reasons: excessive radiation, due to high latitude Or a decrease in oxygen intake due to prolonged coma, various types of anemia, and the like. As a response to the hypoxia stress of the tissue, the increase in the level of erythropoietin can stimulate the differentiation of red blood cells to increase red blood. The ability to generate balls. When the number of red blood cells in the body is greater than the demand of normal tissues, the level of erythropoietin in the circulatory system is lowered. It is precisely because erythropoietin is critical for the production of red blood cells, such hormones have broad prospects for the treatment and diagnosis of blood diseases characterized by low red blood cell formation and defects. Recent studies have provided the basis for speculating 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). EPO is used in the treatment of AIDS in AIDS patients undergoing chemotherapy and chemotherapy (Danna, RP, Rudnick, SA, Abels, RI, in: MB, edited by Garnick, Erythropoietin in Clinical Applications) (Erythropoietin in Clinical Applications) -An International Perspective.Marcel Dekker; 1990: p301-324).

However, the unmodified erythropoietin currently has a short plasma half-life, is susceptible to protease degradation, and is not highly utilized, and these defects prevent them from achieving the greatest clinical efficacy. Therefore, long-acting erythropoietin has become the focus of research and development by major research institutions and pharmaceutical companies. For example, amgen's long-acting erythropoietin product (aransp) has been added by genetic engineering. The number of bit points of the base, which in turn increases the degree of glycosylation, once every two weeks, improves the half-life of erythropoietin in the body, but this product still cannot avoid the enzymatic hydrolysis of protease in the body, so The extent of prolonging the half-life in the body is limited and the production cost is high.

It is an object of the present invention to provide a polyethylene glycol modified erythropoietin conjugate having better biological activity and higher bioavailability and a method for preparing the conjugate.

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

The invention discloses a novel polyethylene glycol modified erythropoietin conjugate having the structural formula P-NH-CH ₂ -X-S-Y-(OCH ₂ CH ₂ ) _m1 -OCH ₃ said conjugate is a methoxy polyethylene glycol group via the formula -CH ₂ -X-S-Y- is -CH ₂ - groups erythropoietin amino group formed -NH-CH ₂ - bond Linked to, wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and m1 is selected from 100 An integer between 2000 and Y, selected from: Wherein the number of m, n is each independently selected from 2 to 10.

The recombinant human erythropoietin is preferably recombinant human erythropoietin alpha or beta, especially alpha. The preparation of erythropoietin is obtained via techniques disclosed in the art.

In the present scheme, the preferred integer of m, n is 2.

In the present scheme, wherein X is -(CH ₂ ) _k -, k is selected from 2 to 10.

In the present scheme, the number of k is preferably from 2 to 4, more preferably 2.

In the present embodiment, the average molecular weight of the methoxypolyethylene glycol group is preferably from 5,000 to 40,000 Daltons, more preferably 20,000 Daltons.

In the present embodiment, the structural formula of the conjugate corresponding to a preferred embodiment is: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and the number of m and n is selected from 2 Up to 10, m _{1 is} selected from an integer between 100 and 2000.

Wherein X is preferably -(CH ₂ ) _k -, and the number of k may more preferably be from 2 to 4, and most preferably 2.

More preferably, the structural formula corresponding to one embodiment of the present invention is: m _{1 is} selected from an integer of from 450 to 600, wherein P means recombinant human erythropoietin, more preferably recombinant human erythropoietin α or β, most preferably α. The invention also provides a conjugate of the following structural formula: Wherein P refers to a recombinant human erythropoietin glycoprotein, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and the number of m is selected from 2 Up to 10, m _{1 is} selected from an integer between 100 and 2000.

More preferably, in the formula, P means recombinant human erythropoietin α ; X is -(CH ₂ ) _k -, wherein k is 2; the number of m is selected from 2; m _{1 is} selected from an integer of 450 to 600 .

The invention also provides a conjugate of the following structural formula: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and m _{1 is} selected from 100 to 2000. An integer between.

More preferably, in the formula, P means recombinant human erythropoietin α; X is -(CH ₂ ) _k -, wherein k is 2; and m _{1 is} selected from an integer of 450 to 600.

The invention also provides a conjugate of the following structural formula: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and m _{1 is} selected from 100 to 2000. An integer between.

More preferably, in the formula, P means recombinant human erythropoietin α; X is -(CH ₂ ) _k -, wherein k is 2; and m _{1 is} selected from an integer of 450 to 600.

The invention also provides a conjugate of the following structural formula: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and the number of m and n is selected from 2 Up to 10, m _{1 is} selected from an integer between 100 and 2000.

More preferably, in the formula, P means recombinant human erythropoietin α; X is -(CH ₂ ) _k -, wherein k is 2; m, n is selected from 2; m _{1 is} selected from 450 to 600 The integer.

The invention also provides a conjugate of the following structural formula: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and m _{1 is} selected from 100 to 2000. An integer between.

More preferably, in the formula, P means recombinant human erythropoietin α ; X is -(CH ₂ ) _k -, wherein k is 2; and m _{1 is} selected from an integer of 450 to 600.

The invention also provides a conjugate of the following structural formula: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and the number of m and n is selected from 2 Up to 10, m _{1 is} selected from an integer between 100 and 2000.

More preferably, in the formula, P means recombinant human erythropoietin α ; X is -(CH ₂ ) _k -, wherein k is 2; m, n is selected from 2; m _{1 is} selected from 450 to 600 The integer.

The conjugate disclosed in the present invention is a erythropoietin which is covalently linked to a methoxypolyethylene glycol group via a linker (-CH ₂ -X-S-Y-) and is modified, and the key is to promote red blood cell production. The conjugate and the linker are linked in the form of a -NH ₂ -CH ₂ - bond, and the conjugate provided by the present invention can increase plasma residence time and circulating half-life, and reduce clearance rate compared to unmodified erythropoietin. It has higher biological activity in the body and has the same use as erythropoietin. Compared with the PEGylated erythropoietin conjugate which forms the -NH-CO- bond, it has the advantages of more single reaction site and more controllable quality. The PEGylated erythropoietin conjugate provided by the present invention can be used to treat diseases characterized by a deficiency or deficiency of erythropoietin or red blood cell population.

The invention also discloses a preparation method of the above conjugate, comprising the steps of: firstly, reductive amination reaction of erythropoietin and an aldehyde group containing a protected sulfhydryl group to form a bond via a -NH-CH ₂ - bond; The erythropoietin is activated, and then the activated erythropoietin is deprotected, coupled with a reactive methoxypolyethylene glycol derivative, and further obtained by further separation and purification.

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

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

Preparation of erythropoietin

The preparation of erythropoietin is a method of initiating expression of a protein via an endogenous gene, and is a technique well known in the art. Its preparation and therapeutic applications are described in detail in U.S. Patent Nos. 5,594,933, 5, 612, 1080 and 5,955, 422, EP-B 0 209 539 and EP-B 0 148 605 and EP-B 0 209 539, and Huang, SL, Proc. Natl. Acad. Sci. USA ( 1984) 2708-2712 and so on.

Purification of recombinant erythropoietin is generally carried out using hydroxyapatite gel, ion exchange chromatography, gel chromatography, etc., as is well known in the art, and is disclosed in U.S. Patent Nos. 5,457,933, 562, 1080 and 5,955,422. A detailed description is given; Nobuo, I. et al., J. Biochem 107 (1990) 352-359 also describes a purification method for recombinant EPO.

Preparation of PEGylated erythropoietin conjugate

The present invention provides a process for preparing a PEGylated erythropoietin conjugate, the steps of which comprise: (1) preparing a small molecule aldehyde compound of the formula I, such as thiol, via techniques well known to those skilled in the art. The Michael addition reaction of acetic acid with an alkenal compound: Wherein the number of k is selected from 2 to 10, preferably 2; (2) reacting the small molecule aldehyde compound of structural formula I with erythropoietin in a buffer, and adding a reducing agent to obtain activation of the structural formula II Erythropoietin Wherein P refers to recombinant human erythropoietin α or β , preferably recombinant human erythropoietin α , the pH of the buffer is selected from 4.0 to 6.0, preferably 6.0, and the reducing agent may be sodium borohydride or cyanide. Sodium borohydride, preferably sodium cyanoborohydride; (3) a deprotecting agent is added to a buffer containing activated erythropoietin by a technique known in the art, and the structural formula (II) is removed. The acetamino protecting group of the erythropoietin is activated, followed by the PEGylation reaction of the activated methoxypolyethylene glycol of the formula III.

The deprotecting agent to be added is preferably hydroxylamine; the pH of the buffer system is selected from 5.0 to 7.0, preferably pH 6.2; and the AG is selected from the group consisting of: Wherein the number of m, n is selected from 2 to 10, preferably 2; (4) the purification method of the PEGylated erythropoietin conjugate is carried out by techniques well known to those skilled in the art, such as ion exchange chromatography and coagulation. Glue chromatography.

Biological activity test

The biological activity of erythropoietin or the PEGylated erythropoietin conjugate provided by the present invention can be determined by various tests well known in the art. In vivo activity test The subcutaneous injection of erythropoietin and the PEGylated erythropoietin conjugate provided by the present invention were administered to mice for three consecutive days, and then the mice were sacrificed, and whole blood was taken for peripheral blood cell and reticulocyte count. The blood cell count was counted using a fully automated blood cell counter. The pharmacodynamic study of cynomolgus monkey injection was performed at a single dose of 1.35 mg/kg, and the erythropoietin used as a comparative drug was administered at a dose of 240 μg/kg three times a week for six weeks for blood samples. Analysis of relevant hematological indicators.

The test data of the PEGylated erythropoietin conjugate provided by the present invention indicates that the PEGylated pro-erythropoietin provided by the present invention The conjugates can significantly stimulate the increase of peripheral blood reticulocyte counts in mice, indicating that they stimulate red blood cell production, and can also greatly extend the half-life of conjugates in vivo. The PEGylated erythropoietin conjugate had no significant effect on mature red blood cells, hematocrit, and hemoglobin content, and had no significant effect on peripheral blood leukocyte count.

Preparation of pharmaceutical compositions

Pharmaceutical compositions suitable for injection can be made by 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 may be formulated in 10 mM sodium phosphate/potassium buffer pH 7 containing 132 mm sodium chloride. The pharmaceutical composition may contain a preservative as needed. The pharmaceutical composition may contain varying amounts of erythropoietin, preferably from 10 to 1000 micrograms per milliliter.

Example 1: Preparation of acetyl hydrazinopropanal

11.2 g (20 mmol) of acrolein and dry 100 ml of THF were added to the reaction flask, cooled to 0 ° C, and then a mixed solution of 1.52 g (0.2 mol) of thioacetic acid / 20 ml of THF was slowly added dropwise. After the dropwise addition, the reaction was allowed to stand for 2 hours. The excess acrolein was removed by concentration under reduced pressure at 35 °C. Then, it was subjected to flash column chromatography (eluent pure n-hexane: n-hexane / ethyl acetate = 50/1), and the product was collected and concentrated to dryness to give an oily liquid.

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

20 g (1 mmol) of mPEG-OH (20 kD) was placed in a 200 ml single-mouth bottle, 100 ml of toluene was added, and refluxed for 2.5 hr; then toluene was distilled off, cooled to room temperature, and then 100 ml of DCM was added, followed by the addition of 1.18 g (4 mmol). The triphosgene was stirred at room temperature overnight; the next day of treatment: the reaction solution was poured into 200 ml of anhydrous diethyl ether in a ventilated kitchen, filtered, and dried in vacuo to give a white solid 15 g. 15 g of the above white solid was placed in a 200 ml single-mouth bottle, 100 ml of Toluene/DCM (2:1) solution was added, 0.25 g of HOSu was added, followed by 0.3 g of triethylamine, and the reaction was stirred at room temperature for 4 hr (or overnight). After the reaction is completed, the reaction solution is filtered, and the filtrate is directly poured into 100 ml of anhydrous diethyl ether, filtered, and dried in vacuo to give a white solid 14 g, which is SC-m PEG (20 kD); 1.4 g of anhydrous ethylenediamine with 50 ml DCM Dissolved in a 200 ml reaction flask, and then dissolved in 14 ml of SC-mPEG (20 kD) dissolved in 100 ml of DCM, added to the above ethylenediamine solution, and allowed to react overnight; the reaction was stopped the next day and filtered, and the filtrate was washed with 500 ml of saturated brine. The organic layer was separated, and the aqueous layer was extracted with EtOAc EtOAc EtOAcjjjjjjjjjj 13 g of white solid, ie mPEG-NHCH ₂ CH ₂ NH ₂ (20 kD); 1.9 g of MAL-ONP was dissolved with 50 mL of DCM, 0.04 g of triethylamine was added, and 13 g of mPEG-NHCH ₂ CH ₂ NH _{2 was added.} (20kD) Dissolve with 100ml of freshly opened DCM, then add MAL-ONP in DCM solution, the reaction was carried out at room temperature overnight; DCM was concentrated under reduced pressure on the next day, and the residue was added to 200 ml of anhydrous diethyl ether. The solid was precipitated and dried in vacuo to give a white solid (12.5 g, m. 20kD).

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

2.0 g of MAL-ONP was dissolved in 50 mL of DCM, 0.05 g of triethylamine was added, and 15 g of mPEG-NH ₂ (20 kD) was dissolved in 100 ml of freshly opened DCM, and then added to the above MAL-ONP in DCM solution. The reaction was carried out at rt.

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

0.5 g of MPPS was dissolved in 50 ml of DCM, followed by the addition of 0.05 g of triethylamine and 20 g of mPEG-NH ₂ (20 kD), and the reaction was stirred at room temperature overnight, and DCM was evaporated to dryness. Filtration and drying in vacuo gave 19 g of a white solid, m.p.

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

0.5 g of MPPS was dissolved in 50 ml of DCM, followed by the addition of 0.05 g of triethylamine and 20 g of mPEG-NHCH ₂ CH ₂ NH ₂ (20 kD), and the reaction was stirred at room temperature overnight, and DCM was evaporated to dryness. The solid was precipitated, filtered and dried in vacuo to give a white solid, 18.5 g, m.p.

Example 6: Preparation of activated erythropoietin

Take erythropoietin stock solution 60 mg, protein concentration 1.5 mg / ml, a total of 40 ml, the system is 0.1 M sodium phosphate buffer, pH 6.0; take 2.5 mg of ethyl decyl mercaptan dissolved in 80 μl of acetonitrile After adding the above protein solution; weighed 50 mg of sodium cyanoborohydride into the above reaction solution, and reacted under slow stirring, and the reaction temperature was controlled at 10 ° C for 24 hours in an ice bath; then the reaction solution was transferred to dialysis. Bag (molecular weight cutoff 3500), dialysis against 0.1M sodium phosphate buffer, containing 2mM EDTA, pH6.25, in order to remove excess small molecule aldehyde, and then add hydroxylamine to remove the ethyl thiol group to obtain activated erythropoiesis Prime.

Example 7: Preparation of HH-EPO-014A

10 mg of activated erythropoietin (1.4 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) obtained in Example 6 was added, and 100 mg of mPEG-MAL-01 (20 kD) was added. The reaction was stirred for 60 minutes; N-methyl maleimide was further added to a concentration of 5 mM, and reacted at room temperature for 30 minutes to remove the remaining sulfhydryl groups on the protein; then the reaction solution was dialyzed against a 20 mM acetic acid-acetate buffer system. .

The purification of the reaction solution was carried out by ion exchange chromatography (SP Sepharose H.P.) and gel chromatography (Superdex 200) to obtain PEGylated erythropoietin (HH-EPO-014A), about 5 mg.

Example 8: Preparation of HH-EPO-014B

10 mg of activated erythropoietin (1.4 mg/ml, 0.1 M sodium phosphate buffer containing 2 mM EDTA, pH 6.25) containing free sulfhydryl group obtained in Example 6, and 100 mg of mPEG-MAL-02 (20 kD) was added. . Stir the reaction for 60 minutes (25 ° C); add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes to remove the remaining sulfhydryl groups on the protein; then buffer the reaction solution in 20 mM acetic acid-acetate The liquid system is dialyzed.

The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) and gel chromatography (Superdex 200) to obtain PEGylated erythropoietin (HH-EPO-014B), which was about 5.5 mg.

Example 9: Preparation of HH-EPO-014C

To a solution of 10 mg of activated erythropoietin (1.4 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) obtained in Example 6, 100 mg of mPEG-MAL (20 KD) was added, and the reaction was stirred for 60 minutes. (25 ° C), then add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes to remove the remaining sulfhydryl groups on the protein; then the reaction solution was dialyzed against a 20 mM acetic acid-acetate buffer system .

The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) and gel chromatography (Superdex 200) to obtain PEGylated erythropoietin (HH-EPO-014C), which was about 6.2 mg.

Example 10: Preparation of HH-EPO-014D

Add 10 mg of mPEG-OPPS (20 KD) to a solution of 10 mg of activated erythropoietin (1.4 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) obtained in Example 6, and stir the reaction for 60 minutes. (25 ° C), then add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes to remove the remaining sulfhydryl groups on the protein; then the reaction solution was dialyzed against a 20 mM acetic acid-acetate buffer system .

The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) and gel chromatography (Superdex 200) to obtain PEGylated erythropoietin (HH-EPO-014D), which was about 6.0 mg.

Example 11: Preparation of HH-EPO-014E

Add 10 mg of mPEG-OPPS-01 (20 KD) to a solution of 10 mg of activated erythropoietin (1.4 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) obtained in Example 6. Stir the reaction. 60 minutes (25 ° C), then add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes to remove the remaining sulfhydryl groups on the protein; then the reaction solution in a 20 mM acetic acid-acetate buffer system Perform dialysis.

The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) and gel chromatography (Superdex 200) to obtain PEGylated erythropoietin (HH-EPO-014E), which was about 5.6 mg.

Example 12: Preparation of HH-EPO-014F

To the solution of 10 mg of activated erythropoietin (1.4 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) obtained in Example 6, 100 mg of mPEG-OPPS-02 (20 KD) was added, and the reaction was stirred. 60 minutes (25 ° C), then add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes to remove the remaining sulfhydryl groups on the protein; then the reaction solution in a 20 mM acetic acid-acetate buffer system Perform dialysis.

Purification of the reaction solution using ion exchange chromatography (SP Sepharose) H.P) and gel chromatography (Superdex 200) gave PEGylated erythropoietin (HH-EPO-014F), about 5.6 mg.

Experimental example Experimental Example 1: Effect of PEGylated erythropoietin conjugate on mice

Objective: To evaluate and compare the effects of PEGylated erythropoietin conjugate and erythropoietin protein on erythrocyte production in mice.

Materials and methods: PEGylated erythropoietin conjugates HH-EPO-014A, HH-EPO-014B, HH-EPO-014C, HH-EPO-014D, HH-EPO-014E, HH-EPO-014F Provided by Jiangsu Haosen Pharmaceutical Co., Ltd.; erythropoietin (positive control): purchased from Shenyang Sansheng Pharmaceutical Co., Ltd.; Kunming mice, purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences, weighing 25 to 30g, ♀ The number of animals in each group: 10 animals.

The mice were injected subcutaneously with PEGylated erythropoietin conjugate and erythropoietin for three consecutive days, then the mice were sacrificed, whole blood was taken for peripheral blood cells and reticulocyte count, and the blood cell count was controlled by automatic blood cell counter. count.

RESULTS AND DISCUSSION: According to the current dosage regimen, PEGylated erythropoietin conjugate and erythropoietin can significantly stimulate the increase of peripheral blood reticulocyte count in mice, indicating that they stimulate red blood cell production (see Table I). The PEGylated erythropoietin conjugate had no significant effect on mature red blood cells, hematocrit, and hemoglobin content (see Table 2), and had no significant effect on peripheral blood leukocyte counts (see Table 3).

Experimental Example 2: Effect of PEGylated erythropoietin conjugate on macaque

Objective: To evaluate the effect of PEGylated erythropoietin conjugate on erythrocyte production in macaques

Materials and Methods: PEGylated erythropoietin conjugate HH-EPO-014A, provided by Jiangsu Haosen Pharmaceutical Co., Ltd.; erythropoietin (positive control): purchased from Shenyang Sansheng Pharmaceutical Co., Ltd. the company. Dilute with normal saline containing 0.1% BSA before use.

Rhesus monkeys, weighing 5.5 to 8.5 kg, are not limited to males and females. They were purchased from Suzhou Xishan Zhongke Experimental Animal Center. Rhesus monkeys were grouped according to basal hemoglobin, three in each group. HH-EPO-014A, 1.35 mg/kg, intravenous-dose; EPO 240 μ/kg, three times/week, continuous administration for 6 weeks, and 1 to 2 hematological parameters were measured weekly.

RESULTS AND DISCUSSION: Single intravenous injection of HH-EPO-014A resulted in increased peripheral blood hemoglobin content and increased hematocrit in rhesus monkeys, indicating that HH-EPO-014A stimulated hemoglobin production, which reached a peak after 35 days of administration, followed by slowing. Decreased, the stimulating effect on hemoglobin is about 33%. The positive control erythropoietin also increased the peripheral blood hemoglobin content of the macaque and increased the hematocrit, and its effect slowly weakened after stopping the drug. According to the current dosing regimen, a single intravenous injection of HH-EPO-014A and multiple consecutive intravenous injections of erythropoietin have comparable stimulatory effects on rhesus hemoglobin production (see Figures 1 and 2).

Figure 1 is the effect of PEGylated erythropoietin conjugate (HH-EPO-014A) on hematocrit in rhesus monkeys.

Figure 2 is the effect of PEGylated erythropoietin conjugate (HH-EPO-014A) on the hemoglobin content of macaques.

Claims (30)

A PEGylated erythropoietin conjugate having the structural formula P-NH-CH ₂ -XSY-(OCH ₂ CH ₂ ) _m1 -OCH ₃ conjugate of methoxypolyethylene glycol The group is obtained by linking a -CH ₂ - group in the formula -CH ₂ -XSY- with an amino group of erythropoietin to form a -NH-CH ₂ - bond, wherein P means recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, m1 is selected from an integer between 100 and 2000, and Y is selected from: Wherein the number of m, n is each independently selected from 2 to 10. The conjugate of claim 1, wherein the recombinant human erythropoietin is recombinant human erythropoietin α or β. The conjugate of claim 2, wherein the recombinant human erythropoietin is recombinant human erythropoietin alpha. The conjugate of claim 1, wherein m=2, n=2. The conjugate of claim 1, wherein X is -(CH ₂ ) _k -, and k is selected from 2 to 10. The conjugate of claim 5, wherein k is selected from 2 to 4. The conjugate of claim 6 wherein k is 2. The conjugate of claim 1, wherein the methoxypolyethylene glycol group has an average molecular weight of from 5,000 to 40,000 Daltons (Dalton). The conjugate of claim 8 wherein the methoxypolyethylene glycol group has an average molecular weight of 20,000 Daltons. The conjugate of claim 1 wherein the structural formula of the conjugate is: , P means recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, m, n is selected from 2 to 10 m _{1 is} selected from an integer between 100 and 2000. The conjugate of claim 10, wherein X is -(CH ₂ ) _k -. The conjugate of claim 10, wherein k is selected from 2 to 4. The conjugate of claim 12, wherein k is 2. The conjugate of claim 10, wherein the structural formula of the conjugate is Wherein m _{1 is} selected from an integer of from 450 to 600. The conjugate of claim 10, wherein the recombinant human erythropoietin is recombinant human erythropoietin α or β. The conjugate of claim 15, wherein the recombinant human erythropoietin is recombinant human erythropoietin alpha. For example, in the conjugate of claim 1 of the patent scope, the structural formula is: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and m is selected from 2 to 10. m _{1 is} selected from an integer between 100 and 2000. The conjugate of claim 17, wherein P refers to recombinant human erythropoietin α, wherein k is 2; m is 2; and m ₁ is an integer selected from 450 to 600. For example, in the conjugate of claim 1 of the patent scope, the structural formula is: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, k is selected from 2 to 10, m is selected from 2 to 10, m _{1 is} selected An integer from 100 to 2000. The conjugate of claim 19, wherein P means recombinant human erythropoietin α; X is -(CH ₂ ) ₂ -; m is 2; and m _{1 is} selected from an integer of 450 to 600. q The conjugate of claim 1 of the patent scope, wherein the structural formula of the conjugate is: Its qP is q recombinant human q erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, k is selected from 2 to 10, and m _{1 is} selected from 100 to 2000. Integer. The conjugate of claim 21, wherein P means recombinant human erythropoietin α; X is -(CH ₂ ) ₂ -; m _{1 is} selected from an integer of 450 to 600. The conjugate of claim 1 wherein the structural formula of the conjugate is: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10; m is selected from 2 to 10; m _{1 is} selected from an integer between 100 and 2000. The conjugate of claim 23, wherein P means recombinant human erythropoietin α; X is -(CH ₂ ) ₂ -; m is 2, and m _{1 is} selected from an integer of 450 to 600. For example, in the conjugate of claim 1 of the patent scope, the structural formula is: Wherein P refers to recombinant human erythropoietin, X is -(CH ₂ ) _k - or -CH ₂ (OCH ₂ CH ₂ ) _k -, the number of k is selected from 2 to 10, and m and n are selected from 2 to 10 m _{1 is} selected from an integer between 100 and 2000. The conjugate of claim 25, wherein P means recombinant human erythropoietin α; X is -(CH ₂ ) ₂ -; m, n is 2; and m _{1 is} selected from an integer of 450 to 600 . A method for preparing a conjugate according to any one of claims 1 to 26, comprising the steps of: (1) reductive amination of erythropoietin and an aldehyde group having a protected sulfhydryl group, forming a pass -NH-CH ₂ - linkage activated erythropoietin; (2) the activated erythropoietin deprotected with active methoxy polyethylene glycol derivative conjugate. A pharmaceutical composition comprising: (1) a therapeutic amount of a PEGylated erythropoietin conjugate as described in any one of claims 1 to 26, and (2) a pharmaceutically acceptable drug Carrier. Use of a PEGylated erythropoietin conjugate of any one of claims 1 to 26 for the treatment of a disease characterized by a deficiency or deficiency of erythropoietin or red blood cell population Drug. For example, the use of the scope of claim 29, wherein the lack of erythropoietin or red blood cell population deficiency or defects characterized by end stage renal failure or dialysis; AIDS-related anemia, autoimmune disease, or malignant tumor; cystic Fibrosis; early maturity anemia; anemia associated with chronic inflammatory disease; spinal cord injury; acute blood loss; aging and neoplastic disease with abnormal red blood cell production.