TW201002350A - Polyethylenenized erythropoietin conjugate, preparation thereof and its use - Google Patents

Abstract

The present invention discloses pegylated erythropoietin conjugates, which are obtained by reacting -CH2- group in formula -CH2-X-S-Y- of methoxy polyethylene glycol group with amino group of erythropoietin to form a bond of-NH2-CH2-. The present invention also discloses the method of preparing above conjugates, i.e. the conjugates are prepared by the reductive amination of reacting erythropoietin with aldehydes containing protected mercaptos to form activated erythropoietin connected by -NH2-CH2-; the activated erythropoietin further were coupled to methoxy polyethylene glycol derivatives to obtain the pegylated erythropoietin conjugates. The method has the advantages of being more specific of the reaction sites and the quality being more controllable. The pegylated erythropoietin conjugates or the pharmaceutical composition containing the same provided by the present invention can be used widely for the treatment of diseases characterized in the deficiency of erythropoietin and the lack or defect of erythrocyte group.

Description

201002350 IX. Description of the invention: [Technical field of the invention] The present invention relates to a PEGylated erythropoietin conjugate, which has the biological activity of increasing the self-content of red blood eggs and the number of red blood cells in the body. The field of application of the invention relates to the treatment of biochemistry, medicinal chemistry and human diseases. [Prior Art] Erythropoietin (EPO) is a glycoprotein hormone with a molecular weight of about 34 kD. The erythropoietin present in plasma consists of 165 amino acids with a high degree of glycosylation and a predominantly sialic acid. Depending on the carbohydrate content, naturally occurring erythropoiesis is divided into two types, alpha containing 34% carbohydrate and p type containing 26% carbohydrate. Both types are identical in biological properties, antigenicity, and clinical application. The human erythropoietin gene is located in the 22nd region of chromosome 7. In 1985, its cDNA was successfully cloned and used to generate recombinant human erythropoietin (rHuEP〇), which is widely used in clinical practice. The use of recombinant DNA technology has biosynthesized erythropoietin JC, Strickland, TW, Lane, J et al (986) Immunobiology (lmmun〇bi〇1) 72·213-224), which is inserted into the ovary of Chinese hamsters. A product of a cloned human erythropoietin gene expressed in a tissue cell (CH〇 cell). The naturally occurring human erythropoietin is first translated into a polypeptide chain containing 166 amino acids and the 166th position is arginine. In the post-translational modification, the arginine at position 166 was cleaved with no peptidase. There is no glycosylation of human Ep〇 more than 94338 5 201002350 The chain of the knife in the chain l8236Da in the complete erythropoietin octapeptide II ^ Tangji accounted for about 40% of the entire molecular weight (JBic) 1.chem erythropoietin is the earliest The cytokine applied to the clinic is the hemoglobin preparation which has the most single-function and the most reliable shirt. For kidney blood, aplastic anemia, multiple osteosarcoma and paroxysmal nocturnal hematuria - have a certain effect, in addition, the application of EP0 can also reduce the degree of surgery in the second to some extent corrected by malignant tumors, Chemotherapy and rheumatoid arthritis are negative blood caused by fire. Since erythropoietin is mainly produced by the renal tubules, the anemia caused by renal diseases is the inflammatory factor of erythropoietin, and the effect of correcting renal anemia is almost the same, but it is not changed to sigma. The treatment of erythropoietin is safe, effective, and suitable for (4) treatment, and can also avoid blood shortage. In the annual global biotechnology market, the recombinant drug that promotes erythropoietin accounts for I-100 million US dollars, and has a huge market capacity. ~, (EP〇: 2, 1 field 989 Yuenian 'US FDA It officially approved the treatment of recombinant human erythropoietin in renal anemia, but it was not published in China until 1992. The annual incidence of chronic nephritis in mainland China is about 0.25%, and some patients will eventually become kidney. Decline, 3 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Medication, the mainland market capacity is called (a) Italian yuan or even period, the average weight is calculated according to 5GKg). Since the 2nd century 9G era > * month EPO has m 1 -» China's key cities hospitals selling drugs, 2003 94338 201002350 In the sample hospitals of 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 mainland China increased to 80.49 million yuan, a year-on-year increase of 3〇〇/0. It acts on bone marrow hematopoietic cells, promotes the proliferation and differentiation of erythroid progenitor cells, and finally matures endocrine hormones. It plays an important role in regulating the oxygen supply of organisms. In the early stage of embryos, Ep〇 is produced by the liver and then gradually metastasized to the kidneys. It is mainly secreted by tubulointerstitial cells. ° In the process of erythropoietin-induced red cell differentiation, globulin is induced, which enables cells to absorb more iron and synthesize functional red blood eggs. Sexual hemoglobin can bind to oxygen in mature red blood cells, so red blood cells and hemoglobin play an extremely important role in providing organic oxygen. This process is between erythropoietin and surface receptors of red blood cells. § caused by interaction § When the human body is in a healthy state, the tissue can absorb enough oxygen from the existing red: when the erythropoietin in the body is concentrating on you, the normal lower red blood cells Generate "degrees can be stabbed." Promote red blood cells that are normally lost due to age problems. #Red blood cells for oxygen delivery II, when hypoxia occurs, erythropoietin is in the body ^ : 加因? = hypoxia can be caused by the following reasons: excessive (four) heart rate or long-term coma caused by oxygen intake Reducing, each sputum is in response to the fibrination, and the increase in erythropoietin level can stimulate the differentiation of red blood cells to increase the ability of red blood 94438 7 201002350. When the number of red blood cells in the body is greater than normal tissue At the demand, the level of erythropoietin in the circulatory system is reduced. It is precisely because erythropoietin is crucial for the production of red blood cells, so these hormones are characterized by low red blood cell formation and defects for treatment and diagnosis. There is a wide range of prospects for blood diseases. Recent studies have provided the basis for speculating the utility of erythropoietin therapy in a variety of diseases, disorders, and ▲ fluid 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 for AIDS and cancer patients undergoing chemotherapy (Danna, RP, Rudnick, SA, Abels, RI, in: MB, edited by Garnick, International Prospects for 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. These defects prevent them from achieving maximum Clinical efficacy. Therefore, the long-acting red-promoting jk pheromone has become the focus of research and development by major research institutions and pharmaceutical companies. For example, amgen's long-acting erythropoietin product (aransp) is listed through the gene. The engineering means increase the position of the glycosylation 娄i: to improve the glycosylation process. Degree, reaching once every two weeks, improves the half-life of erythropoietin in the body, but this product is still unable to avoid the enzymatic hydrolysis of the protease in the body, so the degree of half-life in the body is limited, and the production cost is relatively low. [Inventive content] 8 94338 201002350 The present invention aims to provide a biologically active, bioavailable anabolic erythropoietin conjugate and = The purpose of the present invention is to provide a pharmaceutical composition comprising the modified erythropoietin conjugate for treating a disease characterized by a deficiency or deficiency of erythropoietin or a red blood cell population. A novel polyethylene glycol-modified erythropoietin conjugate ′ having the structural formula P-NH-CH2-XSY-(〇CH2CH2)ml-OCH3. The conjugate is composed of a decyloxy polyethylene glycol group. The group is linked to the amino group of erythropoietin by the -CH2. group in the formula _CH2_x_s_y_ to form -νη/η〗-bonding to obtain 'wherein Ρ means recombinant human erythropoietin, χ is (CH2)k- or _CH2(OCH2CH 2) k-, k is selected from 2 to l〇, mi is selected from an integer between 100 and 2000, and Y is selected from:

94338 9 201002350 The number of octagonal η is each independently selected from 2 to hemogenin, preferably recombinant human erythropoietin. . The preparation of erythropoietin is via the art in the present scheme, wherein m, n preferably has an integer of two. In the present scheme 'where X is -(CH2)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, and the 〇〇〇 Dalton, more preferably, 〇 (8) Dalton. In this embodiment, the chond of the preferred embodiment corresponds to: 13

八中P疋拍 recombinant human erythropoietin, X is or -CH2(〇CH2CH2)k-, the number of k is selected from 2 to 10, and the number of m and η is selected from 2 to 10' selected from 1 to An integer between 2000. Wherein X is preferably _(CH2)k-, and the number of k may more preferably be from 2 to 4, most preferably 2. More preferably, the structural formula corresponding to one embodiment of the present invention is:

0 is an integer selected from 450 to 600, where P refers to recombinant human erythrocyte growth 10 94338 201002350

^中中p refers to recombinant human erythropoietin glycoprotein, x*_(cH2)k_ or -CH2(〇CH2CH2)k-, the number of k is selected from 2 to 1〇, and the number of melons is selected from 2 to 10. Mi is selected from an integer between 1 〇〇 and 2 。. Further: In the structural formula, P refers to recombinant human erythropoietin α 'X疋-(CH2)k·, where let 2; 111 is selected from:: an integer selected from 450 to 600 . The invention also provides a conjugate of the following structural formula

八中p refers to recombinant human erythropoietin, χ is or -CH2(〇CH2CH2)k-, the number of k is selected from ^1〇, the number of m is selected from 2 to 10, and is selected from 1〇〇 to 2000 An integer between. More preferably, 'P in the structural formula refers to recombinant human erythropoietin α 'X疋-(CH2)k- ' where k is 2; the number of m is selected from 2; !^ is selected from an integer of 450 to 600. The present invention also provides a conjugate of the following structural formula: p-nh-ch2-x-s-s-cH3CH2_^〇CH2CH2|__〇ch 11 94338 201002350 wherein p is a recombinant human erythropoietin, x is The number of _(CH2)k_ or -CH2(OCH2CH2)k·,k is selected from 2 to 1 〇, which is an integer selected from 1 〇〇 to 2 00. More preferably, in the structural formula, P means recombinant human erythropoietin α, X $_(CH2)k-, wherein k is 2; m is selected from an integer of 450 to 000. The invention also provides a conjugate of the following structural formula:

P-NH-^_X_S_S

Wherein Ρ refers to recombinant human erythropoietin, χ is _(CH2)k_ or -CH2(OCH2CH2)k-, the number of k is selected from 2 to 1〇, m] is selected from 1〇〇 to 2000 Integer. More preferably, in the formula, P means recombinant human erythropoietin α, X is -(CHA- 'where k is 2; mi is selected from an integer of 45 〇 to _. The present invention also provides the following structural formula Conjugate: X-S-S-^CH2j~ H2 p-NH-c

The number of -CH2(OCH2CH2)k-,k is selected from . , 2 to 1 日, the number of m, η is selected from 2 to 10, and mi is selected from an integer between 100 and 2 。. More preferably, in the structural formula ρ县> in _ Λ, y 叭 叭 T 疋 疋 重组 重组 重组 重组 重组 重组 重组 重组 重组 , , , , , , , , , , , , , , lc lc lc lc lc lc lc lc lc lc lc lc lc lc lc lc lc lc lc ; T 疋 2, the number of m, η is selected from 2; im is selected from an integer of 450 to 600. 94338 12 201002350 The conjugate disclosed in the present invention is a erythropoietin which is covalently linked to a τ-oxypolyethylene glycol group via a hydrazine and is further decorated with a erythropoietin conjugate to form π 氐. _CH bond, connected and compared with erythropoietin which is not repaired: the conjugate provided by this method can increase plasma residence time and circulatory half-life, reduce clearance rate, and have more biological activity in vivo. It is high and has the same use as erythropoietin. Compared with the form of the polyethylene conjugated pheromone conjugate, it has the advantage of a single reaction site. The PEGylated erythropoiesis-deficient τ provided by the present invention is used to treat diseases characterized by lack of erythropoietin or red blood cell population defects or defects. The invention also discloses a preparation method of the above conjugate, comprising: first: erythropoietin and an aldehyde containing a protected sulfhydryl group, X-reductive amination reaction, formation via N, red blood cell m ^ The activation of the bond is coupled with the activity of the activated erythropoietin deprotected roll-based polyglycol derivative, and further obtained by further separation and purification. ^J Knife, above, also discloses a pharmaceutical composition comprising: (1) a therapeutic amount of a drug: a body 7 alcoholated erythropoietin conjugate; (2) a pharmaceutically acceptable polymerization having any of the above therapeutic amounts Ethylene glycolation promotes less red blood cells or defects: 4 士 = treatment for lack of erythropoietin or red blood cell population deficiency or permeation: disease. In particular, the following diseases are treated: end-stage renal function 'AIDS-related anemia, autoimmune disease, or 94238 13 201002350 'malignant tumor; cystic fibrosis; early maturity anemia; anemia associated with chronic inflammatory disease; spinal cord injury Acute blood loss; aging and tumor diseases associated with abnormal red blood cells. Preparation of erythropoietin The preparation method 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, 542, 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., and is purified by methods well known in the art, and is specialized in U.S. Patent Nos. 5,457,933, 562, 1080 and 5,955,422. This is described in detail in Nobuo, I., et al., J. Biochem 107 (1990) 352-359, which also describes methods for the purification of recombinant EPO. Preparation of PEGylated erythropoietin conjugate The present invention provides a method for preparing a PEGylated erythropoietin conjugate, the steps of which include: (1) via techniques well known to those skilled in the art Preparation of a small molecule aldehyde compound of the formula I', such as a Michael addition reaction of thioacetic acid with an aldehyde compound: Η

〇

CH 14 94338 201002350 wherein the number of k is selected from 2 to 1 Torr, preferably 2. The small molecular disk compound of buffer type 1 and erythropoietin are used to obtain an activated red blood erythropoietin of structural formula 11 in a reducing agent; ' -CH, -N-4c2Jps-

II :, " refers to the recombinant human erythropoietin α or 10,000, the selection of hemopoietin α' buffer ". to 6.0: better: the cyanide nano; minus, reduce Wei (four), Preferably, the cyano group (3) is added to the buffer containing the deprotecting agent by a technique known in the art, and the hematopoietic erythropoietin is produced by the erythrocyte-forming erythropoietin. The poly-incorporation structure...the living AG is in the middle of the ά λ in : the deprotecting agent is preferably added to 7.0, preferably pH 6.2; the AGii| ^ 糸 糸 yang is selected from 5 94338 15 201002350

Cn4"2^·

m u

Wherein the number of m and n is selected from 2 to 1 〇, preferably 2; (4) PEGylation of erythropoietin is coupled by a method known to those skilled in the art, and the method of deuteration Separation. Techniques, such as ion exchange chromatography, gel layer biological activity testing, can be measured by various tests well known in the art. The PEGylated erythropoietin conjugated physicochemical activity provided by the present invention. The test for in vivo activity was carried out by subcutaneous injection of erythropoietin in mice and the PEGylated erythropoietin conjugate provided by the present invention. The mice were sacrificed for two days, and the whole blood was taken for peripheral blood cells and reticulation. Red blood cell counts, blood cell counts were counted using an automated blood cell counter. Pharmacodynamic study of cynomolgus monkey intravenous injection, single dose * Hg / kg, as a comparative drug used erythropoietin dose of 24 ' heart three times a week, continuous administration for six weeks, blood samples were collected for correlation Analysis of hematology indicators. The test data of the PEGylated erythropoietin conjugate provided by the present invention indicates that the PEGylated erythrocyte-producing 16 94338 201002350 f-conjugate provided by the present invention can significantly stimulate the peripheral blood reticulocyte of mouse peripheral blood. The rise in n indicates that they stimulate red blood cell production and also greatly extend the half-life of the conjugate in the body. The PEGylated erythropoietin conjugate had no significant effect on the mature red blood cells, hematocrit, and hemoglobin content. There was no significant effect on the peripheral blood leukocyte count. For the preparation of a pharmaceutical composition, a pharmaceutical composition suitable for injection can be prepared by a method known in the art using a pharmaceutically acceptable carrier or excipient. A well-acceptable carrier for formulating the products of the present invention is human serum albumin, human plasma protein and the like. The compound of the present invention can be formulated in 10 mM sodium phosphate/potassium buffer pH 7 containing 132 mm of sodium chloride. The pharmaceutical composition may contain a preservative as needed. The pharmaceutical composition may contain varying amounts of erythropoiesis-forming hormone, preferably 10-1000 micrograms per milliliter. EXAMPLES Example 1: Preparation of Ethyl Mercaptopropionaldehyde

11.2 g (20 mmol) of compenic acid and dry 1 〇〇 mi thf were added to the reaction flask, cooled to 0 ° C, and then a mixed solution 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, the column was chromatographed (the eluent was purely hexane-supplemented, and the acetic acid was used to be 50/1). The product was collected and concentrated to dryness to give an oily liquid. Example 2: Preparation of mPEG-MAL-01 (20 kD) 94338 17 201002350

mPEG-OH(20KD) triphosgene 〇 〇 HOSU Tea"

5C-mPEG (20KD)

NH2

〇mPEG-NHCH2CH2NH2 (20KD) mPEG-MAL-01 (20KD) 20 20g (lmmol) mPEG-OH (20kD) was put into a 200ml single-mouth bottle, 1 〇〇ml of hydrazine was added, and the reaction was refluxed for 2.5 hr; Then, toluene was distilled off, cooled to room temperature, and then 100 ml of DCM was added, followed by 1.18 g (4 mmol) of triphosgene, and the reaction was allowed to stand overnight at room temperature; the second reaction was carried out: the reaction solution was poured into a ventilated cloud. In 200 ml of anhydrous B, it was filtered and dried in vacuo to give 15 g of white solid. 15 g of the above white solid was placed in a 200 ml single-mouth bottle, a solution of 10 ml of Toluene/DCM (2:1) was added, and then 25 g of HOSu was added, followed by the addition of 0.3 g of triethylamine, and the mixture was stirred at room temperature. After the reaction is completed, the reaction solution is filtered, and the filtrate is directly filtered into 100 ml of anhydrous diethyl ether, filtered, and dried in vacuo to give 14 g of white solid as SC-m PEG (20 kD); Diamine was dissolved in 2 ml of reaction flask with 50 ml of DCM, and 14 g of SC-mPEG (20 kD) was dissolved in 10 ml of DCM to dissolve 18 94338 201002350, and then added to the above ethylenediamine solution for overnight reaction; The reaction was stopped and filtered, and the filtrate was added to 500 ml of saturated brine for washing: the organic layer was separated, and the aqueous layer was extracted three times (20 〇 1111 < 3) with 0 〇], and the organic layers were combined and dried over anhydrous sodium sulfate. Filtration, the filtrate was concentrated to 100 ml under reduced pressure, and the solid was precipitated in 500 ml of anhydrous diethyl ether. The solid was filtered and dried in vacuo to give 13 g of white solid as m.sup.---------------- 〇.〇4g of triethylamine, then 13g of mPEG-NHC H2CH2NH2 (20kD) was dissolved in 100 ml of fresh DCM, then added to the above MAL-ONP in DCM solution, and allowed to react overnight at room temperature; DCM was concentrated under reduced pressure on the next day, and the residue was added to 200 ml of anhydrous diethyl ether to precipitate solids, vacuum Drying a white solid 12.5 g is mPEG-MAL_01 (20 kD). Example 3: Preparation of mPEG-MAL-02 (20 kD)

mPEG-MAL-02 (20KD) Dissolve 2.0g of MAL-ONP with 50mL of DCM, add 5g of triethylamine, and then dissolve 15g of mPEG-NH2 (20kD) with 100ml of newly opened DCM, then add The above-mentioned MAL-ONP in DCM solution was reacted at room temperature overnight; DCM was concentrated under reduced pressure on the next day, and the residue was added to 200 ml of 19 94338 201002350 of anhydrous diethyl ether to precipitate a solid which was dried in vacuo to give a white solid 13 g, m. -02 (20kD). Example 4: Preparation of mPEG-OPPS-01 (20 kD)

Η

〇mPEG-OPPS-01 (20KD) 0.5 g of MPPS was dissolved in 50 ml of DCM, followed by 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 concentrated under reduced pressure on the next day, and the residue was added. 200 ml of anhydrous diethyl ether, a solid precipitated, which was filtered and dried in vacuo to give a white solid, 19 g, m.p. Example 5: Preparation of mPEG-OPPS-02 (20 kD)

mPEG-NHCH2GH2NH2 (20KD)

mPEG-OPPS-02 (20KD) 0.5 g of MPPS was dissolved in 50 ml of DCM, followed by addition of 0.05 g of triethylamine and 20 g of mPEG-NHCH2CH2NH2 (20 kD), and the reaction was stirred at room temperature for 20 94338 201002350 'night, and DCM was concentrated under reduced pressure on the next day. The residue was added to 200 ml of anhydrous diethyl ether to precipitate a solid, which was filtered and dried under vacuum to give a white solid, 18.5 g, which is mPEG-〇PPS-02 (2〇kD) ° Example 6: Preparation of activated erythropoietin to promote red blood cells The product is 60 mg, the protein concentration is 1.5 mg/ml, a total of 40 ml, the system is 0.1 M sodium phosphate buffer, pH 6.0; 2.5 mg of acetoyl-propionic propionic acid 1 is dissolved in 8 μL of acetonitrile After adding the above protein to dissolve the wave; weighed 50 mg of cyano hydride and added the above reaction solution, and reacted under slow stirring. The reaction temperature was controlled at 10 ° C for 24 hours by an ice bath; then the reaction solution was then reacted. Transfer to dialysis bag (molecular weight cutoff 3500), dialysis against 0.1M sodium salt buffer, containing 2 mM EDTA, pH 6.25, in order to remove excess small molecule acid 1 'and then add amine to deacetylate 'It is activated to promote erythropoietin. - Example 7: Preparation of HH-EPO-014A 10 mg of activated erythropoietin (1.4 mg/ml, 0.1 M sodium acetate buffer 'containing 2 mM EDTA, pH 6.25) obtained from Example 6 Add 1 mg of mPEG-MAL-01 (20 kD). The reaction was stirred for 60 minutes; N-decylmaleimide was added to a concentration of 5 mM, and reacted at room temperature for 3 minutes to remove the remaining Wei group on the protein; then the reaction solution was applied to a 20 ml V1 acetic acid monoacetate 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-014A), about 5 mg. Example 8: Preparation of HH-EPO-014B 21 94338 201002350 1 mg of activated erythropoietin (1.4 mg/ml, 0.1 M sodium phosphate buffer containing 2 mM EDTA) containing free Wei group obtained via Example 6. pH 6.25), 100 mg of mPEG-MAL-02 (20 kD) was added. The reaction was stirred for 60 minutes (25 ° C); N-decylmaleimide was 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 treated with 20 mM acetic acid monoacetate. The buffer system was 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 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, 1 mg was added. mPEG-MAL (20KD), stir the reaction for 60 minutes (25 ° C), add N-mercaptomaleimide to a concentration of 5 mM, react at room temperature for 30 minutes to remove the remaining sulfhydryl groups on the protein; then react The solution was dialyzed against a 20 mM acetate-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 100 mg of the 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. Mg mPEG-OPPS (20KD), stir reaction for 60 minutes r 22 94338 201002350 (25 ° C), then add N-mercaptomaine to a concentration of 5 mM, react at room temperature for 30 minutes to remove the remaining protein Sulfhydryl; the reaction was then dialyzed against a 20 mM acetate-acetate buffer system. Purification of the reaction solution was carried out by using ion-exchanged 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 1 Omg of activated erythropoietin (1.4 mg / liter, 0.1 M sodium phosphate buffer, containing 2 mM hydrazine), ρ Η 6.25) solution obtained in Example 6 Add 100 mg of mPEG-OPPS-01 (20KD), stir the reaction for 60 minutes (25 ° C), add N-mercaptomaine to the concentration of 5 mM, and react at room temperature for 30 minutes to remove the remaining protein. Sulfhydryl; the reaction was then dialyzed against a 20 mM acetate-acetate buffer system. The purification of the reaction solution can be carried out by using _ ion exchange chromatography (SP Sepharose HP) and gel chromatography (Superdex 200) to obtain PEGylated red erythropoietin (HH-EPO-014E), about 5.6 mg. . Example 12: Preparation of HH-EPO-014F Add 100 mg of the solution of 10 mg of activated erythropoietin (1.4 mg / liter, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) obtained in Example 6. Mg mPEG-OPPS-02 (20KD), stir the reaction for 60 minutes (25 ° C), add N-methyl maleimide to a concentration of 5 mM, and react at room temperature for 30 minutes to remove the remaining sulfhydryl groups on the protein; The reaction solution was dialyzed against a 20 mM acetate-acetate buffer system. The reaction solution is purified by ion exchange chromatography (SP Sepharose 23 94338 201002350 HP) and gel chromatography (Superdex 2〇〇) to obtain PEGylated erythropoietin (HH-EPO-014F). 5.6 mg. Experimental Example Experimental Example 1. Effect of PEGylated erythropoietin conjugate on mice Objective: To evaluate and compare PEGylated erythropoietin conjugate and erythropoietin protein to mice The effect of red blood cell formation. Materials and Methods: PEGylated erythropoietin conjugate HH_Ep〇_〇i4A, HH-EPO-014B . HH-EPO-OHC . HH-EPO-014D , ΗΗ·ΕΡ〇·〇14E, HH-EPO -G14F is provided by Jiangsu Haosen Pharmaceutical Co., Ltd.; erythropoietin (positive control): purchased from Panyang Sansheng Pharmaceutical Co., Ltd.: Kunming mice, purchased from the Chinese Academy of Sciences Shanghai experimental money ~, weight 25 Up to 30g, early, the number of animals in each group: 1 〇 only. 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 dosing regimen, PEGylated erythropoietin conjugate and erythropoietin can significantly stimulate the rise of peripheral blood reticulocyte counts in mice, indicating that they stimulate red blood cell production ( See Table 1). The PEGylated erythropoietin conjugate had no significant effect on mature red blood cells and blood cell pressure 94438 24 201002350 'product and hemoglobin content (see Table 2), and had no significant effect on peripheral blood leukocyte counts (see Table 3). . Table 1. Effect of PEGylated erythropoietin conjugate on mouse reticulocyte formation. Group mice (only) Dosage and protocol reticulocyte count (x109/L, x±SD) Control group 10 0.1% BSA in NS 177.6±55.2 HH-EPO-014A 10 5pg/kg, sc, dl -3 826.1±25.1 HH-EPO-014B 10 5pg/kg, sc, dl -3 810.2±58.3 HH-EPO-014C 10 5pg/kg, sc, dl -3 765.3±67.5 HH-EPO-014D 10 5pg/kg, sc, dl-3 652.1±81.3 HH-EPO-014E 10 5pg/kg, sc, dl -3 647.5±24.1 HH- EPO-014F 10 5pg/kg, sc, dl -3 586_0±32.4 EPO 10 5pg/kg, sc, dl-3 360.6±27.0 Table 2. Pegylated erythropoietin conjugates on mouse erythropoiesis, Effects of hematocrit, blood and erythroprotein content on mice (only) Dosage and protocol red blood cell count (χ106/μί, x soil SD) Hematocrit (%) Hemoglobin (%) control ,10 0.1%BSAinNS 9 ·6±0·5 48.2+3.0 14.8±0.7 HH-EPO-014A 10 5pg/kg,sc,dl-3 9_9±0·4 52.111.6 15.7±0.6 HH-EPO-014B 10 5pg/kg,sc, Dl-3 9.3±0·1 53.0±1.5 15.7±0.7 HH-EPO-014C 10 5gg/kg, sc, dl-3 9.6±0.3 5 3.2+1.4 15.1±0.5 HH-EPO-014D 10 5pg/kg, sc, dl-3 9·7±0_1 50.0+1.9 14.7+0.7 HH-EPO-014E 10 5pg/kg, sc, dl-3 9.1±0.5 55.4+1.2 16.5+0.9 HH-EPO-014F 10 5pg/kg, sc, dl-3 9.2±0.6 56.5+1.8 16.3±0.7 EPO 10 5pg/kg, sc, dl-3 9.0+0.6 46.2+2.7 14.3±0.7 25 94338 201002350 Table 3. Effects of PEGylated erythropoietin conjugates on platelet and leukocyte production in mice Grouped mice dosed platelet leukocyte _(t) and protocol (χ103/μΙ〇 (xloVpL)

Control HH-EPO-014 A HH-EPO-014B HH-EPO-014C HH-EPO-014D HH-EPO-014E HH-EPQ-014F EPO ο ο ο ο ο ο ο 1* 1J ΙΑ 11 11 11 11 ο 1Χ 0.1% BSA in NS 1078.0±151.2 5.1±1.5 5pg/kg, sc, dl-3 1372·5±135·4·2±1.5 5pg/kg, sc, dl-3 1350.8±327 4.2±1.1 5eg/kg, Sc,dl-3 1207±237 5·0±2·2 5pg/kg,sc,dl-3,1325±223 5.5±1.2 5pg/kg,sc,dl-3 1457.6±247.6 4.2±1.2 5pg/kg, Sc, dl-3 1186.8±218.6 4.1±1.2 5ug/kg, sc, dl-3 1306.8±170. 4.0±0.9 Experimental Example 2: Effect of PEGylated erythropoietin conjugate on macaques Purpose: Evaluation Effect of PEGylated erythropoietin conjugate on erythrocyte production of rhesus monkeys Materials and methods: I Ethyl alcoholated erythropoietin conjugate HH-EPO-014A, provided by Jiangsu Haosen Pharmaceutical Co., Ltd.; promote red blood cells Producin (positive control). Purchased from Shenyang Ersheng Pharmaceutical Co., Ltd. Dilute with physiological saline containing 0.1% BSA before use. - Rhesus monkeys, weighing 55 to 85 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. EPO 〇i4A, i .35mg/kg ' intravenous injection; Ep〇, twice/week, continuous administration for 6 weeks 'weekly measurement】 to 2 times of gray liquidity index. RESULTS AND DISCUSSION: 94338 26 201002350 HH-EPO-014A single intravenous injection resulted in increased peripheral blood hemoglobin content and increased hematocrit in rhesus macaques, indicating that HH_Ep〇_〇14A stimulated hemoglobin production, which reached a peak 35 days after administration. Then, it slowly decreased, and the stimulating effect on hemoglobin was about 33%. The positive control promoted, 'hematopoietic stimulating hormone also increased the peripheral blood hemoglobin content of the monkey, 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 hemoglobin production in rhesus monkeys (see Figures 1 and 2). [Simple illustration of the diagram] Figure 1 shows the effect of polyethylated erythropoietin conjugate (HH-EP〇-〇 14 A) on the hematocrit of the monkey.苐2 is the effect of PEGylated erythropoietin conjugate (HH-EPO-014A) on the hemoglobin content of macaques. [Main component symbol description] None 94338 27

Claims (1)

201002350 X. Patent application scope: 1. A PEGylated erythropoietin conjugate, the structural formula of which is P-NH-CH2-XSY-(OCH2CH2)ml-〇CH3. The conjugate is composed of methoxy The polyglycol group is obtained by a bond between the -CH2. group in S_CH2_x_s_y_ and the amino group of erythropoietin, wherein P means recombinant human erythropoietin, χ is _(CH2)k• or - The number of CH2(〇CH2CH2)k-,k is selected from 2 to 1〇, ml is selected from an integer between 1 〇〇 and 2〇〇〇, and γ is selected from: 0 -f|-NH-CH2CH2- - _ -- , 5 CH2CH2 wherein the number of m, n is each independently selected from 2 to 1 Å. 2. The conjugate of claim 1, wherein the recombinant human erythropoietin is recombinant human erythropoietin alpha or stone. The conjugate of claim 2, wherein the recombinant human erythropoietin is recombinant human erythropoietin alpha. The conjugate of claim 1, wherein m = 2 and η = 2. m CH^ nh-ch2ch2 -S-I-CH b)-^-NH-(cH2)-m n CH2f NH-jy 28 94338 201002350 The conjugate of item 1 wherein X is _(CH2)k-, The group has an average molecular weight of 5, 〇〇〇 to 40, and Dalton. 5. If the patent application scope k is selected from 2 to 1 〇. 6. The conjugate of claim 8, wherein the decyloxypolyethylene glycol group has an average molecular weight of 20,000 Daltons, as claimed in claim 7. 1. The conjugate of claim 1, wherein the conjugate has the structural formula: 'P refers to recombinant human erythropoietin, X is _(CH2)k_biting-CH2(〇CH2CH2)k-, the number of k is selected from 2 to l〇, m, r^| 2 to 1〇' An integer selected from 1〇〇 to 2000. The conjugate of claim </RTI> wherein the X is _(CIi2)k_. 12. The conjugate of claim 10, wherein k is selected from 2 to 4. 13. The conjugate of claim 12, wherein k is 2. U. The conjugate of claim 10, wherein the conjugate has a dry configuration of 〇CH2CH2f〇〇n , 7ml 29 201002350 wherein an integer selected from 450 to 600 is selected. 15. The conjugate of claim 1 wherein the recombinant agent promotes '..X hematopoietin 疋 recombinant human erythropoietin^ or cold. 16. The conjugate according to claim 15, wherein the recombinant human erythropoietin is recombinant human erythropoietin. 17. The conjugate of the ninth aspect of the patent application is characterized by ·· Wherein P refers to recombinant human erythropoietin, χ is _(匸4)^ or -CH2(OCH2CH2)k·, the number of k is selected from 2 to i〇, and m is selected from 2 to 10 'π^ An integer between 1〇〇 and 2〇〇〇. 18. The conjugate of claim 17, wherein p refers to recombinant human erythropoietin alpha, wherein let 2; 111 is 2; and is selected from an integer selected from 450 to 600. 19. The conjugate of claim i, wherein the structural formula is P~NH-CH2—X—s”々丨X (CH2/~C_NHtCH2j ^0CH2CH less OCH; wherein P refers to recombinant human erythropoietin, χ is _(匸^) wide or -CH2(OCH2CH2)k- , k is selected from 2 to 1 , and m is selected from 2 to 1 , and is an integer selected from 100 to 2000. 1 20. The conjugate of claim 19, wherein p is a recombinant Erythropoietin α; again _((:112)2_; 111 is 2; is an integer selected from 94238 30 201002350 4 5 0 to 600. 21 · conjugate as claimed in claim 1 Wherein, the age of the conjugate is: ',' mouth P-NH-CH〇-X- _s-CH2CH2-|-〇CH2CH2〇CH, wherein P refers to recombinant human erythropoietin, x is _ (匚 only) Ge-CH2(OCH2CH2)k-, k is selected from 2 to 10, and mi is selected from an integer between 1 and 2 to 22. The conjugate of claim 21, wherein p is a heavy human erythropoietin alpha; Χ is -(CH2)2·; mi is selected from the group consisting of 45 〇, and an integer of 600. Structure to the conjugate 23. As in the scope of claim 1 Linkage, where the formula is: Wherein P refers to recombinant human erythropoietin, χ is _(CH^-CH2(〇CH2CH2)k_, the number of k is selected from 2 to 1〇; m is selected from 2 or 10; IHi is selected from 1〇〇 to 2 An integer between 〇〇〇. 24. The conjugate of claim 23, wherein P is heavy* human erythropoietin α; X is -(CH2)2-; m is 2, called 'Integer of 450 I to 600. From 254. For example, the conjugate of claim 1 of the patent scope has the structural formula: 94338 201002350 9 ,, P, privately recombinant human erythropoietin, X is _(CH2)k_ or -CH2(〇CH2CH2)k, k is selected from 2S10, m, n is selected from 2 to 10, m ! is selected from an integer between 1〇〇 and 2000. 26. For example, the conjugate of claim 25, wherein? Refers to recombinant human erythropoietin alpha; X is -(CH2)2-; m, n is selected from 2; mi is selected from an integer from 450 to 600. A method for preparing a conjugate of the ninth to the ninth item of the patent application, comprising the steps of: (2) reductive amination of erythropoietin and an aldehyde containing a protected sulfhydryl group; Forming an activated erythropoietin linked by an even-bond; (2) the activated erythropoietin is protected and coupled with a reactive oxy polyethylene glycol derivative. 28. A pharmaceutical composition comprising: (1) a therapeutic amount of a PEGylated erythropoietin conjugate as described in the above-mentioned patent 丨i %, and (2) a pharmaceutically acceptable pharmaceutical carrier. 29. The use of any of the items 1 to 26 of the patent range of claim 1 for the sputum hemagglutinin conjugate, which is prepared for use in the absence of erythropoietin or a red blood cell group or a disease .曰砀 镟 镟 〇 〇 〇 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 如 申请 , , , , , , , , , , , , , , , , , , , , , , , , , Allergic diseases, 94338 32 201002350 ' 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 cells . 33 94338