WO2009079911A1 - Pegylated erythropoietin conjugate and preparation method and uses thereof - Google Patents

Pegylated erythropoietin conjugate and preparation method and uses thereof Download PDF

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WO2009079911A1
WO2009079911A1 PCT/CN2008/001921 CN2008001921W WO2009079911A1 WO 2009079911 A1 WO2009079911 A1 WO 2009079911A1 CN 2008001921 W CN2008001921 W CN 2008001921W WO 2009079911 A1 WO2009079911 A1 WO 2009079911A1
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erythropoietin
integer
conjugate
recombinant human
human erythropoietin
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PCT/CN2008/001921
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French (fr)
Chinese (zh)
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Aifeng LÜ
Changan Sun
Ruijun Wang
Keran Chen
Yunbo Li
Yali Wang
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Jiangsu Hansen Pharmaceutical Co., Ltd.
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Priority to CN2008800211594A priority Critical patent/CN101687934B/en
Publication of WO2009079911A1 publication Critical patent/WO2009079911A1/en
Priority to HK10104546.9A priority patent/HK1136838A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • erythropoietin has a short plasma half-life, is susceptible to protease degradation, and is not highly utilized, and these defects prevent them from achieving maximum clinical efficacy. Therefore, long-acting erythropoietin has become a hotspot for research and development by major research institutions and pharmaceutical companies. For example, amgen's long-acting erythropoietin product (aransp) has been genetically engineered to increase glycosylation.
  • the present invention also provides a conjugate of the following structural formula -
  • P means recombinant human erythropoietin a;
  • X is -(CH 2 ) k -, wherein k is 2; the number of m is selected from 2; and is selected from an integer of 450 to 600.
  • erythropoietin is a method of activating a protein expressed by 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, US 562, 1080 and 5,955,422, EP-B 0 209 539 and EP-B 0 148 605 and EP-B 0 209 539, and Huang, S., Proc. Natl. Acad. Sci. ) (1984) 2708-2712 and so on.
  • a deprotecting agent added to a buffer containing activated erythropoietin by using a technique known in the art to remove an acetyl protecting group of an activated erythropoietin having a structural formula of ( ⁇ ), followed by addition of a structure.
  • the activated methoxypolyethylene glycol of formula III is subjected to a pegylation reaction.
  • the deprotecting agent to be added is preferably hydroxylamine; the pH of the buffer system is selected from 5.0 to 7.0, preferably ⁇ 6.2; G is selected from the group consisting of:
  • Example 6 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-methylhamalimide was further added to a concentration of 5 mM, and reacted at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; then the reaction solution was dialyzed to a 20 mM acetate-acetate buffer system. .
  • mice dose and protocol red blood cell count hematocrit hemoglobin

Abstract

Provided is a pegylated erythropoietin conjugate having the general formula of rhuEPO-NH-CH2-X-S-Y-mPEG. Also provided is a preparation method of the conjugate comprising carrying out reductive amination on erythropoietin and aldehyde compound containing protected sulfhydryl to form activated erythropoietin linked to -NH-CH2- bond, then carrying out methoxypolyethylene glycol derivative coupling reaction, thus obtaining the conjugate. The conjugate or pharmaceutical composition comprising the conjugate can be used for the treatment of diseases involving erythropoietin insufficiency or red blood cell insufficiency or defect.

Description

聚乙二醇化促红细胞生成素偶联物和其制备方法与用途 技术领域  PEGylated erythropoietin conjugate and preparation method and use thereof
本发明涉及一种聚乙二醇促红细胞生成素偶联物， 该偶联物具有提高体内血 红蛋白含量和网织红细胞数目的生物学活性， 本发明的应用领域涉及生物化学、 药物化学以及人类疾病的治疗。 技术背景  The present invention relates to a polyethylene glycol erythropoietin conjugate having biological activity for increasing hemoglobin content and reticulocyte count in vivo, and the field of application of the present invention relates to biochemistry, medicinal chemistry and human diseases Treatment. technical background
促红细胞生成素 （ erythropoietin, EPO) 是一种糖蛋白激素， 分子量约 34kD。 血浆中存在的促红细胞生成素由 165个氨基酸组成， 糖基化程度很高， 糖基成分 主要是唾液酸。 根据碳水化合物含量不同， 天然存在的促红细胞生成素分为两种 类型， α型含 34%的碳水化合物， β型含 26%的碳水化合物。两种类型在生物学特 性、 抗原性及临床应用效果上均相同。 人类促红细胞生成素基因位于 7号染色体 长 22区。 1985年其 cDNA被成功克隆，并利用基因重组技术开始大批量生产重组 人促红细胞生成素 （recombinant human erythropoietin, rHuEPO)，广泛用于临床。 应用重组 DNA技术已经生物合成出促红细胞生成素 (Egrie, JC,Strickland, TW, Lane, J等 (986)免疫生物学 （lmmunobiol)72: 213-224), 其是插入到中国仓鼠的卵巢组 织细胞 (CHO细胞）中并且表达的克隆的人促红细胞生成素基因的产物。天然存在 的人促红细胞生成素首先翻译成含有 166个氨基酸并且 166位是精氨酸的多肽链。 在翻译后修饰中用羟肽酶裂解掉 166位精氨酸。 没有糖基的人 EPO的多肤链的分 子量是 18236Da .在完整的促红细胞生成素分子中， 糖基占整个分子量的大约 40%( J.Biol.Chem.262: 12059)。  Erythropoietin (EPO) is a glycoprotein hormone with a molecular weight of approximately 34kD. The erythropoietin present in plasma consists of 165 amino acids with a high degree of glycosylation and the glycosylation 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 characteristics, antigenicity, and clinical application. The human erythropoietin gene is located on chromosome 7 in the long 22 region. In 1985, its cDNA was successfully cloned, and recombinant human erythropoietin (rHuEPO) was started to be mass-produced by gene recombination technology, which was 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 166 being arginine. In the post-translational modification, arginine at position 166 was cleaved with hydroxypeptidase. The glycosyl group has a molecular weight of 18236 Da. In the intact erythropoietin molecule, the glycosyl group accounts for about 40% of the entire molecular weight (J. Biol. Chem. 262: 12059).
促红细胞生成素是最早应用于临床的细胞因子， 是迄今所知作用最单一、 且 安全可靠的升血红蛋白制剂。 对于肾贫血、 再生障碍性贫血、 多发性骨髓瘤及阵 发性夜间血尿等均有一定疗效； 此外， 应用 EPO还可减少手术中的输血量， 并能 在一定程度上纠正由恶性肿瘤、 化疗及类风湿性关节炎引起的贫血。 由于促红细 胞生成素主要由肾小管内皮细胞产生， 肾性疾患引起的贫血是促红细胞生成素的 首选适应证； EPO纠正肾性贫血的疗效几乎 100%，但并不能改善肾功能。促红细 胞生成素的治疗安全、 有效， 适合长期治疗， 也能避免血源紧张。 在 2006年的全 球生物技术药市场上， 促红细胞生成素类的重组药物占了 119亿美元， 有巨大的 市场容量。  Erythropoietin is the earliest clinically applied cytokine and is the singlest, safe 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 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 preferred indication 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, erythropoietin-like recombinant drugs accounted for $11.9 billion and had huge market capacity.
早在 1989年， 美国 FDA就正式批准重组人促红素 （EPOGEN)用于肾性贫 血的治疗， 但直到 1992年才在我国上市。 我国慢性肾炎的年发病率约为 0.25%， 其中相当一部分患者最终会转为肾衰， 每年的肾性贫血患者约 50 -60万。根据保 守的用药量估算， 如果按当前的价格 30-40元 /支， 加上癌症相关性贫血等其他病 人的用药， 国内市场容量约 12-16亿元甚至更大 (病人平均体重按 50Kg计算)。 自 20世纪 90年代后期， EPO已进入我国重点城市医院畅销药品行列， 2003年在全 国重点城市样本医院用药金额 6213万元， 排名第 56位。 2004年， 全国重点城市 样本医院购药金额增长到 8049万元， 同比增长了 30%。 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 China until 1992. The annual incidence of chronic nephritis in 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 dose estimation, if the current price is 30-40 yuan / support, plus other diseases such as cancer-related anemia The dosage of human medicine, the domestic market capacity is about 1.2-1.6 billion yuan or even larger (the average patient weight is calculated according to 50Kg). Since the late 1990s, EPO has entered the ranks of best-selling drugs in key city hospitals in China. In 2003, the amount of drugs used in sample hospitals in key cities nationwide was 62.13 million yuan, ranking 56th. In 2004, the amount of medicine purchased by sample hospitals in key cities nationwide increased to 80.49 million yuan, a year-on-year increase of 30%.
促红细胞生成素作为一种作用于骨髓造血细胞， 促进红系祖细胞增生、 分化， 最终成熟的内分泌激素。对机体供氧状况发挥重要 的调控作用。在胚胎早期， EPO 由肝生成， 然后逐渐向肾转移， 出生后主要由肾小管间质细胞分泌。  Erythropoietin acts as an endocrine hormone that acts on bone marrow hematopoietic cells to promote proliferation, differentiation, and eventually maturation of erythroid progenitor cells. It plays an important role in regulating the oxygen supply of the body. 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.
在促红细胞生成素诱导红组细胞分化过程中， 球蛋白被诱导， 这能使细胞吸 收更多的铁合成功能性的血红蛋白， 这种功能性的血红蛋白可以和成熟的红血球 中的氧结合， 因此， 红血球和血红蛋白在提供机体氧方面扮演了极其重要的角色。 这一过程是由促红细胞生成素与红组细胞的表面受体之间的相互作用引起的。  In the process of erythropoietin-induced red blood cell differentiation, globulin is induced, which enables the cells to absorb more iron-synthetic functional hemoglobin, which can bind to oxygen in mature red blood cells. 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 red blood cells.
当人体处于健康状态时， 组织可以从已经存在的红血球中吸收足够多的氧， 此时体内的促红细胞生成素浓度很低， 这种正常的较低的促红细胞生成素浓度完 全可以刺激促进由于年龄的问题而正常损失的红细胞。  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.
当循环系统中的依靠红细胞进行氧输送的水平被降低进而出现缺氧情况时， 促红细胞生成素在体内的数量将会增加， 机体缺氧状态可以由以下原因引起的： 过量的辐射、 因高纬度或长期昏迷造成的氧摄入量减少、 各种类型的贫血等等。 作为对组织处于缺氧压力的应答， 促红细胞生成素水平的提高可以刺激红组细胞 的分化达到提高红细胞生成的能力。 当体内的红细胞的数量大于正常组织的需求 时， 循环系统中促红细胞生成素的水平被降低。 正是由于促红细胞生成素对于红 细胞的生成有着至关重要的作用， 因此这类激素对于治疗和诊断以红细胞生成低 下和缺陷为特征的血液病方面有着很广泛的前景。 最近的研究为推测促红细胞生 成素疗法在多种疾病、 紊乱和血液学异常情况中的效用提供了基础， 这些疾病包 括： 慢性肾衰竭 (CRF)患者贫血症的治疗中使用促红细胞生成素和在艾滋病和接受 化疗的癌症患者贫血症的治疗中使用 EPO(Danna, RP, Rudnick, SA， Abels, RI， 于： MB， Garnick编著， 临床应用中的促红细胞生成素一国际展望（Erythropoietin in Clinical Applications- An International Perspective. Marcel Dekker; 1990:p301-324)。  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 the following reasons: Excessive radiation, high Decreased oxygen intake due to latitude or long-term coma, various types of anemia, etc. As a response to tissue hypoxia stress, an increase in erythropoietin levels can stimulate differentiation of red blood cells to increase erythropoiesis. When the amount 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. Because erythropoietin is critical for the production of red blood cells, these hormones have broad prospects for the treatment and diagnosis of blood diseases characterized by low erythropoiesis 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 and anemia in cancer patients undergoing chemotherapy (Danna, RP, Rudnick, SA, Abels, RI, in: MB, edited by Garnick, Erythropoietin in Clinical Applications) Applications- An International Perspective. Marcel Dekker; 1990: p301-324).
但是当前可获得未被修饰的促红细胞生成素的血浆半衰期短， 容易遭受蛋白 酶降解， 利用率不高， 这些缺陷阻止它们取得最大的临床疗效。 因此获得长效的 促红细胞生成素已经成了各大研究机构及制药企业竞相研发的热点， 如已经上市 的 amgen公司的长效促红细胞生成素产品（aransp)是通过基因工程的手段增加糖 基化的位点数目， 进而提高糖基化的程度， 达到每两周注射一次， 改善了促红细 胞生成素在体内的半衰期， 但是这种产品由于仍然无法避免体内蛋白酶的酶解影 响， 因此延长体内半衰期的程度有限， 并且生产成本较高。 发明内容 本发明的目的在于提供一种生物学活性更好， 生物利用度更高的的聚乙二醇 修饰的促红细胞生成素偶联物以及该偶联物的制备方法。 However, currently available unmodified erythropoietin has a short plasma half-life, is susceptible to protease degradation, and is not highly utilized, and these defects prevent them from achieving maximum clinical efficacy. Therefore, long-acting erythropoietin has become a hotspot for research and development by major research institutions and pharmaceutical companies. For example, amgen's long-acting erythropoietin product (aransp) has been genetically engineered to increase glycosylation. The number of sites, which in turn increases the degree of glycosylation, once every two weeks, improves the half-life of erythropoietin in vivo, but this product is still unable to avoid the enzymatic hydrolysis of proteases in the body, thus prolonging the body The half-life is limited and the production cost is high. Summary of the invention 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.
本发明目的还在于提供一种含有该被修饰的促红细胞生成素偶联物的药物组 合物， 用于治疗以缺乏红细胞生成素或红细胞群缺少或缺陷为特征的疾病。  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 a population of red blood cells.
本发明所公开的一种新的聚乙二醇修饰的促红细胞生成素偶联物，其结构通式 为  A novel polyethylene glycol modified erythropoietin conjugate disclosed by the present invention has a structural formula of
P-NH-CH2-X-S-Y- (OCH2CH2) mi-OCH3 P-NH-CH 2 -XSY- (OCH 2 CH 2 ) m i-OCH 3
所述偶联物是由甲氧基聚乙二醇基团通过式 -C¾- X-S-Y-中的 -CH2-基团与促红 细胞生成素的氨基形成 -NH-CH2-键连接而得到， 其中 P是指重组人促红细胞 生成素， X是 -(CH2)k-或 -CH2(OCH2CH2)k -， k的数目选自 2〜10， ml选自 100〜 2000之间的整数， Y选自： The conjugate is obtained by a methoxypolyethylene glycol group via a -CH 2 - group in the formula -C3⁄4-XSY- and a -NH-CH 2 - linkage of the amino group of erythropoietin. 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 ml is selected from 100 to 2000. The integer, Y is selected from:
Figure imgf000005_0001
Figure imgf000005_0001
其中 m、 n的数目各自独立地选自 2〜10。 其中重组人促红细胞生成素优选重组人促红细胞生成素 α或 β， 特别是 α。 促 红细胞生成素的制备是通过本领域公开的技术而得到。  Wherein the number of m and n are each independently selected from 2 to 10. Wherein recombinant human erythropoietin is preferably recombinant human erythropoietin alpha or beta, in particular alpha. The preparation of erythropoietin is obtained by techniques disclosed in the art.
在本方案中， 其中 m , n优选的整数为 2。  In the present scheme, wherein m and n are preferably an integer of 2.
在本方案中， 其中 X是 -(CH2)k -， k选自 2〜10。 In the present scheme, wherein X is -(CH 2 ) k -, k is selected from 2 to 10.
在本方案中， k的数目优选 2〜4， 进一步优选为 2。  In the present embodiment, the number of k is preferably 2 to 4, and further preferably 2.
在本方案中， 甲氧基聚乙二醇基团的平均分子量优选为 5， 000〜40， 000道 尔顿， 进一步优选为为 20， 000道尔顿。  In the present embodiment, the average molecular weight of the methoxypolyethylene glycol group is preferably from 5,000 to 40,000 Daltons, and further preferably 20,000 Daltons.
在本方案中， 一个优选实施方案所对应的偶联物的结构式为：
Figure imgf000006_0001
In this embodiment, the structural formula of the conjugate corresponding to a preferred embodiment is:
Figure imgf000006_0001
其中 P是指重组人促红细胞生成素， X是 -(CH2)k-或 -CH2(OCH2C¾)k-， k的数 目选自 2〜10， m、 n的数目选自 2〜10， 选自 100〜2000之间的整数。 Wherein P refers to recombinant human erythropoietin, X is -(CH 2 ) k - or -CH 2 (OCH 2 C3⁄4) k -, the number of k is selected from 2 to 10, and the number of m, n is selected from 2~ 10, selected from an integer between 100 and 2000.
其中 X优选为 -(CH2)k-， k的数目可进一步优选自 2〜4， 最优选的是 2。 Wherein X is preferably -(CH 2 ) k -, and the number of k may further preferably be from 2 to 4, and most preferably 2.
进一步优选地， 本发明的一个实施方案对应的结构式为：  Further preferably, the structural formula corresponding to one embodiment of the present invention is:
Figure imgf000006_0002
Figure imgf000006_0002
n 选自 450〜600的整数，其中 P是指重组人促红细胞生成素，可进一步优选 重组人促红细胞生成素 α或 β， 最优选 α。  n is selected from an integer of 450 to 600, wherein P means recombinant human erythropoietin, and further preferably recombinant human erythropoietin α or β, most preferably α.
本发明还提供一种如下结构通式的偶联物-  The present invention also provides a conjugate of the following structural formula -
Figure imgf000006_0003
Figure imgf000006_0003
其中 P是指重组人促红细胞生成素糖蛋白，X是 -(CH2)k-或 -CH2(OCH2CH2)k-， k 的数目选自 2〜10， m的数目选自 2〜10， 选自 100〜2000之间的整数。 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 ~10, selected from integers between 100 and 2000.
进一步优选地， 该结构式中 P是指重组人促红细胞生成素 α; X是 -(CH2)k-， 其中 k是 2; m的数目选自 2; 选自 450〜600的整数。 Further 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; and is selected from an integer of 450 to 600.
本发明还提供一种如下结构通式的偶联物-  The present invention also provides a conjugate of the following structural formula -
Figure imgf000006_0004
Figure imgf000006_0004
其中 P是指重组人促红细胞生成素， X是 -(C¾)k-或 -CH2(OC¾C¾)k-， k的数 目选自 2〜10， m的数目选自 2〜10， 选自 100〜2000之间的整数。 Wherein P refers to recombinant human erythropoietin, X is -(C3⁄4) k - or -CH 2 (OC3⁄4C3⁄4) k -, the number of k is selected from 2 to 10, and the number of m is selected from 2 to 10, selected from 100 An integer between ~2000.
进一步优选， 该结构式中 P是指重组人促红细胞生成素 a; X是 -(CH2)k-， 其 中 k是 2; m的数目选自 2; 选自 450〜600的整数。 Further preferably, in the formula, P means recombinant human erythropoietin a; X is -(CH 2 ) k -, wherein k is 2; the number of m is selected from 2; and is selected from an integer of 450 to 600.
本发明还提供一种如下结构通式的偶联物-
Figure imgf000006_0005
其中 P是指重组人促红细胞生成素， X是 -(CH2)k-或 -CH2(OCH2CH2)k-， k的数 目选自 2〜10， mi选自 100〜2000之间的整数。 The present invention also provides a conjugate of the following structural formula -
Figure imgf000006_0005
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 mi is selected from 100 to 2000. The integer.
进一歩优选地， 该结构式中 P是指重组人促红细胞生成素 a; X是 -(CH2)k -， 其中 k是 2 ; mi选自 450〜600的整数。 Further, in the formula, P means recombinant human erythropoietin a; X is -(CH 2 ) k -, wherein k is 2; mi is selected from an integer of 450 to 600.
本发明还提供一种如下结构通式的偶联物-
Figure imgf000007_0001
The present invention also provides a conjugate of the following structural formula -
Figure imgf000007_0001
其中 P是指重组人促红细胞生成素， X是 -(C¾)k -或 -CH2(OC¾CH2)k-， k的数 目选自 2〜10， 选自 100〜2000之间的整数。 Wherein P refers to recombinant human erythropoietin, X is -(C3⁄4) k - or -CH 2 (OC3⁄4CH 2 ) k -, and the number of k is selected from 2 to 10, and is selected from an integer between 100 and 2000.
进一步优选地， 该结构通式中 P是指重组人促红细胞生成素 α; X是 -(CH2)k -， 其中 k是 2; mi选自 450〜600的整数。 Further preferably, in the structural formula, P means recombinant human erythropoietin α; X is -(CH 2 ) k -, wherein k is 2; mi is selected from an integer of 450 to 600.
本发明还提供一种如下结构通式的偶联物：
Figure imgf000007_0002
The invention also provides a conjugate of the following structural formula:
Figure imgf000007_0002
其中 P是指重组人促红细胞生成素， X是 -(C¾)k -或 -CH2(OCH2CH2)k-， k的数 目选自 2〜10， m、 n的数目选自 2〜10， 选自 100〜2000之间的整数。 Wherein P refers to recombinant human erythropoietin, X is -(C3⁄4) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 2 to 10, and the number of m, n is selected from 2~ 10, selected from an integer between 100 and 2000.
进一步优选地， 该结构式中 Ρ是指重组人促红细胞生成素 α; X是 -(CH2)k -， 其中 k是 2; m、 n的数目选自 2 ; mt选自 450〜600的整数。 Further preferably, in the formula, Ρ refers to recombinant human erythropoietin α; X is —(CH 2 ) k — wherein k is 2; m, n is selected from 2; mt is selected from 450 to 600 integers .
本发明所公开的偶联物是促红细胞生成素通过连接体（-CH2-X-S-Y-) 与甲氧 基聚乙二醇基团共价连接进而被修饰， 关键在于促红细胞生成素与连接体以形成 -NH2-CH2-键形式而连接， 与未被修饰的促红细胞生成素相比， 本发明提供的偶联 物能够提高血浆的停留时间和循环半衰期， 降低清除率， 在体内的生物活性更高， 并且具有和促红细胞生成素一样的用途。与以形成 -NH-CO-键形式的聚乙二醇促红 细胞生成素偶联物相比具有反应位点更单一， 质量更可控的优点。 本发明提供的 聚乙二醇促红细胞生成素偶联物可以被用来治疗以缺乏红细胞生成素或红细胞群 缺少或缺陷为特征的疾病。 本发明还公开了上述偶联物的一种制备方法， 包括以下步骤： 首先是促红细 胞生成素和含有已保护巯基的醛类物质发生还原胺化反应， 形成通过 -NH-CH2-键 连接的活化促红细胞生成素， 然后， 将所述活化促红细胞生成素脱保护， 与活性 甲氧基聚乙二醇衍生物偶联， 通过进一歩的分离纯化而得到。 The conjugate disclosed in the present invention is that erythropoietin is covalently linked to a methoxypolyethylene glycol group via a linker (-CH2-XSY-), and the key is to promote erythropoietin and the linker. The formation of the -NH 2 -CH 2 - bond form, the conjugate provided by the present invention can increase plasma residence time and circulating half-life, reduce clearance rate, and in vivo organisms compared to unmodified erythropoietin. It is more active and has the same use as erythropoietin. Compared with the polyethylene glycol erythropoietin conjugate in the form of a -NH-CO- bond, it has the advantages of a single reaction site and a more controllable quality. The polyethylene glycol erythropoietin conjugate provided by the present invention can be used to treat diseases characterized by a deficiency or deficiency in erythropoietin or red blood cell population. The invention also discloses a preparation method of the above conjugate, comprising the following steps: First, the erythropoietin and the aldehyde group containing the protected sulfhydryl group undergo reductive amination reaction to form a bond through the -NH-CH2- bond. The erythropoietin is activated, and then the activated erythropoietin is deprotected, coupled with a reactive methoxypolyethylene glycol derivative, and obtained by further separation and purification.
本发明还公开了一种药物组合物， 包含： （1 ) 治疗量的上述聚乙二醇化促红 细胞生成素偶联物； （2 )药学可接受的药物载体。  The invention also discloses a pharmaceutical composition comprising: (1) a therapeutic amount of the above PEGylated erythropoietin conjugate; (2) a pharmaceutically acceptable pharmaceutical carrier.
本发明含有任意的上述治疗量的聚乙二醇化促红细胞生成素偶联物用于治疗 以缺乏红细胞生成素或红细胞群缺少或缺陷为特征的疾病。 特别是治疗下述疾病- 末期肾功能衰竭或透析； AIDS相关性贫血， 自身免疫性疾病， 或恶性肿瘤； 囊性 纤维变性； 早期早熟性贫血； 与慢性炎性疾病相关的贫血； 脊髓损伤； 急性失血； 衰老和伴有异常红细胞产生的肿瘤疾病。 促红细胞生成素的制备 The present invention comprises any of the above therapeutic amounts of a pegylated erythropoietin conjugate for use in therapy A disease characterized by a lack of erythropoietin or a deficiency or defect in the 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 tumors; 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. Preparation of erythropoietin
促红细胞生成素的制备方法是通过内源基因激活表达蛋白质的方法， 都是本 领域公知的技术。其制备及治疗应用详细描述于美国专利 US5547933、 US5621080 和 US5955422, EP-B0209539和 EP-B0148605和 EP-B0209539， 以及 Huang,S丄.， 美国国家科学院院刊 （Proc.Natl.Acad.Sci.USA) (1984)2708-2712等。  The preparation of erythropoietin is a method of activating a protein expressed by 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, US 562, 1080 and 5,955,422, EP-B 0 209 539 and EP-B 0 148 605 and EP-B 0 209 539, and Huang, S., Proc. Natl. Acad. Sci. ) (1984) 2708-2712 and so on.
重组促红细胞生成素的纯化一般使用羟基磷灰石凝胶， 离子交换层析， 凝胶 层析等本领域公知的纯化方法进行纯化， 并在美国专利 No.5547933、 5621080和 5955422 等专利中均有详细描述； Nobuo,I.等， 生物化学杂质 （J.Biochem ) 107(1990)352-359也描述了重组 EPO的纯化方法。 聚乙二醇促红细胞生成素偶联物的制备  Purification of recombinant erythropoietin is generally carried out using hydroxyapatite gel, ion exchange chromatography, gel chromatography, etc., and 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; Nobuo, I., et al., J. Biochem 107 (1990) 352-359 also describes a purification method for recombinant EPO. Preparation of polyethylene glycol erythropoietin conjugate
本发明提供一种制备聚乙二醇促红细胞生成素偶联物的制备方法， 其步骤包 括- The present invention provides a method for preparing a polyethylene glycol erythropoietin conjugate, the steps of which include -
( 1 ) 通过本领域技术人员所公知的技术制备结构通式 I的小分子醛化合物，如将 硫代乙酸与烯醛类化合物进行迈克尔加成反应：
Figure imgf000008_0001
(1) A small molecule aldehyde compound of the formula I is prepared by techniques well known to those skilled in the art, such as a Michael addition reaction of a thioacetic acid with an aldehyde compound:
Figure imgf000008_0001
I  I
其中 k的数目选自 2〜10， 优选 2;  Wherein the number of k is selected from 2 to 10, preferably 2;
(2)将结构通式 I的小分子醛化合物与促红细胞生成素在缓冲液里反应， 并 加入还原剂得到结构通式为 II的活化促红细胞生成素；
Figure imgf000008_0002
(2) reacting a small molecule aldehyde compound of the formula I with erythropoietin in a buffer, and adding a reducing agent to obtain an activated erythropoietin of the formula II;
Figure imgf000008_0002
II  II
其中 P是指重组人促红细胞生成素 α或 β， 优选重组人促红细胞生成素 α， 缓 冲液的 ρΗ选自 4.0〜6.0， 优选 6.0， 还原剂可以是硼氰化钠、 氰基硼氰化钠， 优 选氰基硼氰化钠；  Wherein P refers to recombinant human erythropoietin alpha or beta, preferably recombinant human erythropoietin alpha, 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 cyanogenic boron cyanide. Sodium, preferably sodium cyanoborohydride;
(3 )利用本领域公知的技术在含有活化促红细胞生成素的缓冲液里加入脱保 护剂， 脱去结构通式为 （Π) 的活化促红细胞生成素的乙酰基保护基， 随后加入结 构通式为 III的活化甲氧基聚乙二醇进行聚乙二醇化反应。 其中加入的脱保护剂优选羟胺； 缓冲体系的 ρΗ选自 5.0〜7.0， 优选 ρΗ6.2; G选自： (3) adding a deprotecting agent to a buffer containing activated erythropoietin by using a technique known in the art to remove an acetyl protecting group of an activated erythropoietin having a structural formula of (Π), followed by addition of a structure. The activated methoxypolyethylene glycol of formula III is subjected to a pegylation reaction. The deprotecting agent to be added is preferably hydroxylamine; the pH of the buffer system is selected from 5.0 to 7.0, preferably ρ 6.2; G is selected from the group consisting of:
Figure imgf000009_0001
Figure imgf000009_0001
其中 m、 n的数目选自 2〜10， 优选 2;  Wherein the number of m, n is selected from 2 to 10, preferably 2;
(4)聚乙二醇促红细胞生成素偶联物的纯化方法采用本领域技术人员公知的 技术， 如离子交换层析、 凝胶层析。 生物学活性的测试  (4) A method for purifying a polyethylene glycol erythropoietin conjugate is carried out by a technique known to those skilled in the art, such as ion exchange chromatography or gel chromatography. Biological activity test
通过本领域公知的各种测试可以测定促红细胞生成素或本发明提供的聚乙二 醇促红细胞生成素偶联物的生物学活性。 体内活性的测试通过小鼠皮下注射促红 细胞生成素及本发明提供的聚乙二醇促红细胞生成素偶联物， 连续三天， 然后处 死小鼠， 取全血进行外周血细胞及网织红细胞计数， 血细胞计数采用全自动血球 计数仪计数。 对猕猴静脉注射进行药效学研究， 单次给药剂量 1.35mg/kg， 作为对 比药物使用的促红细胞生成素给药剂量为 240μ/1¾， 每周三次， 连续给药六周， 采 集血样进行相关血液学指标分析。  The biological activity of erythropoietin or the polyethylene glycol erythropoietin conjugate provided by the present invention can be determined by various tests well known in the art. In vivo activity test The mouse was injected subcutaneously with erythropoietin and the polyethylene glycol erythropoietin conjugate provided by the present invention 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 an automatic blood cell counter. A pharmacodynamic study was conducted on the intravenous injection of rhesus monkeys at a single dose of 1.35 mg/kg. The erythropoietin used as a comparator was administered at a dose of 240 μ/13⁄4, three times a week for six weeks, and blood samples were taken. Analysis of relevant hematological indicators.
通过对本发明提供的聚乙二醇促红细胞生成素偶联物的测试数据表明:本发明 提供的聚乙二醇促红细胞生成素偶联物能够明显刺激小鼠外周血网织红细胞计数 的升高， 说明它们刺激红细胞生成， 同时还能够大大延长偶联物在体内的半衰期。 聚乙二醇促红细胞生成素偶联物对成熟的红细胞、 血细胞压积、 血红蛋白含量没 有明显的影响， 对外周血白细胞计数液没有明显影响。 药物组合物的制备  The test data of the polyethylene glycol erythropoietin conjugate provided by the present invention indicates that the polyethylene glycol erythropoietin conjugate provided by the invention can significantly stimulate the increase of peripheral blood reticulocyte count in mice. , indicating that they stimulate red blood cell production, while also greatly extending the half-life of the conjugate in the body. The polyethylene glycol 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
可以通过本领域公知的方法用药学可接受载体或赋形剂制成适合注射的药物 组合物。 用于配制本发明的产物的优选的药学可接受载体是人血清白蛋白， 人血 浆蛋白质等。 本发明的化合物可以在含有 132mm氯化钠的 10mM磷酸钠 I钾缓冲 液 pH7中配制。 任选地， 药物组合物可以含有防腐剂。 药物组合物可以含有不同 量的促红细胞生成素， 优选 10-1000微克 /毫升。 附图说明 Pharmaceutical compositions suitable for injection can be made by pharmaceutically acceptable carriers or excipients by methods known in the art. A preferred pharmaceutically acceptable carrier for formulating the products of the invention is human serum albumin, human blood Pulp protein and so on. The compounds of the invention may be formulated in 10 mM sodium phosphate potassium phosphate buffer pH 7 containing 132 mm sodium chloride. Optionally, the pharmaceutical composition may contain a preservative. The pharmaceutical composition may contain varying amounts of erythropoietin, preferably from 10 to 1000 micrograms per milliliter. DRAWINGS
图 1 : 聚乙二醇化促红细胞生成素偶联物 （HH-EPO- 014A)对猕猴血细胞压 积的影响。  Figure 1: Effect of PEGylated erythropoietin conjugate (HH-EPO-014A) on hematocrit in rhesus monkeys.
图 2: 聚乙二醇化促红细胞生成素偶联物 （HH-EPO-014A)对猕猴血红蛋白 含量的影响。 具体实施方式 实施例 1: 乙酰基巯基丙醛的制备
Figure imgf000010_0001
将 11.2 g (20mmol) 丙烯醛与千燥的 100 ml THF加入到反应瓶中， 冷却至 0°C， 然后缓慢滴加 1.52g (0.2 mol)硫代乙酸 /20ml THF的混合溶液。滴加完毕保 温反应 2小时后。 35°C减压浓缩除去过量的丙烯醛。 然后快速上柱 （洗脱液纯正 己烷→正己烷 /乙酸乙酯 =50/1 )， 合并收集产物点， 减压浓缩至干得油状液体 0.6g。 实施例 2: mPEG-MAL-01(20kD)的制备 Figure 2: Effect of PEGylated erythropoietin conjugate (HH-EPO-014A) on hemoglobin content in macaques. DETAILED DESCRIPTION OF THE INVENTION Example 1: Preparation of Acetylmercaptopropanal
Figure imgf000010_0001
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, the column was quickly applied (eluent pure n-hexane → n-hexane / ethyl acetate = 50/1), and the product was collected. Example 2: Preparation of mPEG-MAL-01 (20 kD)
Figure imgf000011_0001
Figure imgf000011_0001
mPEG-NHCH2CH2NH2(20KD)  mPEG-NHCH2CH2NH2 (20KD)
Figure imgf000011_0002
将 20g(lmmol)mPEG-OH(20kD)投入到 200ml的单口瓶中， 加入 100ml甲苯， 回流分水反应 2.5hr; 然后蒸出甲苯， 冷却至室温， 再加入 lOOmlDCM, 随后加入 1.18g(4mmol)的三光气 (triphosgene), 室温密闭搅拌反应过夜； 次日处理： 将反应 液于通风厨中冲析入 200ml的无水乙醚中， 过滤后真空干燥得白色固体 15g。 将 15g上步白色固体投入到 200ml的单口瓶中，加入 100ml Toluene/DCM(2:l)的溶液， 再加入 0.25g的 HOSu, 随后加入 0.3g三乙胺， 室温密闭搅拌反应 4hr (或过夜）； 反应结束后， 将反应液过滤， 滤液直接冲析入 100ml的无水乙醚中， 过滤， 真空 干燥得白色固体 14g， 即为 SC-mPEG(20kD);
Figure imgf000011_0002
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 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 top 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 mixture 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 14 g of white solid as SC-m PEG (20 kD);
将 1.4g 无水乙二胺用 50ml DCM 于 200ml 反应瓶中溶解， 再取 14g SC-mPEG(20kD)溶解于 100ml的 DCM溶解后加入到上述乙二胺溶液中， 反应过 夜； 次日停止反应过滤， 滤液加入 500ml的饱和食盐水洗涤， 分出有机层， 水层 用 DCM提取三次（200mlx3 )， 合并有机层， 无水硫酸钠干燥， 过滤， 滤液减压浓 缩至 100ml,于 500ml无水乙醚中沉降析出固体，过滤，真空干燥得白色固体 13g， 即为 mPEG-NHCH2CH2NH2(20kD); 1.4 g of anhydrous ethylenediamine was dissolved in a 200 ml reaction flask with 50 ml of DCM, and 14 g of SC-mPEG (20 kD) was dissolved in 100 ml of DCM and dissolved in the above ethylenediamine solution, and allowed to react overnight; the reaction was stopped the next day. Filtration, the filtrate was washed with 500 ml of saturated brine, and the organic layer was evaporated. EtOAcjjjjjjjjjjjjjjj settling precipitated solid was filtered and dried in vacuo to give a white solid 13g, that is, mPEG-NHCH 2 CH 2 NH 2 (20kD);
将 1.9g的 MAL-ONP用 50mL DCM溶解， 加入 0.04g的三乙胺， 再将 13g的 mPEG-NHCH2CH2NH2(20kD)用 100ml 的新开的 DCM 溶解， 然后加入上述 MAL-ONP的 DCM溶液中， 室温反应过夜； 次日减压浓缩出 DCM， 残余物加入 200ml 的无水乙醚中， 沉降析出固体， 真空干燥得白色固体 12.5g， 即为 mPEG-MAL-01(201dD 实施例 3: mPEG-MAL-02(20kD)的制备 1.9 g of MAL-ONP was dissolved in 50 mL of DCM, 0.04 g of triethylamine was added, and 13 g of mPEG-NHCH 2 CH 2 NH 2 (20 kD) was dissolved in 100 ml of freshly opened DCM, and then the above MAL-ONP was added. In a solution of DCM, the mixture was stirred at room temperature overnight; DCM was concentrated under reduced pressure, and the residue was added to ethyl ether (200 ml). mPEG-MAL-01 (201dD Example 3: Preparation of mPEG-MAL-02 (20kD)
Figure imgf000012_0001
Figure imgf000012_0001
mPEG-MAL-02(20KD) 将 2.0g的 MAL-ONP用 50mL DCM溶解， 加入 0.05g的三乙胺， 再将 15g的 mPEG-N¾(20kD)用 100ml的新幵的 DCM溶解，然后加入上述 MAL-ONP的 DCM 溶液中，室温反应过夜；次日减压浓缩出 DCM,残余物加入 200ml的无水乙醚中， 沉降析出固体， 真空干燥得白色固体 13g， 即为 mPEG-MAL-02(20kD)。 实施例 4: mPEG-OPPS-01(20kD)的制备  mPEG-MAL-02 (20KD) 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-N3⁄4 (20 kD) was dissolved with 100 ml of neodymium DCM, and then added MAL-ONP in DCM solution was allowed to react 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 13 g of white solid, m. ). Example 4: Preparation of mPEG-OPPS-01 (20 kD)
Figure imgf000012_0002
Figure imgf000012_0002
mPEG-OPPS-01 (20KD)  mPEG-OPPS-01 (20KD)
将 0.5g MPPS 用 50mlDCM 溶解， 随后加入 0.05g 三乙胺和 20g mPEG-NH2(201<D),室温搅拌反应过夜，次日减压浓缩出 DCM，残余物加入 200ml 的无水乙醚， 析出固体， 过滤， 真空干燥得白色固体 19g， 即为 mPEG-OPPS-01(20kD 实施例 5： mPEG-OPPS-02(20kD)的制备
Figure imgf000013_0001
Dissolve 0.5 g of MPPS in 50 ml of DCM, then add 0.05 g of triethylamine and 20 g of mPEG-NH 2 (201<D), stir the reaction at room temperature overnight, concentrate DCM under reduced pressure on the next day, and add 200 ml of anhydrous diethyl ether to precipitate. Solid, filtered, dried in vacuo to give a white solid, 19 g, m.p.
Figure imgf000013_0001
mPEG-NHCH2CH2NH2(20KD)  mPEG-NHCH2CH2NH2 (20KD)
Figure imgf000013_0002
Figure imgf000013_0002
mPEG-OPPS-02(20KD) 将 0.5g MPPS 用 50mlDCM 溶解， 随后加入 0.05g 三乙胺和 20g mPEG-NHCH2CH2NH2(201cD) , 室温搅拌反应过夜， 次日减压浓缩出 DCM， 残余 物加入 200ml 的无水乙醚， 析出固体， 过滤， 真空干燥得白色固体 18.5g， 即为 mPEG-OPPS-02(20kD)。 实施例 6: 活化促红细胞生成素的制备 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-NHCH 2 CH 2 NH 2 (201 cD), and the reaction was stirred at room temperature overnight, and DCM was concentrated under reduced pressure on the next day. The residue was added to 200 ml of dry diethyl ether. The solid was crystallised, filtered, and dried in vacuo to give a white solid (18.5 g, m.p.). Example 6: Preparation of activated erythropoietin
取促红细胞生成素原液 60毫克， 蛋白浓度为 1.5毫克 /毫升， 共 40毫升， 体 系为 0.1M磷酸钠盐缓冲液， pH6.0; 取 2.5毫克乙酰基巯基丙醛溶于 80微升乙腈 后加入以上蛋白溶液； 再称取 50毫克氰基硼氢化钠加入上述反应液， 并慢速搅拌 反应， 冰浴控制反应温度在 10°C， 反应 24小时； 然后将反应液转入透析袋（截留 分子量 3500)，对 0.1M磷酸钠盐缓冲液，含 2mM EDTA,pH6.25进行透析， 以除掉 过量的小分子醛， 然后再加入羟胺脱去乙酰基， 得活化促红细胞生成素。 实施例 7: HH-EPO-014A的制备  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 acetyl mercaptopropionaldehyde dissolved in 80 μl of acetonitrile Add the above protein solution; add 50 mg of sodium cyanoborohydride to the above reaction solution, and stir the reaction slowly, control the reaction temperature at 10 ° C for 24 hours in an ice bath; then transfer the reaction solution to a dialysis bag (retained Molecular weight 3500), dialysis against 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25, to remove excess small molecule aldehyde, and then add hydroxylamine to deacetylate to obtain activated erythropoietin. Example 7: Preparation of HH-EPO-014A
通过实施例 6得到的 10mg活化促红细胞生成素 （1.4毫克 /毫升， 0.1M磷酸钠 盐缓冲液， 含 2mM EDTA,pH6.25)， 加入 100毫克 mPEG-MAL- 01(20kD)。 搅拌反 应 60分钟； 再加入 N-甲華马来酰亚胺至浓度为 5mM， 室温反应 30分钟， 以反应 掉蛋白上剩余的巯基； 然后将反应液透析至 20mM乙酸一乙酸盐缓冲液体系。  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-methylhamalimide was further added to a concentration of 5 mM, and reacted at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; then the reaction solution was dialyzed to a 20 mM acetate-acetate buffer system. .
反应液的纯化依次使用离子交换层析（SP Sepharose H.P)和凝胶层析（Superdex 200)， 即可得到聚乙二醇化促红细胞生成素 （HH-EPO-014A)，约 5mg。 实施例 8: HH-EPO-014B的制备  Purification of the reaction solution Using ion exchange chromatography (SP Sepharose H.P.) and gel chromatography (Superdex 200), PEGylated erythropoietin (HH-EPO-014A), about 5 mg, was obtained. Example 8: Preparation of HH-EPO-014B
通过实施例 6得到的含有游离巯基的 1 Omg活化促红细胞生成素（ 1.4毫克 /毫升， 1 Omg of activated erythropoietin (1.4 mg / ml, containing free sulfhydryl groups) obtained in Example 6
0.1M磷酸钠盐缓冲液，含 2mM EDTA,pH6.25)，加入 100毫克 mPEG-MAL-02(20kD)。 搅拌反应 60分钟 （25°C) ; 再加入 N-甲基马来酰亚胺至浓度为 5mM, 室温反应 30 分钟， 以反应掉蛋白上剩余的巯基； 然后将反应液透析至 20mM乙酸一乙酸盐缓冲 液体系。 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25), 100 mg of mPEG-MAL-02 (20 kD) was added. The reaction was stirred for 60 minutes (25 ° C); N-methyl maleimide was further added to a concentration of 5 mM, and reacted at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; then the reaction solution was dialyzed to 20 mM acetic acid-B. Acid buffer Liquid system.
反应液的纯化依次使用离子交换层析（SP Sepharose H.P)和凝胶层析（Superdex 200), 即可得到聚乙二醇化促红细胞生成素 （HH-EPO-014B)，约 5.5mg。 实施例 9: HH-EPO-014C的制备  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
向实施例 6得到的 10mg活化促红细胞生成素 （1.4毫克 /毫升， 0.1M磷酸钠盐 缓冲液， 含 2mM EDTA,pH6.25 )溶液中加入 100毫克 mPEG-MAL(20KD),搅拌反应 60分钟 (25°C)，再加入 N-甲基马来酰亚胺至浓度为 5mM, 室温反应 30分钟， 以 反应掉蛋白上剩余的巯基； 然后将反应液透析至 20mM乙酸一乙酸盐缓冲液体系。  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, 100 mg of mPEG-MAL (20 KD) was added, and the reaction was stirred for 60 minutes. (25 ° C), further add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; then dialyzate the reaction solution to 20 mM acetate-acetate buffer solution system.
反应液的纯化依次使用离子交换层析（SP Sepharose H.P)和凝胶层析（Superdex The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) and gel chromatography (Superdex).
200), 即可得到聚乙二醇化促红细胞生成素 （HH-EPO-014C) ，约 6.2mg。 实施例 10: HH-EPO-014D的制备 200), PEGylated erythropoietin (HH-EPO-014C), about 6.2 mg. Example 10: Preparation of HH-EPO-014D
向实施例 6得到的 10mg活化促红细胞生成素 （1.4毫克 /毫升， 0.1M磷酸钠盐 缓冲液，含 2mM EDTA,pH6.25 )溶液中加入 100毫克 mPEG-OPPS(20KD),搅拌反应 60分钟 （25°C) ，再加入 N-甲基马来酰亚胺至浓度为 5mM， 室温反应 30分钟， 以 反应掉蛋白上剩余的巯基； 然后将反应液透析至 20mM乙酸一乙酸盐缓冲液体系。  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-OPPS (20 KD) was added, and the reaction was stirred for 60 minutes. (25 ° C), add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; then dialyzate the reaction solution to 20 mM acetate-acetate buffer solution system.
反应液的纯化依次使用离子交换层析（SP Sepharose H.P)和凝胶层析（Superdex 200), 即可得到聚乙二醇化促红细胞生成素 （HH-EPO-014D)，约 6.0mg。 实施例 11: HH-EPO-014E的制备  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
向实施例 6得到的 10mg活化促红细胞生成素 （1.4毫克 /毫升， 0.1M磷酸钠盐 缓冲液， 含 2mM EDTA,pH6.25)溶液中加入 100毫克 mPEG-OPPS-01(20KD),搅拌 反应 60分钟（25°C)，再加入 N-甲基马来酰亚胺至浓度为 5mM，室温反应 30分钟， 以反应掉蛋白上剩余的巯基;然后将反应液透析至 20mM乙酸一乙酸盐缓冲液体系。  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-OPPS-01 (20 KD) was added, and the reaction was stirred. 60 minutes (25 ° C), additional N-methyl maleimide to a concentration of 5 mM, reaction at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; then the reaction solution was dialyzed to 20 mM acetate-acetate Buffer system.
反应液的纯化依次使用离子交换层析（SP Sepharose H.P)和凝胶层析（Superdex 200), 即可得到聚乙二醇化促红细胞生成素 （HH-EPO-014E)，约 5.6mg。 实施例 12: HH-EPO-014F的制备  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
向实施例 6得到的 lOmg活化促红细胞生成素 （1.4毫克 /毫升， 0.1M磷酸钠盐 缓冲液， 含 2mM EDTA,pH6.25 )溶液中加入 100毫克 mPEG-OPPS- 02(20KD),搅拌 反应 60分钟（25°C)，再加入 N-甲基马来酰亚胺至浓度为 5mM， 室温反应 30分钟， 以反应掉蛋白上剩余的巯基;然后将反应液透析至 20mM乙酸一乙酸盐缓冲液体系。  To 100 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), and then added N-methyl maleimide to a concentration of 5 mM, reacted at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; then dialyzed the reaction solution to 20 mM acetate-acetate Buffer system.
反应液的纯化依次使用离子交换层析（SP Sepharose H.P)和凝胶层析（Superdex 200)， 即可得到聚乙二醇化促红细胞生成素 （HH-EPO-014F) ,约 5.6mg。 实验例 实验例 1: 聚乙二醇促红细胞生成素偶联物对小鼠的作用 Purification of the reaction solution Using ion exchange chromatography (SP Sepharose HP) and gel chromatography (Superdex 200) in this order, PEGylated erythropoietin (HH-EPO-014F) was obtained, which was about 5.6 mg. Experimental example Experimental Example 1: Effect of polyethylene glycol erythropoietin conjugate on mice
实验目的： Purpose:
评价并比较聚乙二醇促红细胞生成素偶联物及促红细胞生成素蛋白对小鼠红 细胞生成的影响。 材料及方法：  The effects of polyethylene glycol erythropoietin conjugate and erythropoietin protein on erythropoiesis in mice were evaluated and compared. Materials and methods:
聚乙二醇促红细胞生成素偶联物 HH-EPO-014A、 HH-EPO-014B、 HH-EPO-014C, HH-EPO-014D、 HH-EPO-014E, HH-EPO-014F由江苏豪森药业股 份有限公司提供； 促红细胞生成素（阳性对照)： 购自沈阳三生制药有限责任公司； 昆明种小鼠， 购自中科院上海实验动物中心， 体重 25〜30g， ？, 各组动物数： 10 只。  Polyethylene glycol erythropoietin conjugates HH-EPO-014A, HH-EPO-014B, HH-EPO-014C, HH-EPO-014D, HH-EPO-014E, HH-EPO-014F by Jiangsu Haosen Provided by 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 25~30g, ? , the number of animals in each group: 10 only.
小鼠皮下注射聚乙二醇促红细胞生成素偶联物及促红细胞生成素， 连续三天， 然后处死小鼠， 取全血进行外周血细胞及网织红细胞计数， 血细胞计数用全自动 血球计数仪计数。 结果与讨论：  The mice were injected subcutaneously with polyethylene glycol erythropoietin conjugate and erythropoietin for three consecutive days, then the mice were sacrificed, whole blood was taken for peripheral blood cell and reticulocyte count, and blood cell count was performed with automatic blood cell counter. count. Results and discussion:
按照目前的给药方案， 聚乙二醇促红细胞生成素偶联物及促红细胞生成素均 能明显剌激小鼠外周血网织红细胞计数的升高， 说明它们剌激红细胞生成 (见表 一)。 聚乙二醇促红细胞生成素偶联物对成熟的红细胞、 血细胞压积、 血红蛋白含 量没有明显的影响 （见表二）， 对外周血白细胞计数液没有明显影响 （见表三）。 表一、 聚乙二醇促红细胞生成素偶联物对小鼠网织红细胞生成的影响  According to the current dosage regimen, both polyethylene glycol erythropoietin conjugate and erythropoietin can significantly stimulate the increase of peripheral blood reticulocyte count in mice, indicating that they stimulate erythropoiesis (see Table 1). ). Polyethylene glycol erythropoietin conjugates 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). Table 1. Effect of polyethylene glycol erythropoietin conjugate on reticulocyte production in mice
分组 小鼠 给药剂量和方案 网织红细胞计数  Group mice dose and protocol reticulocyte count
(只） （X 109 L,x±SD) control 10 0.1%BSA in NS Π7.6±55. 2 (only) (X 10 9 L, x ± SD) control 10 0.1% BSA in NS Π 7.6 ± 55. 2
HH-EPO-014A 10 5 g/kg,sc,dl-3 826.1 ±25. 1  HH-EPO-014A 10 5 g/kg, sc, dl-3 826.1 ± 25.1
HH-EPO-014B 10 5 g/kg,sc, n-3 810.2±58. 3  HH-EPO-014B 10 5 g/kg, sc, n-3 810.2±58. 3
HH-EPO-014C 10 5 g/kg,sc,dl-3 765·3±67. 5  HH-EPO-014C 10 5 g/kg, sc, dl-3 765·3±67. 5
HH-EPO-014D 10 5 g/kg,sc，dl- 3 652.1 ±81. 3  HH-EPO-014D 10 5 g/kg, sc, dl- 3 652.1 ±81. 3
HH-EPO-014E 10 5 g/kg,sc,dl-3 647.5 ±24. 1  HH-EPO-014E 10 5 g/kg, sc, dl-3 647.5 ± 24.1
HH-EPO-014F 10 5ng/kg,sc,dl-3 586.0±32. 4  HH-EPO-014F 10 5ng/kg, sc, dl-3 586.0±32. 4
EPO 10 5 g/kg，sc,d 3 360·6±27· 0 表二、 聚乙二醇促红细胞生成素偶联物对小鼠红细胞生成、 血细胞压积、 血 红蛋白含量的影响  EPO 10 5 g/kg, sc, d 3 360·6±27· 0 Table 2. Effect of polyethylene glycol erythropoietin conjugate on erythropoiesis, hematocrit and hemoglobin in mice
分组 小鼠 给药剂量和方案 红细胞计数 血细胞压积 血红蛋白  Group mice dose and protocol red blood cell count hematocrit hemoglobin
(只） ( X 106 iL， x ( %) ( % ) 土 SD) (only) (X 10 6 iL, x ( %) ( % ) Soil SD)
control 10 0.1%BSA inNS 9.6±0. 5 48.2±3. 0 14.8±0. 7Control 10 0.1%BSA inNS 9.6±0. 5 48.2±3. 0 14.8±0. 7
HH-EPO-014A 10 5ng/kg,sc,dl-3 9.9±0. 4 52.1 ±1. 6 15.7±0. 6HH-EPO-014A 10 5ng/kg, sc, dl-3 9.9±0. 4 52.1 ±1. 6 15.7±0. 6
HH-EPO-014B 10 5 g/kg,sc,dl-3 9.3 ±0. 1 53.0± 1. 5 15.7±0. 7 HH-EPO-014C 10 5ng/kg,sc,dl-3 9.6±0. 3 53.2± 1. 4 15.1 ±0. 5HH-EPO-014B 10 5 g/kg, sc, dl-3 9.3 ±0. 1 53.0± 1. 5 15.7±0. 7 HH-EPO-014C 10 5ng/kg, sc, dl-3 9.6±0. 3 53.2± 1. 4 15.1 ±0. 5
HH-EPO-014D 10 5ng/kg,sc,dl-3 9.7±0. 1 50.0± 1. 9 14.7±0. 7HH-EPO-014D 10 5ng/kg, sc, dl-3 9.7±0. 1 50.0± 1. 9 14.7±0. 7
HH-EPO-014E 10 5 g/kg,sc,dl-3 9.1 ±0. 5 55.4± 1. 2 16.5±0. 9HH-EPO-014E 10 5 g/kg, sc, dl-3 9.1 ±0. 5 55.4± 1. 2 16.5±0. 9
HH-EPO-014F 10 5^ig/kg,sc,dl-3 9.2±0· 6 56.5± 1. 8 16.3 ±0. 7HH-EPO-014F 10 5^ig/kg, sc, dl-3 9.2±0· 6 56.5± 1. 8 16.3 ±0. 7
EPO 10 5 g/kg,sc, U-3 9.0 + 0. 6 46.2 + 2. 7 14.3+0. 7 表三、 聚乙二醇促红细胞生成素偶联物对小鼠血小板、 白细胞生成的影响 分组 小鼠 给药剂量和方案 血小板 （X ] 034iL: ) 白细胞 （Χ 103/μ EPO 10 5 g/kg, sc, U-3 9.0 + 0. 6 46.2 + 2. 7 14.3+0. 7 Table 3. Effect of polyethylene glycol erythropoietin conjugate on platelet and leukocyte production in mice Grouped mice administered doses and regimens platelets (X ] 0 3 4iL: ) white blood cells (Χ 10 3 /μ
(只）  (only)
control 10 0.1%BSA inNS 1078.0±151.2 5.1±1.5 Control 10 0.1%BSA inNS 1078.0±151.2 5.1±1.5
HH-EPO-014A 10 5 g/kg,sc,dl-3 1372.5±135 4.2±1.5  HH-EPO-014A 10 5 g/kg, sc, dl-3 1372.5±135 4.2±1.5
HH-EPO-014B 10 5ng/kg,sc,dl-3 1350.8±327 4.2±1.1  HH-EPO-014B 10 5ng/kg, sc, dl-3 1350.8±327 4.2±1.1
HH-EPO-014C 10 5 g/kg,sc,dl-3 1207±237 5.0±2.2  HH-EPO-014C 10 5 g/kg, sc, dl-3 1207±237 5.0±2.2
HH-EPO-014D 10 5ng/kg,sc,dl-3 1325±223 5.5±1.2  HH-EPO-014D 10 5ng/kg, sc, dl-3 1325±223 5.5±1.2
HH-EPO-014E 10 5^ig/kg,sc,dl-3 1457.6土 247.6 4.2±1.2  HH-EPO-014E 10 5^ig/kg, sc, dl-3 1457.6 soil 247.6 4.2±1.2
HH-EPO-014F 10 5^ig/kg,sc,dl-3 1186.8±218.6 4.1±1.2  HH-EPO-014F 10 5^ig/kg, sc, dl-3 1186.8±218.6 4.1±1.2
EPO 10 5^ig/kg,sc,dl-3 1306.8±170. 4.0±0.9 实验例 2: 聚乙二醇促红细胞生成素偶联物对猕猴的作用  EPO 10 5^ig/kg, sc, dl-3 1306.8±170. 4.0±0.9 Experimental Example 2: Effect of polyethylene glycol erythropoietin conjugate on macaque
实验目的： Purpose:
评价聚乙二醇促红细胞生成素偶联物对猕猴红细胞生成的影响 材料及方法：  Evaluation of the effect of polyethylene glycol erythropoietin conjugate on erythrocyte production of macaques Materials and methods:
聚乙二醇促红细胞生成素偶联物 HH-EPO-014A, 由江苏豪森药业股份有限公 司提供； 促红细胞生成素（阳性对照）： 购自沈阳三生制药有限责任公司。 使用前 以含 0.1 %BSA的生理盐水稀释。  Polyethylene glycol erythropoietin conjugate HH-EPO-014A, provided by Jiangsu Haosen Pharmaceutical Co., Ltd.; erythropoietin (positive control): purchased from Shenyang Sansheng Pharmaceutical Co., Ltd. Dilute with normal saline containing 0.1% BSA before use.
猕猴， 体重 5.5〜8.5kg， 雌雄不限， 购自苏州西山中科实验动物中心。猕猴根 据基础血红蛋白分组， 每组三只。 HH-EPO-014A ,1.35nig/kg， 静脉注射一次； EPO 240μ/1¾， 三次 /周， 连续给药 6周， 每周测 1〜2次血液学指标 结果及讨论：  Macaque, weighing 5.5~8.5kg, male or female, purchased from Suzhou Xishan Zhongke Experimental Animal Center. Macaques are grouped according to basal hemoglobin, three in each group. HH-EPO-014A, 1.35nig/kg, intravenous injection; EPO 240μ/13⁄4, three times/week, continuous administration for 6 weeks, 1~2 times of hematology test per week Results and discussion:
HH-EPO-014A单次静脉注射导致猕猴外周血血红蛋白含量上升， 血细胞压积 升高， 说明 HH-EPO-014A刺激血红蛋白生成， 该刺激作用在给药 35天后达到顶 峰， 随后缓慢下降， 对血红蛋白的剌激作用大约为 33 %。 阳性对照促红细胞生成 素同样升高猕猴外周血血红蛋白含量， 升高血细胞压积， 其作用在停药后缓慢减 弱。按照目前的给药方案， 单次静脉注射 HH-EPO-014A和多次连续静脉注射促红 细胞生成素对猕猴血红蛋白生成的刺激作用相当 （见附图 1、 2)。  HH-EPO-014A single intravenous injection caused increased peripheral blood hemoglobin content and increased hematocrit in rhesus macaques, indicating that HH-EPO-014A stimulated hemoglobin production, which reached a peak after 35 days of administration, followed by a slow decline to hemoglobin. The stimulating effect is about 33%. The positive control erythropoietin also increased the peripheral blood hemoglobin content of the macaque and increased the hematocrit, which was slowly reduced 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).

Claims

权利要求书: Claims:
1. 一种聚乙二醇化促红细胞生成素偶联物， 其结构通式为 A PEGylated erythropoietin conjugate having the structural formula
P-NH-CH2-X-S-Y- (OCH2CH2) mi-OCH3 P-NH-CH 2 -XSY- (OCH 2 CH 2 ) m i-OCH 3
所述偶联物是由甲氧基聚乙二醇基团通过式 -CHrX-S-Y-中的 -CH2-基团与促红 细胞生成素的氨基形成 -NH-CH2-键连接而得到， 其中 P是指重组人促红细胞 生成素， X是 -(CH2)k -或 -CH2(OCH2CH2)k-， k的数目选自 2〜10的整数， ml 选自 100〜2000之间的整数， Y选自： The conjugate is obtained by a methoxypolyethylene glycol group by a -CH 2 - group in the formula -CHrX-SY- and a -NH-CH 2 - linkage of the amino group of erythropoietin. 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 an integer from 2 to 10, and ml is selected from 100 to 2000. The integer between, Y is selected from:
Figure imgf000017_0001
Figure imgf000017_0002
Figure imgf000017_0001
Figure imgf000017_0002
其中 m、 n的数目各自独立地选自 2〜10的; 根据权利要求 1所述的偶联物， 其特征在于所述重组人促红细胞生成素是 重组人促红细胞生成素 α或 β， 优选重组人促红细胞生成素 α。 根据权利要求 1所述的偶联物， 其特征在于 Υ中的 m=2， n=2。 根据权利要求 1所述的偶联物， 其特征在于 X是 -(CH2)k-， k选自 2〜10 的整数， 优选 2〜4的整数， 最优选为 2。 根据权利要求 1所述的偶联物， 其特征在于所述甲氧基聚乙二醇基团的平 均分子量为 5， 000〜40， 000道尔顿， 优选为 20， 000道尔顿。 Wherein the number of m, n is each independently selected from 2 to 10; the conjugate according to claim 1, characterized in that the recombinant human erythropoietin is recombinant human erythropoietin alpha or beta, preferably Recombinant human erythropoietin alpha. The conjugate according to claim 1, characterized in that m = 2 and n = 2 in the oxime. The conjugate according to claim 1, wherein X is -(CH 2 ) k -, k is selected from an integer of 2 to 10, preferably an integer of 2 to 4, most preferably 2. The conjugate according to claim 1, characterized in that the methoxypolyethylene glycol group has an average molecular weight of from 5,000 to 40,000 Daltons, preferably 20,000 Daltons.
6. 根据权利要求 1所述的偶联物， 其特征在于偶联物的结构式为: 6. The conjugate according to claim 1, wherein the structural formula of the conjugate is:
Figure imgf000018_0001
Figure imgf000018_0001
其中 P是指重组人促红细胞生成素；  Wherein P refers to recombinant human erythropoietin;
X是 -(CH2)k-或 -CH2(OCH2CH2)k-， 优选是 -(C¾)k-; X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, preferably -(C3⁄4) k - ;
k的数目选自 2〜10的整数， 优选 2〜4的整数， 最优选为 2;  The number of k is selected from an integer of 2 to 10, preferably an integer of 2 to 4, and most preferably 2;
m、 n选自 2〜10的整数；  m, n are selected from an integer of 2 to 10;
选自 100〜2000之间的整数。  It is selected from an integer between 100 and 2000.
7. 根据权利要求 1 所述的偶联物， 其特征在于偶联物的结构式 7. The conjugate according to claim 1, characterized by the structural formula of the conjugate
Figure imgf000018_0002
Figure imgf000018_0002
其中 选自 450〜600的整数。  Among them, an integer selected from 450 to 600 is selected.
8. 根据权利要求 6所述的偶联物， 其特征在于所述重组人促红细胞生成素是 重组人促红细胞生成素 01或 ， 优选重组人促红细胞生成素 α。 The conjugate according to claim 6, characterized in that the recombinant human erythropoietin is recombinant human erythropoietin 01 or, preferably recombinant human erythropoietin.
9. 根据权利要求 1所述的偶联物， 其结构式为- 9. The conjugate according to claim 1, which has the structural formula -
Figure imgf000018_0003
Figure imgf000018_0003
其中 P是指重组人促红细胞生成素， 优选重组人促红细胞生成素 α;  Wherein P refers to recombinant human erythropoietin, preferably recombinant human erythropoietin alpha;
X是 -(CH2)k-或 -C¾(OCH2CH2)k-; X is -(CH 2 ) k - or -C3⁄4(OCH 2 CH 2 ) k - ;
k的数目选自 2〜10的整数， 优选为 2; The number of k is selected from an integer of 2 to 10, preferably 2 ;
m选自 2〜10的整数， 优选为 2;  m is selected from an integer of 2 to 10, preferably 2;
选自 100〜2000之间的整数， 优选自 450〜600的整数。  It is selected from an integer of from 100 to 2000, preferably from an integer of from 450 to 600.
10.根据权利要求 1所述的偶联物， 其结构式为: 10. The conjugate according to claim 1, which has the structural formula:
Figure imgf000018_0004
其中 P是指重组人促红细胞生成素， 优选是重组人促红细胞生成素 α; X是 -(CH2)k-或 -CH2(OCH2CH2)k-，优选是 X是 -(CH2)2-；
Figure imgf000018_0004
Wherein P refers to recombinant human erythropoietin, preferably recombinant human erythropoietin alpha; X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, preferably X is -(CH 2 ) 2 -;
k选自 2〜10的整数；  k is selected from an integer of 2 to 10;
m选自 2〜10， 优选为 2 ;  m is selected from 2 to 10, preferably 2;
选自 100〜2000之间的整数， 优选自 450〜600的整数。  It is selected from an integer of from 100 to 2000, preferably from an integer of from 450 to 600.
11.根据权利要求 1所述的偶联物， 其特征在于偶联物的结构式为:
Figure imgf000019_0001
11. A conjugate according to claim 1 wherein the structural formula of the conjugate is:
Figure imgf000019_0001
其中 P是指重组人促红细胞生成素， 优选是重组人促红细胞生成素 α; X是- (CH2)k-或 -C¾(OCH2C¾)k-，优选是 X是 -(C¾)2-; Wherein P refers to recombinant human erythropoietin, preferably recombinant human erythropoietin alpha; X is -(CH 2 ) k - or -C3⁄4(OCH 2 C3⁄4) k -, preferably X is -(C3⁄4) 2 - ;
k选自 2〜10的整数；  k is selected from an integer of 2 to 10;
Π 选自 100〜2000之间的整数， 优选自 450〜600的整数。  Π is selected from an integer between 100 and 2000, preferably from an integer of from 450 to 600.
12. 根据权利要求 1所述的偶联物， 其特征在于偶联物的结构式为：
Figure imgf000019_0002
12. The conjugate according to claim 1, wherein the structural formula of the conjugate is:
Figure imgf000019_0002
其中 P是指重组人促红细胞生成素， 优选是重组人促红细胞生成素 α; Wherein P refers to recombinant human erythropoietin, preferably recombinant human erythropoietin alpha;
X是 -(C¾)k-或 -CH2(OCH2CH2)k-，优选是 X是 -(CH2)2-; X is -(C3⁄4) k - or -CH 2 (OCH 2 CH 2 ) k -, preferably X is -(CH 2 ) 2 - ;
k的数目选自 2〜10的整数；  The number of k is selected from an integer of 2 to 10;
m选自 2〜10的整数， 优选为 2; m is selected from an integer of 2 to 10, preferably 2 ;
选自 100〜2000之间的整数， 优选自 450〜600的整数。  It is selected from an integer of from 100 to 2000, preferably from an integer of from 450 to 600.
13.根据权利要求 1所述的偶联物， 其结构式为：
Figure imgf000019_0003
13. The conjugate of claim 1 having the structural formula:
Figure imgf000019_0003
其中 P是指重组人促红细胞生成素， 优选是重组人促红细胞生成素 α; Wherein P refers to recombinant human erythropoietin, preferably recombinant human erythropoietin alpha;
X是 -(CH2)k-或 -CH2(OCH2CH2)k-，优选是 X是 -(C¾)2-; X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, preferably X is -(C3⁄4) 2 - ;
k的数目选自 2〜10的整数；  The number of k is selected from an integer of 2 to 10;
m、 n选自 2〜10的整数， 优选为 2;  m, n is selected from an integer of 2 to 10, preferably 2;
选自 100〜2000之间的整数， 选自 450〜600的整数。  An integer selected from 100 to 2000, selected from an integer of 450 to 600.
14.一种制备权利要求 1〜13任意一项所述偶联物的方法， 包括以下步骤- ( 1 )促红细胞生成素和含有已保护巯基的醛类物质发生还原胺化反应，形成 通过 -NH-CH2-键连接的活化促红细胞生成素； 14. A process for the preparation of a conjugate according to any one of claims 1 to 13, comprising the steps of - (1) reductive amination of erythropoietin and an aldehyde containing a protected thiol group, forming Activated erythropoietin linked by a -NH-CH2- linkage;
(2)所述活化促红细胞生成素脱保护， 与活性甲氧基聚乙二醇衍生物偶联。  (2) The activated erythropoietin is deprotected and coupled to a reactive methoxypolyethylene glycol derivative.
15.一种药物组合物， 包含： 15. A pharmaceutical composition comprising:
( 1 ) 治疗量的如权利要求 1〜13任意一项所述的聚乙二醇化促红细胞生成 素偶联物， 和  (1) A therapeutic amount of the PEGylated erythropoietin conjugate according to any one of claims 1 to 13, and
(2)药学可接受的药物载体。  (2) A pharmaceutically acceptable pharmaceutical carrier.
16.根据权利要求 1〜13任意一项所述的偶联物在制备用于治疗以缺乏红细胞 生成素或红细胞群缺少或缺陷为特征的疾病的药物中的用途。 16. Use of a conjugate according to any one of claims 1 to 13 for the manufacture of a medicament for the treatment of a disease characterized by a deficiency or deficiency of erythropoietin or a population of red blood cells.
17.根据权利要求 15所述的药物组合物在制备用于治疗以缺乏红细胞生成素 或红细胞群缺少或缺陷为特征的疾病的药物中的用途。 17. Use of a pharmaceutical composition according to claim 15 in the manufacture of a medicament for the treatment of a disease characterized by a deficiency or deficiency of erythropoietin or a population of red blood cells.
18.根据权利要求 16或 17所述的用途， 其特征在于缺乏红细胞生成素或红细 胞群缺少或缺陷为特征的疾病是末期肾功能衰竭或透析； AIDS相关性贫 血， 自身免疫性疾病， 或恶性肿瘤； 囊性纤维变性； 早期早熟性贫血； 与 慢性炎性疾病相关的贫血； 脊髓损伤； 急性失血； 衰老和伴有异常红细胞 产生的肿瘤疾病。 The use according to claim 16 or 17, characterized in that the disease characterized by lack of erythropoietin or red blood cell population deficiency or defect is end stage renal failure or dialysis; AIDS-related anemia, autoimmune disease, or malignancy 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.
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