WO2007082483A1 - Medicament for treatment of tumor and the use thereof - Google Patents

Medicament for treatment of tumor and the use thereof Download PDF

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Publication number
WO2007082483A1
WO2007082483A1 PCT/CN2007/000204 CN2007000204W WO2007082483A1 WO 2007082483 A1 WO2007082483 A1 WO 2007082483A1 CN 2007000204 W CN2007000204 W CN 2007000204W WO 2007082483 A1 WO2007082483 A1 WO 2007082483A1
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Prior art keywords
endostatin
complex
polyethylene glycol
group
seq
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PCT/CN2007/000204
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English (en)
French (fr)
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WO2007082483B1 (en
Inventor
Yongzhang Luo
Qing Han
Qingxin Lei
Guodong Chang
Yan Fu
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Tsinghua University
Protgen Ltd
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Tsinghua University
Protgen Ltd
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Priority to CA2637698A priority Critical patent/CA2637698C/en
Priority to AU2007207268A priority patent/AU2007207268B2/en
Priority to EP07702136.8A priority patent/EP1985302B1/en
Priority to JP2008550619A priority patent/JP5687823B2/ja
Priority to US12/161,477 priority patent/US20100285103A1/en
Priority to CN200780010122.7A priority patent/CN101405019B/zh
Application filed by Tsinghua University, Protgen Ltd filed Critical Tsinghua University
Publication of WO2007082483A1 publication Critical patent/WO2007082483A1/zh
Publication of WO2007082483B1 publication Critical patent/WO2007082483B1/zh
Anticipated expiration legal-status Critical
Priority to NO20083287A priority patent/NO20083287L/no
Priority to US16/025,315 priority patent/US20180360922A1/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the present invention relates to a complex and sustained release preparation containing endostatin having biological activity and metabolic stability and a process for the preparation thereof.
  • the present invention also provides a pharmaceutical combination comprising the vascular endostatin complex and the sustained release preparation, and a kit containing the same.
  • the present invention also provides a method for preventing, diagnosing, treating a tumor, and preparing an antitumor drug using the endostatin complex, the sustained release preparation, the pharmaceutical combination and the kit of the present invention.
  • Neovascularization refers to the formation of new capillaries on the original blood vessels. Tumor growth and migration depend on the formation of new blood vessels, and microvascular endothelial cells in tumors as a target for cancer treatment provide a treatment for tumors (Folkman J. N Engl J Med 1971; 285: 1182-1186) .
  • Endostatin is a cleavage product of a molecular weight of 20 ldDa at the carboxy terminus of collagen X.
  • Professor Judah Folkmaii of Harvard University and others found this protein in the culture medium of hemangioendothelial cells, which has the activity of inhibiting vascular endothelial cell proliferation, migration, and in vivo angiogenesis. Further experiments have shown that recombinant endostatin can inhibit the growth and metastasis of various tumors in mice, and even completely cure the tumor, and does not produce drug resistance.
  • the mechanism of its activity is that it inhibits the growth of vascular endothelial cells, inhibits the formation of new blood vessels near the tumor tissue, makes the tumor tissue unable to obtain the large amount of nutrients and oxygen necessary for growth, and finally stops growth or necrosis (Folkman J. Et al. Cell 1997; 88:277-285; Folkman J. et al. Nature 1997; 390:404-407).
  • Recombinant human endostatin prepared by genetic engineering can be used as a therapeutic drug for tumors, and its clinical tests show that it can effectively inhibit tumor growth. In China, clinical trials with non-small cell lung cancer as the main indication have shown that it has a prominent therapeutic effect on tumors.
  • Peptide and protein drugs have less toxic and side effects than chemical drugs, and do not produce drug resistance. Point.
  • protein drugs are usually administered intravenously.
  • the half-life in vivo after intravenous administration is usually short.
  • an important aspect is that small protein proteins can be quickly eliminated from the body by renal filtration.
  • proteins with a hydraulic radius exceeding albumin or having a molecular weight greater than about 66,000 daltons (66 kDa) are generally stably retained in the circulatory system.
  • small molecules are quickly eliminated from the blood by glomerular filtration.
  • Endothelin a protein drug
  • Endothelin also has a shortcoming of a short half-life and a high clearance rate in the body.
  • the current clinical medications are mainly frequent intravenous or drip, usually daily, and require long-term medication, which makes the patient's mental and economic burden relatively large.
  • the object of the present invention is to improve the in vivo metabolic characteristics of the protein, to have higher stability and longer in vivo half-life, and even higher therapeutic effect, thereby reducing the frequency of administration, reducing the cost of medication, and reducing the economy of patients. The purpose of the burden.
  • Modification of proteins with high molecular polymers is a common method for altering and controlling the kinetic properties of drugs such as half-life, immunological characteristics, and toxicological properties.
  • the high molecular weight polymer used to modify the protein has the characteristics of good water solubility, good biocompatibility, and no or weak immunogenicity.
  • polyethylene glycol (PEG) is the most commonly used protein modification molecule.
  • Polyethylene glycol molecules are amphiphilic, soluble in water, soluble in most organic solvents, non-toxic, non-immunogenic, highly soluble in aqueous solution, etc. Polymers approved for drug preparation approved by drug regulatory agencies in many countries, including China's SFDA.
  • Coupling proteins with hydrophilic polymers such as polyethylene glycol increases protein stability and reduces non-specific adsorption and antigenicity. When the conjugate reaches a certain molecular weight, it can greatly reduce the elimination efficiency of the kidney, and is an effective method for prolonging the half-life of the protein drug in vivo (Frokjaer S. et al. Nat. Rev. Drug Discov. 2005 Apr 4(4): 298-306 Harris JM. et al. Nat. Rev. Drug Discov. 2003 Mar 2(3): 214-21).
  • the initial polyethylene glycol modification uses an amino group as a reaction site, mainly including The N-terminal ⁇ -amino group of the protein and the ⁇ -amino group on the side chain of the lysine residue.
  • the product of this type of reaction is a protein molecule coupled to one or more PEG molecules. Modifications on the ⁇ -amino group of the side chain of the lysine residue are also often due to the unspecificity of the reaction site to produce a modified isomer.
  • a polyethylene glycol modification reagent which is only modified for the protein end is newly developed, so that the modification sites are identical.
  • the modified product has a uniform composition.
  • Another protein site that can be used for polyethylene glycol modification is the thiol group of cysteine.
  • cysteine can now be introduced at any part of the protein as a specific modification site.
  • the introduction of cysteine as a modification site also has certain limitations, because some proteins with cysteine itself may cause mismatch or irreversibility of disulfide bonds.
  • the carboxyl group of the protein is also a commonly used modification site (Veronese FM et al. Drug Discov. Today. 2005 Nov l; 10(21): 1451-8.) o Polyethylene glycol modification technology has been successfully applied in many For proteinaceous drugs, more successful include: PEG-asparaginase (Graham ML Adv. Drug Deliv. Rev.
  • blood proteins or fragments thereof as a carrier, coupled with or expressed by a pharmaceutical protein, thereby increasing the molecular weight of the protein is another method for prolonging the half-life of the drug.
  • the Fc fragment of an immunoglobulin is coupled with a protein of interest to extend its half-life, for example, the method is used in the Notch 1 receptor protein (Kitajewsky Jan et al. WO2005111072), erythropoietin (EPO) (Gillies) T N2007/000204
  • Serum albumin is also a common coupling to a carrier, can be used in the antibiotics, anti-inflammatory, antioxidant and other proteins (Ezrin Alan M et al. WO0117568 , Otagiri Masaki et al. EP1571212) 0
  • the protein drug in addition to being coupled to a drug protein in vitro, the protein drug may be modified in vitro to have a chemical reaction activity or a high affinity for other molecules in the body. It can react with certain components of the blood after entering the body to form a macromolecule or complex with a long half-life.
  • This technique is the application of this technique to the antiviral small peptide molecule anti-RSV, which is modified with a maleimide-active group that reacts with blood proteins or sulfhydryl groups in cell surface proteins ( Bridon Miguel P et al. WO0069902).
  • Another example is the use of an acylation reaction to introduce fatty acids on the amino acid residues on the surface of the protein, increasing the affinity of the protein for albumin in the body, allowing the protein to form a larger complex with albumin after entering the blood, thereby extending the half-life.
  • this method has been used on long-acting insulin (Kurtzhals P et al. Biochem. J. (1995) 312, 725-731; Frokjaer S et al. Nat Rev Drug Discov. 2005 Apr; 4(4): 298 -306).
  • Another way to extend the in vivo half-life of a protein is to make the protein into a sustained release dosage form.
  • the protein drug is placed in a pharmaceutically acceptable carrier such that the protein is slowly released from the carrier to maintain a stable drug concentration in the body.
  • Commonly used methods are hydrogels, microcapsules, microspheres, micro-osmotic pumps, liposomes, etc. (Peppas NA et al. Eur. J. Pharm. Biopharm. 50, 27-46 (2000); Packhaeuser CB et al. Eur. J. Pharm. Biopharm. 58, 445-455 (2004); Metselaar JM et al. Mini Rev. Med. Chem.
  • 0 liposome is a bilayer membrane structure Ultra-microscopic particles in the shape of an air sphere.
  • the bilayer membrane is composed of an amphiphilic molecule mostly composed of phospholipids and having an aqueous cavity.
  • the hydrophilic protein drug is encapsulated in the inner cavity of the liposome and slowly released in the body, which can maintain the blood concentration of the protein and increase the half-life.
  • Some examples include use of nerve growth factor (Hou Xinpu et al. CN1616087), hemoglobin (Farmer Martha C et al. US4911929), and the like. Summary of invention
  • the present invention provides a complex formed by a modification with endostatin, wherein the modification
  • the substance can prolong the in vivo half-life of endostatin.
  • the modifications used in the present invention may be linked to endostatin by a covalent bond. These modifications may be selected from high molecular weight polymers, protein molecules or fragments thereof, peptide chains, small molecules or other chemicals.
  • the high molecular polymer which can be used in the present invention is, for example, polyethylene glycol having an average molecular weight of between 1,000 and 100,000 Daltons, preferably between 5,000 and 40,000 Daltons.
  • the complex is characterized by a vascular endostatin molecule coupled to one or more polyethylene glycol molecules.
  • the site of the coupling is the N-terminal a-amino group of endostatin, the ⁇ -amino group of the side chain of the lysine residue, and a half. a thiol group of a side chain of a cystine residue, a carboxyl group of a side chain of an aspartic acid residue, or a carboxyl group of a side chain of a glutamic acid residue or a combination thereof.
  • the coupling site is an a-amino group of the end of vascular endostatin.
  • the endostatin molecule is coupled to one or more polyethylene glycol molecules, and the site of the coupling is endostatin alpha-amino or wild-type human vascular endothelium.
  • the endostatin molecule is coupled to one or more polyethylene glycol molecules by a method of attaching a cyst to the endothelium or C-terminus of the endostatin molecule.
  • a peptide or a cysteine-containing peptide chain such that the coupling site is a thiol group on the side chain of an additional cysteine residue.
  • the endostatin molecule is coupled to one or more polyethylene glycol molecules, and the site of the coupling is the endostatin or glutamic acid residue side of the endostatin. a carboxyl group on the chain.
  • the protein used in the complex of the present invention is selected from the group consisting of albumin, immunoglobulin, thyroid binding protein, transthyretin, transferrin, fibrinogen or fragments thereof.
  • the complex is an albumin-coupled endostatin characterized by an endostatin molecule coupled to one or more albumin, the conjugate being chemistry The modified, or obtained by fusion, or obtained by other methods, wherein the albumin is preferably human serum albumin or a fragment thereof.
  • the complex is an immunoglobulin Fc fragment-conjugated endostatin, characterized in that a endostatin molecule is coupled to one or more immunoglobulin Fc fragments, and the conjugate can be passed The chemically modified, or obtained by fusion expression, or obtained by other methods, wherein the Fc fragment is preferably a fragment of the Fc region in human immunoglobulin IgG.
  • the complex of the present invention also includes a small molecule or a small peptide or other compound modified endostatin, which is characterized in that the complex has an activity of reacting or binding with other molecules or components in the body, so that the polymer can be in vivo. Forms a larger complex with other ingredients.
  • the reactive group can react with the amino group, the hydroxyl group, and the thiol group of the blood component to form a covalent bond.
  • These reactive groups are, for example, maleimides which react with blood components such as sulfhydryl groups in albumin.
  • the complex of the present invention will have a strong affinity with certain components of blood, such as albumin and immunoglobulin, and can form larger complexes with each other.
  • the complex of the present invention also includes other molecules or small peptide-modified endostatin, such as a product in which endostatin is glycosylated, phosphorylated or acylated, wherein the modified site is present in the original protein sequence.
  • endostatin is glycosylated, phosphorylated or acylated, wherein the modified site is present in the original protein sequence.
  • the complex formed by the modification of the present invention and endostatin may also be a complex formed by a non-covalent interaction between the modification and the endostatin, wherein the modification may be a protein, a small molecule or other chemical substance. .
  • the complex of the present invention can form a sustained release preparation with a biocompatible carrier.
  • the sustained release preparation may be in the form of a microcapsule, a hydrogel, a microsphere, a micro osmotic pump or a liposome.
  • the endostatin used in the complex or sustained release preparation of the present invention may be derived from human, mouse, or other mammals.
  • the endostatin contained in the complex or the sustained release preparation is wild type human endostatin, and the natural form thereof has the sequence shown by SEQ ID NO.
  • the endostatin contained in the complex or sustained-release preparation of the present invention when expressed by Escherichia coli, has a wild-type recombinant human endostatin having the sequence of SEQ ID N0.2, wherein the N-terminal Met is in the large intestine
  • the liver bacteria are sometimes partially deleted when expressed, and thus the corresponding recombinant human endostatin has the sequence of SEQ ID N0.1.
  • endostatin may be vascular endothelial inhibition An active fragment, mutant, derivative, isomer, or a combination thereof.
  • the endostatin fragment is a peptide chain having the sequence of SEQ ID N0.3, SEQ ID 0.4, or SEQ ID NO.
  • the endostatin derivative in the complex or sustained-release preparation is a peptide having a length of 1 to 15 amino acids attached to the N-terminus or C-terminus of wild-type endostatin. Chain formed.
  • the endostatin derivative is a His-tag-containing peptide chain MGGSHHHHH which is composed of a 9-amino acid group at the N-terminus of wild-type human endostatin.
  • the invention also provides a pharmaceutical composition comprising a complex or sustained release formulation of the invention and a pharmaceutically acceptable carrier.
  • the invention also includes kits comprising the complexes, compositions or sustained release formulations of the invention and instructions for use.
  • the present invention also provides a process for the preparation of the above complex according to the claims, characterized in that the activated polyethylene glycol is mixed with endostatin and reacted at a suitable solution, temperature, pH and molar ratio.
  • the pH used in the process is pH 3-10.
  • the coupling product can be purified, for example, with a cation column or with a molecular sieve.
  • the present invention also encompasses the use of the above-described complex, sustained release preparation, pharmaceutical composition or kit for preventing, diagnosing or treating a tumor, and its use in preventing, diagnosing or treating other diseases characterized by a newborn Abnormal vascular formation leads to pathological changes in human tissues or organs.
  • the present invention also provides a method of prolonging the half-life of vascular endostatin, the method comprising the step of forming a complex with a variant capable of prolonging the in vivo half-life of endostatin and comprising an endothelin or a blood vessel
  • the complex of endostatin and the biocompatible material form a step of a sustained release formulation.
  • Figure 2 shows the amino acid sequence of a wild-type recombinant human endostatin expressed by E. coli SEQ ID N0.2. Here the Met at the N-terminus is sometimes partially deleted after expression. If this occurs, the recombinant human endostatin after deletion of the M-terminus at the N-terminus will have the sequence of SEQ ID NO.
  • Figure 3 Sequence showing an active endostatin fragment SEQ ID N0.3.
  • Figure 4 Sequence showing an active endostatin fragment SEQ ID N0.4.
  • Figure 5 Sequence showing an active endostatin fragment SEQ ID N0.5.
  • Figure 6 Amino acid sequence of recombinant human endostatin showing an active N-terminal additional amino acid SEQ ID NO. 6 (Fig. 6A). Here, the Met at the N-terminus is sometimes partially deleted after expression. If this occurs, the recombinant human endostatin after deletion of the M-terminus at the N-terminus will have the sequence of SEQ ID NO. 7 (Fig. 6B;).
  • Figure 7 Coupling of 20 kDa polyethylene glycol with the N-terminus of recombinant human vascular endostatin with the sequence of SEQ ID NO. The solution before and after the reaction was detected by SDS-PAGE, lane 1: 20 kDa polyethylene glycol modified endostatin; lane 2: unmodified endostatin; lane 3: protein molecular weight standard.
  • Figure 8 Coupling of 20 kDa polyethylene glycol with the N-terminus of recombinant human vascular endostatin with the sequence of SEQ ID NO. The solution before and after the modification was detected by SDS-PAGE, lane 1 : unmodified endothelin; lane 2: 20 kDa polyethylene glycol modified endostatin; lane 3: protein molecular weight standard.
  • Figure 9 20 kDa polyethylene glycol modified N-terminus of recombinant human vascular endostatin having the sequence of SEQ ID NO. 2 and purified with a cation column SP.
  • the protein was adsorbed by a cationic column SP, and then eluted with a sodium chloride gradient solution, and finally purified by SDS-PAGE.
  • Lane 1 before purification
  • Lane 2 solution for penetrating the sample
  • Lane 3 ⁇ 10 Distribute the collected eluate.
  • Figure 10 20 kDa polyethylene glycol modified N-terminus of recombinant human endostatin with the sequence of SEQ ID N0.6 followed by purification with a cation column SP.
  • the protein was adsorbed by a cationic column SP, and then eluted with a sodium chloride gradient solution, and finally the purification effect was examined by reducing SDS-PAGE.
  • Lane 1 before purification
  • Lane 2 Solution-penetrating solution
  • Lanes 3 to 8 Distribute the collected eluate.
  • Figure 11 40 kDa polyethylene glycol modified N-terminus of recombinant human vasopressin with the sequence of SEQ ID NO. 2 and purified with a cation column SP.
  • the protein was adsorbed by a cationic column, and then eluted with a sodium chloride gradient solution. Finally, the purification effect was examined by reduction SDS-PAGE. Lane 1: before purification; Lane 2: solution for penetrating the solution; Lanes 3 to 10: Distribute the collected eluate.
  • Figure 12 The activity of 20 kDa polyethylene glycol coupled with the N-terminus of recombinant human endostatin inhibits the migration of vascular endothelial cells.
  • SEQ2 Endostatin having SEQ ID N0.2; PEG-SEQ2: 20 kDa polyethylene glycol modified endostatin having SEQ ID NO. 2; SEQ6: endostatin having SEQ ID NO. 6; PEG-SEQ 6: 20 kDa polyethylene glycol modified endostatin with SEQ ID NO.
  • Figure 13 The product of 20 kDa polyethylene glycol coupled to the N-terminus of recombinant human endostatin inhibits the activity of mouse S180 sarcoma.
  • SEQ2 Endostatin with SEQ ID N0.2 administered daily
  • PEG-SEQ2-1 20 kDa polyethylene glycol modified endostatin with SEQ ID N0.2 sequence administered daily
  • PEG-SEQ 2- 2 20 kDa polyethylene glycol modified endostatin with sequence of SEQ ID N0.2 administered every two days
  • PEG-SEQ 2-3 20 kDa polyethylene glycol modified blood vessel with sequence of SEQ ID NO.
  • Endostatin is administered every three days; SEQ6: Endostatin with SEQ ID N0.6 is administered daily; PEG-SEQ6-1: 20 kDa polyethylene glycol modified endostatin with SEQ ID N0.6 sequence per day Administration; PEG-SEQ 6-2: 20 kDa polyethylene glycol modified endostatin with the sequence of SEQ ID N0.6 administered every two days; PEG-SEQ 6-3: 20 kDa polyethylene glycol modified Endostatin with the sequence of SEQ ID N0.6 is administered every three days.
  • Figure 14 20 kDa polyethylene glycol modified ⁇ -amino group of the side chain of the recombinant human vasopressin lysine residue having the sequence of SEQ ID NO. Lane 1: Loading; Lanes 2 to 10: Distribute the collected eluent.
  • Figure 15 20 kDa polyethylene glycol modified ⁇ -amino group of the side chain of the recombinant human endostatin lysine residue having the sequence of SEQ ID NO: 0.6 and purified with a cation column CM. Lane 1: Loading; Lane 2: Penetration; Lanes 3 to 8: Distribute the collected eluent. Detailed description of the invention
  • the present invention provides a vascular endostatin product having a longer half-life which has higher metabolic stability and longer in vivo half-life than unmodified endostatin while maintaining angiogenesis-inhibiting activity.
  • the endostatin of the present invention is preferably human endostatin.
  • a particular embodiment of the invention relates to a complex of a modification with endostatin.
  • the term "complex" as used in the present invention refers to a modified endostatin. Endostatin is linked to the modification by a covalent bond or by a non-covalent bond to form a stable complex. Therefore, the term "modification" can be either a covalent bond or a non-covalent bond.
  • the modification may be linked to endostatin by chemical coupling or may be linked to endostatin by fusion expression. In a preferred embodiment, such modification is site specific and does not affect the biological activity of endostatin, such as binding to a receptor.
  • the modified product has anti-angiogenic activity but is metabolically more stable than unmodified endostatin, has a longer blood half-life, and can be used as an anti-angiogenic or anti-tumor drug.
  • the molecule used for the modification may be one or more biocompatible polymers, protein molecules or fragments thereof, peptide chains, or other chemicals that increase the blood half-life of endostatin.
  • the high molecular polymer here may have its own biological activity or no biological activity.
  • Suitable polymers include, but are not limited to, polyenol compounds, polyether compounds, polyvinylpyrrolidone, polyamino acids, copolymers of divinyl ether and maleic anhydride, N-(2-hydroxypropyl-methyl) N-(2-Hydroxypropyl)-methacrylamide polysaccharides, polyoxyethylated polyols, heparin or heparin fragments, polyalkyl glycols and their derivatives, polyalkyl glycols and Copolymer of its derivatives, polyvinyl ether, poly(hydroxyethyl)-poly-(hydroxyethyl)-polyamide (polycarboxylates), ethylene oxide and formaldehyde Polyoxyl-oxymethylenes.
  • the above polyenol compounds include, but are not limited to, polyethylene glycol (including monomethyl polyethylene glycol, monohydroxy polyethylene glycol, etc.), polyvinyl alcohol, polypropylene alcohol, polybutenol, and the like. Derivatives.
  • the modification is polyethylene glycol.
  • the polyethylene glycol is monomethylpolyethylene glycol.
  • the above polyether compounds include, but are not limited to, poly(ethylene glycol) (HO((CH 2 ) x O) n H), polypropylene glycol, polyethylene oxide (HO((CH 2 ) 2 0) n H), Polyvinyl alcohol ((CH 2 CHOH) n ) and the like and derivatives thereof.
  • rate amino acids include, but are not limited to, polymers of the same amino acid, copolymers of two or more amino acids, such as polyalanine.
  • polysaccharides include, but are not limited to, dextran and dextran derivatives such as dextran sulfate, cellulose and cellulose derivatives (including methyl cellulose and carboxymethyl cellulose), starch and Starch derivatives, polysucrose, and the like.
  • dextran and dextran derivatives such as dextran sulfate, cellulose and cellulose derivatives (including methyl cellulose and carboxymethyl cellulose), starch and Starch derivatives, polysucrose, and the like.
  • Protein molecules suitable for use as modified vectors herein are preferably naturally occurring proteins or fragments thereof, including but not limited to thyroxine-binding proteins, transthyretin, transferrin (fibrinogen, immunized) Globulin, albumin, etc. and fragments thereof.
  • the carrier protein described above is preferably of human origin.
  • a fragment of the above protein refers to any moiety that is smaller than the protein but retains the function of the carrier.
  • Endostatin is covalently linked directly or indirectly to the above modification, and direct ligation means that an amino acid of endostatin is directly linked to an amino acid of the carrier protein, and may be through a peptide bond or a disulfide bond.
  • Indirect linkage means that endostatin and a carrier protein are linked by a certain chemical group or a combination of several groups.
  • this multi-point modification of the protein causes the conformation of certain internal amino acids to change. causes a decrease in protein activity.
  • the homogeneity of the product is very important for the toxicity of the drug and the reduction of the immunological prototype.
  • quality control it is difficult to ensure that the quality of each batch of the drug is uniform and the drug efficacy is stable. Therefore, it is necessary to ensure that the drug component is fixed, the conformation is uniform, and the activity is stable, which is an important consideration and an urgent problem to be solved in designing and producing the drug.
  • a particular embodiment of the invention relates to a polyethylene glycol modified endostatin, characterized in that a endostatin molecule is coupled to one or more polyethylene glycol molecules.
  • the coupled site is the N-terminal ⁇ -amino group of the protein, the ⁇ -amino group of the side chain of the lysine residue, the thiol group of the cysteine, the carboxyl group of the side chain of the aspartic acid residue, and the glutamic acid residue side.
  • All of the polyethylene glycol molecules used for coupling may be linear or bifurcated, having a molecular weight between 1,000 and 100,000 Daltons, preferably between 5,000 and 40,000 Daltons.
  • the molecular weight of the polyethylene glycol described in the present invention means an approximate average molecular weight.
  • polyethylene glycol modification has the advantage of changing the metabolic properties of the drug, usually this modification also causes the biological activity of the macromolecular drug to decrease, mainly due to the change of the structure of the drug itself and the active site at different modification sites. Shield [Veronese F., et al. Drug Discovery Today, 10, 21, 2005].
  • different modification methods lead to a decrease in the activity of the modified interferon, and the optimal solution only achieves 71% of the original protein activity, and some modification methods Even the protein is almost inactive (Filpula D., et al. Bioconjugate Chem. 2006, 17, 618-630).
  • the recombinant human endostatin is modified using a protein N-terminal specific polyethylene glycol modification reagent (mPEG-ButyrALD, Nektar).
  • the product is a polyethylene glycol linked to a recombinant human endostatin, and the junction site is the N-terminal a-amino group of the protein.
  • the in vitro activity of the N-terminally modified peginterferon ⁇ -lb was only the original protein. About 70% of the activity.
  • the N-terminally modified recombinant human endostatin product inhibits the migration of endothelial cells twice or more than the unmodified protein in in vitro cytology experiments, both proteinaceous organisms. The activity has been greatly improved, this in vitro activity The apparent increase has not been reported.
  • the in vitro activity of the multi-modified endostatin obtained by Qi Guoying et al. (2005) was significantly reduced as the modification reaction progressed.
  • a specific embodiment of the invention relates to the coupling of endostatin and a protein or peptide chain, characterized in that endostatin is directly or indirectly linked to one or more proteins or peptide chains, when linked to a plurality of carrier proteins
  • the selected carrier proteins may be the same or different.
  • the carrier protein therein is preferably a naturally occurring protein or a fragment thereof.
  • a carrier protein for modification in a preferred embodiment is human serum albumin or a fragment thereof.
  • the carrier protein is an Fc fragment of human immunoglobulin IgG.
  • the modification in another embodiment of the present invention is a small molecule or a small peptide or other compound characterized by the ability of the complex containing endostatin to react with certain components of blood or with blood. Some ingredients have strong affinity.
  • An embodiment of the product is that it has a reactive group that reacts with the amino, hydroxyl, and sulfhydryl groups of the blood component to form a covalent bond.
  • Another reactive product of the specific product is a maleimide which reacts with thiol groups in blood proteins such as albumin.
  • Another specific product has a strong affinity with certain components of blood, such as albumin, immunoglobulin, etc., and can form a larger complex with it.
  • the complex according to another embodiment of the present invention is an endothelin modified by other small molecules or small peptides, and may be a product obtained by glycosylation, phosphorylation, acylation or the like of endostatin.
  • the site to be modified may be an amino acid present in the original protein sequence, or may be an amino acid residue produced by mutation.
  • the complex does not have a large molecular weight, the half-life is prolonged because the modification changes the properties of endostatin.
  • Another specific embodiment of the present invention relates to a complex comprising endostatin, which is a complex molecule having a specific composition formed by a non-covalent bond between endostatin and other molecules, wherein the modification may It is a protein, a small molecule, etc.
  • This complex has an activity of inhibiting angiogenesis and has a longer half-life than endostatin in vivo.
  • Another specific embodiment of the present invention relates to a sustained release preparation containing endostatin which is formed by endostatin or any of the above complexes and a biocompatible substance. Endostatin in this composition is still biologically active, but can rely on the carrier to alter drug metabolism characteristics for extended retention time in the body.
  • the sustained release preparations are, but not limited to, microcapsules, hydrogels, microspheres, micro osmotic pumps, liposomes, and the like.
  • the present invention also provides a pharmaceutical composition comprising the above-described complex or sustained release preparation containing endostatin and a pharmaceutically acceptable carrier.
  • the present invention also provides a kit comprising the above-described vascular endostatin-containing complex or sustained-release preparation and instructions for use.
  • a pharmaceutically acceptable carrier for use herein includes a pharmaceutically acceptable carrier, excipient, or stabilizer which is non-toxic to the cells or mammals to which it is administered at the dosages and concentrations employed.
  • a generally physiologically acceptable carrier is an aqueous pH buffer solution.
  • physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid, low molecular weight (up to 10 residues) polypeptides; Proteins such as albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides And other sugars including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or such as TWEEN®, polyethylene glycol Nonionic surfactants such as (PEG), and PLU OMCS®.
  • buffers such as phosphates, citrates, and other organic acids
  • antioxidants including ascorbic acid, low molecular weight (up to 10 residues) polypeptides
  • Proteins such
  • the excipients are preferably sterile and generally free of undesirable materials. These compositions can be sterilized by conventional well known sterilization techniques.
  • the invention also relates to a method of preparing the above-described endostatin product.
  • a method for preparing a polyethylene glycol modified endostatin which is to mix activated polyethylene glycol with endostatin, react at a suitable solution, temperature, pH, molar ratio, and use a cation column or The molecular sieves are used to purify the coupled product.
  • the pH of the reaction is preferably between pH 3 and 10.
  • the present invention also provides a use of the above-described complex, sustained-release preparation, pharmaceutical composition or kit containing an endostatin for preventing, diagnosing or treating a tumor.
  • Suitable cancers for this method include, but are not limited to, lung cancer, neuroendocrine tumor, colon cancer, bone cancer, liver cancer, gastric cancer, pancreatic cancer, oral cancer, breast cancer, prostate cancer, lymphoma, esophageal cancer, oral cancer, nasopharyngeal cancer, Cervical cancer, sarcoma, kidney cancer, biliary cancer, malignant melanoma tumors, etc.
  • the present invention also provides the use of the above-described complex, pharmaceutical composition or sustained release preparation containing endostatin for the preparation of an antitumor drug.
  • the present invention also provides a use of the above-described complex, sustained-release preparation, pharmaceutical composition or kit containing an endostatin, for preventing, diagnosing or treating other diseases and preparing a medicament for treating these diseases, characteristics of the disease
  • a tissue or organ lesion of a human is caused by, but not limited to, abnormal angiogenesis.
  • the administration method is selected from the group consisting of intravenous injection, intravenous drip, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal absorption, subcutaneous embedding, hepatic artery injection, oral administration, and nasal spray.
  • the present invention also provides a method for prolonging the half-life of vascular endostatin, which comprises the steps of forming a complex with a vascular endostatin and preparing a sustained release preparation containing an endostatin.
  • a product of the present invention a polyethylene glycol and a recombinant human endostatin N-terminal complex
  • the activity is obviously improved, and in vivo pharmacokinetic studies show that the modified product can effectively slow down the metabolism of the drug in the body and prolong the half-life in the body.
  • the endostatin mentioned in the present invention may be human, mouse, or other mammals, unless otherwise specified, and a preferred embodiment thereof is human endostatin. Mentioned in the invention Endostatin, unless otherwise specified, includes wild-type endostatin, a naturally occurring form in vivo, or an active mutant, fragment, isomer, derivative, etc., or a combination thereof.
  • the source of endostatin may be, but is not limited to, expressed by animal cells, or may be fermentatively purified from yeast or E. coli.
  • the amino acid sequence of the wild type human endostatin derived from animal cells is represented by SEQ ID NO.
  • the recombinant human angiostatin derived from yeast expression has the sequence of SEQ ID N0.1, or is herein
  • the N-terminus of the sequence has an increase or deletion within 10 amino acids
  • the recombinant human endostatin derived from the expression of Escherichia coli has the sequence of SEQ ID N0.2, but the N-terminal Met is sometimes partially deleted after expression. . If this occurs, the recombinant human endostatin after deletion of the M-terminus at the N-terminus will have the sequence of SEQ ID NO.
  • a mutant of endostatin refers to an endostatin molecule obtained by substitution, deletion, and addition of an amino acid.
  • a fragment of endostatin refers to any smaller portion of the sequence belonging to SEQ ID NO. 1 or SEQ ID N0.2, which may be, but is not limited to, obtained by enzymatic cleavage, or genetically engineered, or by polypeptide Synthetic method obtained.
  • SEQ ID N0.3, SEQ ID N0.4, SEQ ID NO. 5 has been reported to have activity in inhibiting solid tumors in mice (Cattaneo MG et al. Experimental Cell Research 283 (2003) 230-236; Tjin Tham Sjin RM et al.
  • the isomer of endostatin refers to the same amino acid sequence or molecular formula as wild-type endostatin, but has a different conformation, including differences in secondary or tertiary structure of the protein or local amino acid rotation.
  • the source of the isomer can be a naturally occurring mutation or obtained by artificial design.
  • a derivative of endostatin refers to a product modified based on wild-type endostatin, wherein the modification refers to covalent attachment of one or more other small molecules, such as a phosphate molecule, a sugar molecule, etc., to the protein. , or a small peptide chain within 20 amino acids, the attachment site may be any amino acid in the protein.
  • a human endostatin comprising a His-tag-containing peptide chain MGGSHHHHH consisting of a 9-amino acid sequence having the sequence of SEQ ID N0.6 (Luo Yongzhang et al. US2004110671) may be added to the N-terminus.
  • the N-terminal Met When expressed by the large intestine, the N-terminal Met is sometimes partially deleted after expression. If this occurs, the recombinant human endostatin derivative after deletion of the M-terminus at the N-terminus will have the sequence of SEQ ID N0.7.
  • a combination of a mutant, a fragment, an isomer, and a derivative in which endostatin is active refers to a product having a change of two or more of the above characteristics at the same time, for example, but not limited to, modification of a mutant of a fragment, and modification of a mutant Product, etc.
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID NO. 2 and SEQ ID N0.6. Specifically, recombinant human endostatin (Beijing Proji Biotechnology Development Co., Ltd.) was dialyzed into a 30 mM sodium acetate solution of pH 5.5. The protein concentration was measured at 280 nm using an ultraviolet spectrophotometer (Agilent Technologies) and then adjusted to 2 mg/ml.
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID N0.2 and SEQ ID N0.6.
  • DETAILED DESCRIPTION OF THE INVENTION A 20 kDa polyethylene glycol modified endostatin was purified by SP column (Amersham). The pH of the mixture after the reaction was adjusted to 6. The column was equilibrated with an equilibration buffer containing 10 mM phosphate, pH 6.0 and loaded. After loading, it was eluted with a gradient of elution buffer containing 10 mM phosphate, 1 M sodium chloride, pH 6.0.
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID N0.2 and SEQ ID N0.6.
  • DETAILED DESCRIPTION OF THE INVENTION A recombinant human endostatin is dialyzed into a 30 mM sodium acetate solution at pH 5.5. The protein concentration was measured at 280 nm using an ultraviolet spectrophotometer and then adjusted to 2.5 mg/ml.
  • mPEG-ButyrALD 40 kDa, Nektar When coupled with a 40 kDa specific modified protein N-terminal polyethylene glycol (mPEG-ButyrALD 40 kDa, Nektar), take 20 ml of the above protein solution (containing 40 mg of protein) and add 160 mg of 40 gDa polyethylene glycol solid.
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID N0.2 and SEQ ID N0.6.
  • DETAILED DESCRIPTION OF THE INVENTION A 40 kDa polyethylene glycol endostatin conjugate was purified using an SP column (Amersham). The pH of the mixture after the reaction was adjusted to 6. The column was equilibrated with an equilibration buffer containing 10 mM phosphate, pH 6.0 and loaded. After loading, it was eluted with a gradient of elution buffer containing 10 mM phosphate, 1 M sodium chloride, pH 6.0. Unreacted polyethylene glycol appeared in the process of penetration and rinsing due to the little charge.
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID N0.2 and SEQ ID N0.6.
  • the half-life of polyethylene glycol modified and unmodified recombinant human endostatin in mice was measured to examine the long-term benefits of polyethylene glycol modification.
  • Six healthy Kunming mice (Wutton's Experimental Animal Center) of about 25 g body weight were selected and divided into two groups, three in each group, and the unmodified recombinant human endostatin was injected into the tail vein (Beijing Progis).
  • the half-life of recombinant human endostatin with the sequence of SEQ ID N0.6 increased from 7.5 hours to an average of 16 hours after modification. This indicates that coupling high molecular weight polyethylene glycol can effectively increase recombinant human blood vessels.
  • Example 6 The activity of the coupling of 20 kDa polyethylene glycol with recombinant human endostatin N-terminus inhibits vascular endothelial cell migration
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID N0.2 and SEQ ID N0.6.
  • a specific embodiment is to culture human vascular endothelial cells (HMEC) in a logarithmic growth phase in DMEM medium (Hyclone) containing 10% serum. The cells were then starved for 12 hours. After the cells were digested with trypsin (Sigma), they were added to a dish for cell migration, and the number of cells per well was 10,000. The migration conditions were DMEM medium, 1% serum, 10 ng/ml.
  • bFGF (Sigma) and double antibody (streptomycin and ampicillin 10 g/ml, respectively) 0
  • endostatin and modified endostatin were added at a concentration of 30 g/ml.
  • the small dish was taken out, the cells were fixed with 1% glutaraldehyde, the unmigrated cells in the upper layer of the membrane were scraped off, and the lower cells were stained with hematoxylin.
  • the microscope Olympus
  • take the number of cells in the same size field of view Three different fields of view were selected from the same dish, and the inhibition rate was finally calculated.
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID NO. 2 and SEQ ID NO.
  • the in vivo inhibitory activity of 20 kDa polyethylene glycol modified endostatin on mouse S180 tumor was observed.
  • the experimental materials were selected from Kunming mice of 20 g body weight, and the S180 sarcoma cells (Chinese cell bank QK10952) were respectively subsided, and the number of cells per access was 2 X 10 6 .
  • the experimental results showed that: the positive control group had a tumor inhibition rate of 34%, and the administration group had a tumor inhibition rate every three days and every three days. For: 47%, 53%, 40% For recombinant human endostatin with the N-terminal additional amino acid of the sequence of SEQ ID NO.
  • the results of the experiment showed that: the positive control group had a tumor inhibition rate of 40%, and the administration group had daily
  • the tumor inhibition rate per three days for every two days was: 45%, 57%, 50% indicating the modified recombinant person Endostatin has antitumor activity in vivo, and its pharmacological effect is better than unmodified recombinant human endostatin due to its slower metabolism in the body, and it can maintain excellent drug efficiency even in the case of prolonged administration interval.
  • Unmodified recombinant human endostatin The experimental results are shown in Figure 13.
  • Example 8 20 kDa polyethylene glycol modified ⁇ -amino group of the side chain of recombinant human endostatin lysine residue
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID NO. 2 and SEQ ID N0.6.
  • DETAILED DESCRIPTION OF THE INVENTION A recombinant human endostatin was dialyzed into 30 niM phosphate buffer pH 7.8. The protein concentration was measured with an ultraviolet spectrophotometer at a wavelength of 280 mn, and then the concentration was adjusted to 2 mg/ml. A 20 kDa modified lysine residue side chain ⁇ -amino polyethylene glycol (mPEG-SPA 20 kDa, Nektar) molecule was added such that the molar ratio of the polyethylene glycol molecule to the protein was 8:1, respectively.
  • mPEG-SPA 20 kDa, Nektar modified lysine residue side chain ⁇ -amino polyethylene glycol
  • Recombinant human endostatin can be modified faster. After 30 minutes, 80% of vascular endothelial inhibitors can be modified. As the reaction time increases, the yield of the modifier increases, but the reaction rate slows down after 1 hour.
  • the reaction products mainly include monomolecular polyethylene glycol modification and multi-molecular polyethylene glycol modified endostatin. At this time, the reaction solution can be directly used for purification on the upper column.
  • Example 9 20 kDa polyethylene glycol modified ⁇ -amino group of the side chain of recombinant human endostatin lysine residue and purified by molecular sieve
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID N0.2 and SEQ ID N0.6.
  • DETAILED DESCRIPTION OF THE INVENTION A 20 kDa polyethylene glycol endostatin conjugate is purified by molecular sieves. The reaction mixture was adjusted to pH 7.4. The column was equilibrated with equilibration buffer containing 20 mM phosphate, 100 mM sodium chloride, pH 7.4 and loaded. Different separation components can be collected based on UV spectrometry at 280 nm. The purification results of the modified product of endostatin having the sequence of SEQ ID N0.6 are shown in Fig. 14.
  • Example 10 20 kDa polyethylene glycol modified ⁇ -amino group of the side chain of recombinant human endostatin lysine residue and purified by ion column
  • the recombinant human endostatin of the present embodiment includes recombinant human endostatin having the sequences of SEQ ID N0.2 and SEQ ID N0.6.
  • DETAILED DESCRIPTION OF THE INVENTION A 20 kDa polyethylene glycol endostatin conjugate was purified using a CM column. The reaction mixture was adjusted to pH 6.5. The column was equilibrated with an equilibration buffer containing 10 mM phosphate, pH 6.5 and loaded.
  • the recombinant human endostatin of the present embodiment has the recombinant human endostatin of the sequence of SEQ ID N0.6.
  • the in vivo inhibitory activity of 20 kDa polyethylene glycol (mPEG-ButyrALD 20 kDa, Nektar) modified endostatin on mouse S180 tumors was observed under conditions of increased dosing time.
  • the experimental materials were selected from Kunming mice of 20 g body weight, and were subcutaneously connected to S180 sarcoma cells, and the number of cells per access was 2 X 10 6 .
  • mice were sacrificed and weighed. The tumor effect was evaluated by the tumor inhibition rate.
  • the calculation method was: tumor inhibition rate - (negative control group tumor weight - drug group tumor weight) / negative The tumor weight of the control group was 100%.
  • the experimental results showed that: the positive control group had a tumor inhibition of 34%, and the administration group had every 5 days, every 7 days, every 10 days, The tumor inhibition rates per 14 days were: 56.4%, 37.1%, 31.8%, 0%. It is indicated that the modified recombinant human endostatin at this dose still has a certain in vivo antitumor activity for a prolonged period of time to administration every 10 days.

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Description

一种治疗肿瘤的药物及其应用
技术领域
本发明涉及一种具有生物活性和代谢稳定性的含有血管内皮抑制素的 复合物和缓释制剂以及它的制备方法。本发明还提供含有这种血管内皮抑制 素复合物和缓释制剂的药物组合说物以及含有这种血管内皮抑制素复合物的 试剂盒。 本发明还提供使用本发明中的血管内皮抑制素复合物、 缓释制剂、 药物组合和试剂盒来预防、 诊断、 治疗肿瘤， 及制备抗肿瘤药物的方法。
书 背景技术
新生血管形成是指新的毛细血管在原有的血管上的生成。肿瘤的生长和 迁移依赖于新生血管的生成，将肿瘤中的微血管内皮细胞作为癌症治疗的靶 点为治疗肿瘤提供了一种治疗方式 (Folkman J. N Engl J Med 1971; 285: 1182-1186)。
血管内皮抑制素 (Endostatin)是胶原 X 羧基端的一段分子量大小为 20 ldDa的酶切产物。 1997年美国哈佛大学 Judah Folkmaii教授等人在血管内皮 瘤细胞的培养基中发现此蛋白， 它具有抑制血管内皮细胞增殖、 迁移， 和体 内血管生成的活性。进一步实验发现重组血管内皮抑制素可以抑制老鼠体内 多种肿瘤的生长、 转移， 甚至能完全治愈肿瘤， 而且不产生耐药性。 其发挥 活性的机理在于它通过抑制血管内皮细胞的生长，抑制了肿瘤组织附近的新 生血管的生成， 使得肿瘤组织得不到生长所必需的大量营养和氧气， 最后停 止生长或坏死 (Folkman J. et al. Cell 1997; 88:277-285; Folkman J. et al. Nature 1997; 390:404-407)。通过基因工程制备的重组人血管内皮抑制素可以作为肿 瘤治疗药物， 其临床试验表明它可以有效的抑制肿瘤生长。 在中国， 以非小 细胞肺癌为主要适应症的临床试验表明它对于肿瘤的治疗效果比较突出。
多肽和蛋白质类药物相比于化学药具有毒副作用小，不产生耐药性等优 点。 为了达到最高的体内活性， 提高生物利用度， 减少药物体内降解， 通常 蛋白药物采用静脉给药方式。但是对于分子量小的蛋白来说， 通常静脉给药 以后的体内半衰期很短。 除了体内的蛋白降解过程以外， 一个重要的方面是 小分子蛋白能够通过肾过滤的途径从体内很快的被消除。在血液中， 水力半 径超过白蛋白或是分子量大于约 66,000道尔顿（66 kDa)的蛋白通常可以稳 定的保留在循环系统中。但是小分子却会通过肾小球过滤被很快的从血液中 消除。所以， 为了维持小分子量蛋白在血液中的有效治疗浓度， 需要进行频 繁的注射或点滴。这种治疗方式虽然能达到治疗的目的， 但是却给病人带来 了严重的不便与痛苦， 提高了用药成本， 对某些药物长期的使用还有可能产 生一些副作用， 例如免疫反应。
作为蛋白药物的血管内皮抑制素同样存在半衰期短，体内清除率高的缺 点。 目前的临床用药手段主要为频繁的静脉注射或点滴， 通常为每天给药， 而且需要长期用药， 这使得病人的精神和经济负担都比较大。本发明的目的 就在于改善该蛋白的体内代谢特征，使其具有更高的稳定性和较长的体内半 衰期， 甚至具有更高疗效， 从而达到可以减少用药频率， 降低用药成本， 减 轻病人的经济负担的目的。
用高分子聚合物对蛋白进行修饰，它是改变和控制药物的动力学特性如 半衰期、 免疫学特征、 毒理特性的常用方法。 用来修饰蛋白的高分子聚合物 具有水溶性好、 生物相容性好、 无或弱免疫原性等特征。 其中聚乙二醇 (polyethylene glycol, PEG)是应用最为普遍的一种蛋白修饰分子。 聚乙二醇 分子具有两亲性， 既可以溶解于水， 又可以溶解于大多数的有机溶剂， 具有 无毒、无免疫原性、在水溶液中有高溶解性等性质， 是被包括美国 FDA、 中 国 SFDA在内的很多国家的药品管理机构批准的可用于药物制备的高分子。 将蛋白质与亲水性的高分子如聚乙二醇偶联， 可增加蛋白稳定性、减少非特 异性吸附和抗原性。 当偶联物达到一定分子量时， 可大大降低肾脏的排除效 率，是延长蛋白质药物体内半衰期的有效方法 (Frokjaer S. et al. Nat. Rev. Drug Discov. 2005 Apr 4(4): 298-306; Harris JM. et al. Nat. Rev. Drug Discov. 2003 Mar 2(3): 214-21)。最初的聚乙二醇修饰都是以氨基作为反应位点，主要包括 蛋白的 N端 α-氨基和赖氨酸残基侧链上的 ε-氨基。 这一类反应的产物是一 个蛋白分子与一个或多个 PEG分子偶联。 对于赖氨酸残基侧链 ε-氨基上的 修饰也往往因为反应位点不特异而产生修饰后的同分异构体。 目前， 针对蛋 白 Ν端 a-氨基和赖氨酸残基侧链 ε-氨基的等电点差异， 又新开发出了只针 对蛋白 Ν端修饰的聚乙二醇修饰试剂，使得修饰位点一致，修饰产物组成均 一。 另一种可用于聚乙二醇修饰的蛋白位点是半胱氨酸的巯基。 由于巯基在 蛋白中的数目通常少于氨基， 所以这类修饰的位点更加特异。利用基因工程 技术， 现在可以在蛋白任何部位引入半胱氨酸作为特异的修饰位点。但是此 类引入半胱氨酸作为修饰位点也是有一定的局限性， 因为对某些本身带有半 胱氨酸的蛋白可能会造成二硫键的错配或无法复性。另外蛋白的羧基也是一 种常用的修饰位点（Veronese FM et al. Drug Discov. Today. 2005 Nov l;10(21):1451-8.)o 聚乙二醇修饰技术已经成功地运用在许多蛋白类药物上， 比较成功的包括： PEG-天冬酰胺酶 （PEG- asparaginase)(Graham ML Adv. Drug Deliv. Rev. 55, 1293-1302 Avramis Vassilios I. et al. W09939732 and US6689762)、 PEG-腺酐脱氨酶 (PEG- adenosine deaminase)( Levy Y et al. J. Pediatr. 113, 312-317; Davis S et al. ClinExp. Immunol. 46: 649-652; Hershfield MS et al. N Engl J Med 316： 589-596)和 PEG-干扰素 （PEG-interferon a2a、 PEG-interferon a2b等）（Bailon P et al. C. Bioconjug. Chem. 12, 195-202, Wang YS et al. Adv. Drug Deliv. Rev. 54, 547-570， Meng Xiantai et al. WO2005077421, Van Vlasselaer Peter et al. WO2004076474, Ballon Pascal Sebastian et al. US2004030101, Karasiewicz Robert et al. EP0593868) 等等。
其他代表性的高分子修饰剂有葡聚糖、 聚蔗糖、 淀粉、 聚丙氨酸、 乙二 酸和丙二酸的共聚物、 羧甲基纤维素、 聚乙烯吡咯烷酮、 聚 1,3-乙二醇次甲 基醚、 乙烯和顺丁烯二酰肼的共聚物、 多聚唾液酸、 环糊精等。
将血液蛋白或其片段作为载体， 与药用蛋白偶联或融合表达， 从而起到 增加蛋白分子量的目的， 是另一种延长药用半衰期的方法。例如用免疫球蛋 白的 Fc片段和目的蛋白偶联以延长其半衰期，例如将此方法用在 Notch 1受 体蛋白 (Kitajewsky Jan et al. WO2005111072), 促红细胞生成素 (EPO) (Gillies T N2007/000204
Stephen D et al. WO2005063808)、 人生长激素（Kim Young Min et al. WO2005047337)等蛋白上。血浆白蛋白也是一种常用的偶联载体， 可以运用 在抗生素类， 消炎类， 抗氧化物等蛋白上 (Ezrin Alan M et al. WO0117568, Otagiri Masaki et al. EP1571212)0
对于某些血液载体蛋白除了在体外将其与药物蛋白偶联在一起外，还可 以先将蛋白药物在体外进行修饰，使之具有化学反应的活性或是对体内其他 分子具有较高的亲和力， 能够在进入体内以后和血液某些成分进行反应， 形 成半衰期较长的大分子或是复合物。一个实例是将此技术运用在抗病毒的小 肽分子 anti-RSV上，将其修饰上带有马来酰亚胺的活性基团，它可以和血液 蛋白或是细胞表面蛋白中的巯基反应（Bridon Dominique P et al. WO0069902)。 另一个实例是利用酰化反应， 在蛋白表面的氨基酸残基上引 入脂肪酸， 增加蛋白对体内白蛋白的亲和力， 使得蛋白进入血液后可以和白 蛋白形成较大的复合物， 从而达到延长半衰期的目的， 这一方法曾用于长效 胰岛素上 (Kurtzhals P et al. Biochem. J. (1995) 312, 725-731; Frokjaer S et al. Nat Rev Drug Discov. 2005 Apr;4(4):298-306)。
另一种延长蛋白体内半衰期的方法是将蛋白做成缓释剂型。将蛋白药物 放入一种药用载体中， 使得蛋白从载体中缓慢释放， 在体内维持一种稳定的 药物浓度。常用的方法有水凝胶、微胶囊、微球、微型渗透泵、脂质体等 (Peppas NA et al. Eur. J. Pharm. Biopharm. 50, 27-46 (2000)； Packhaeuser CB et al. Eur. J. Pharm. Biopharm. 58, 445-455 (2004)； Metselaar JM et al. Mini Rev. Med. Chem. 4， 319-329 (2002)) 0 脂质体是一种具有双层膜结构的空球形态的超显 微粒子。 该双层膜由两亲分子多为磷脂构成并有水性的内腔。将亲水性的蛋 白药物包裹在脂质体的内腔中，在体内缓慢释放，可以维持蛋白的血药浓度， 达到增加半衰期的作用。 一些实例包括用在神经生长因子 (Hou Xinpu et al. CN1616087)、 血红蛋白 (Farmer Martha C et al. US4911929)等。 发明概述
本发明提供一种修饰物与血管内皮抑制素形成的复合物，其中所述修饰 物能够延长血管内皮抑制素的体内半衰期。用于本发明的修饰物可与血管内 皮抑制素通过共价键连接。 这些修饰物可选自高分子聚合物、 蛋白质分子或 其片段、 肽链、 小分子或其他化学物质。 可用于本发明的高分子聚合物例如 为聚乙二醇， 所述聚乙二醇分子的平均分子量位于 1，000到 100,000道尔顿 之间， 优选 5,000到 40，000道尔顿之间。
根据本发明的一个实施方式，所述复合物的特征为一个血管内皮抑制素 分子和一个或多个聚乙二醇分子偶联。
在本发明的由聚乙二醇与血管内皮抑制素形成的复合物中，偶联的位点 是血管内皮抑制素的 N端 a-氨基、 赖氨酸残基侧链的 ε-氨基、 半胱氨酸残 基侧链的巯基、 天冬氨酸残基侧链的羧基、 谷氨酸残基侧链的羧基中的一种 或是他们的组合。 优选地， 所述偶联位点为血管内皮抑制素 Ν端的 a-氨基。
根据本发明的复合物的一个实施方式，血管内皮抑制素分子与一个或多 个聚乙二醇分子偶联， 偶联的位点是血管内皮抑制素 Ν端 α-氨基或野生型 人血管内皮抑制素 75、 95、 106、 117、 183位的赖氨酸残基侧链上的 ε-氨基 或其组合。
根据本发明的复合物的一个实施方式，血管内皮抑制素分子与一个或多 个聚乙二醇分子偶联，偶联方法为在血管内皮抑制素分子内部或其 Ν末端或 C末端附加半胱氨酸或是含有半胱氨酸的肽链， 使得偶联位点为附加的半胱 氨酸残基侧链上的巯基。
根据本发明的复合物的一个实施方式，血管内皮抑制素分子与一个或多 个聚乙二醇分子偶联， 偶联的位点是血管内皮抑制素天冬氨酸或谷氨酸残基 侧链上的羧基。
用于本发明的复合物的蛋白选自白蛋白、免疫球蛋白、甲状腺结合蛋白、 转甲状腺蛋白、 转铁蛋白、 纤维蛋白原或它们的片段。
根据本发明的一个实施方式，所述复合物是白蛋白偶联的血管内皮抑制 素， 其特征为一个血管内皮抑制素分子和一个或多个白蛋白偶联， 该偶联物 可以是通过化学修饰得到的， 或是通过融合表达得到的， 或是通过其他方法 得到的， 其中的白蛋白优选人血清白蛋白或其片段。 根据本发明的另一个实 施方式， 所述复合物是免疫球蛋白 Fc片段偶联的血管内皮抑制素， 其特征 为一个血管内皮抑制素分子和一个或多个免疫球蛋白 Fc片段偶联， 该偶联 物可以是通过化学修饰得到的， 或是通过融合表达得到的， 或是通过其他方 法得到的， 其中的 Fc片段优选人免疫球蛋白 IgG中的 Fc区域的片段。
本发明的复合物还包括小分子或是小肽或是其他化合物修饰的血管内 皮抑制素， 其特征是该复合物具有同体内其他分子或成分反应或结合的活 性， 使得该聚合物可以在体内和其他成分形成更大的复合物。所述具有反应 活性的基团可以和血液成分的氨基、羟基、 巯基反应形成共价键。这些具有 反应活性的基团例如是是马来酰亚胺， 它可以和血液成分， 例如白蛋白中的 巯基反应。 这样， 本发明的复合物将与血液某些成分， 例如白蛋白、 免疫球 蛋白有较强的亲和力， 能相互形成更大的复合物。
本发明的复合物还包括其他分子或是小肽修饰的血管内皮抑制素，例如 血管内皮抑制素被糖基化、磷酸化或酰基化的产物， 其中修饰的位点是原来 蛋白序列中存在的氨基酸残基， 或是通过突变产生的氨基酸残基。
本发明的修饰物与血管内皮抑制素形成的复合物还可以是修饰物与血 管内皮抑制素通过非共价键相互作用形成的复合物，其中的修饰物可以是蛋 白、 小分子或其他化学物质。
本发明的复合物可与生物相容性载体形成缓释制剂。所述缓释制剂的形 式可选自微胶囊、 水凝胶、 微球、 微型渗透泵或脂质体。
用于本发明的复合物或缓释制剂的血管内皮抑制素可来源于人、 鼠、或 其他哺乳动物。
根据本发明的一个具体实施方式，复合物或缓释制剂中含有的血管内皮 抑制素是野生型的人血管内皮抑制素，其天然形式具有 SEQ ID N0.1所示的 序列。本发明的复合物或缓释制剂中含有的血管内皮抑制素当由大肠肝菌表 达时， 其野生型的重组人血管内皮抑制素具有 SEQ ID N0.2的序列， 其中 N 末端的 Met在大肠肝菌表达时有时会被部分删除，因此相应的重组人血管内 皮抑制素具有 SEQ ID N0.1的序列。
在本发明的复合物或缓释制剂中，血管内皮抑制素可以是血管内皮抑制 素的具有活性的片段、 突变体、 衍生物、 异构体或是它们的组合。 优选地， 在本发明的复合物或缓释制剂中，血管内皮抑制素片段是具有 SEQ ID N0.3、 SEQ ID 0.4, 或 SEQ ID NO.5序列的肽链。 在本发明的一个实施方式中， 所述复合物或缓释制剂中的血管内皮抑制素衍生物是在野生型血管内皮抑 制素的 N端或 C端附加一段长度为 1-15个氨基酸的肽链形成的。 例如， 在 本发明的复合物或缓释制剂中，血管内皮抑制素衍生物是在野生型人血管内 皮抑制素的 N 端附加一段 9 个氨基酸组成的含有 His-tag 的肽链 MGGSHHHHH, 该衍生物具有 SEQ ID N0.6的序列， 当由大肠杆菌表达时 其 N末端的 Met有时会被部分删除， 因此相应的衍生物具有 SEQ ID NO.7 的序列。
本发明还提供药物组合物，其包含本发明的复合物或缓释制剂和药学上 可接受的载体。 本发明还包括试剂盒， 其含有本发明的复合物、 组合物或缓 释制剂和使用说明。
本发明还提供制备权利要求上述复合物的方法，其特征是用活化的聚乙 二醇与血管内皮抑制素混合， 在适当的溶液、 温度、 pH、 摩尔比下反应。 优 选地， 该方法中所用的 pH为 pH 3-10。在上述方法中， 可将偶联产物例如用 阳离子柱纯化或者用分子筛纯化。
本发明也包括上述复合物、 缓释制剂、 药物组合物或试剂盒在预防、 诊 断或治疗肿瘤中的用途， 以及其在预防、 诊断或治疗其他疾病中的用途， 所 述疾病的特征为新生血管异常生成而导致人的组织或器官病变。
本发明还提供延长血管内皮抑制素半衰期的方法，所述方法包含将能够 延长血管内皮抑制素的体内半衰期的修饰物与血管内皮抑制素形成复合物 的步骤或者包含将血管内皮抑制素或含有血管内皮抑制素的复合物与生物 相容的物质形成缓释制剂的步骤。
以下结合附图进一步阐述本发明。应当理解的是， 无论是前面的一般性 描述还是随后的详细描述以及具体的实施例， 均仅用于示例和解释本发明， 而无意于对要求保护的发明进行限制。 附图说明- 图 1 : 显示野生型人血管内皮抑制素的氨基酸序列 SEQ ID N0.1。
图 2: 显示一种大肠杆菌表达的野生型重组人血管内皮抑制素的氨基酸 序列 SEQ ID N0.2。这里 N末端的 Met在表达后有时会被部分删除。若此情 况发生， N末端的 Met删除后的重组人血管内皮抑制素将具有 SEQ ID NO.l 的序列。
图 3: 显示一种具有活性的血管内皮抑制素片段的序列 SEQ ID N0.3。 图 4: 显示一种具有活性的血管内皮抑制素片段的序列 SEQ ID N0.4。 图 5: 显示一种具有活性的血管内皮抑制素片段的序列 SEQ ID N0.5。 图 6:显示一种具有活性的 N端附加氨基酸的重组人血管内皮抑制素的 氨基酸序列 SEQ ID NO. 6(图 6A)。 这里 N末端的 Met在表达后有时会被部 分删除。若此情况发生， N末端的 Met删除后的重组人血管内皮抑制素将具 有 SEQ ID NO.7的序列（图 6B；)。
图 7: 20 kDa聚乙二醇与具有 SEQ ID N0.2序列的重组人血管内皮抑制 素的 N端的偶联。 反应前后溶液用 SDS-PAGE检测， 泳道 1 : 20 kDa聚乙 二醇修饰的血管内皮抑制素； 泳道 2: 未修饰的血管内皮抑制素； 泳道 3: 蛋白分子量标准。
图 8: 20 kDa聚乙二醇与具有 SEQ ID N0.6序列的重组人血管内皮抑制 素的 N端的偶联。修饰前后溶液用 SDS-PAGE检测， 泳道 1 : 未修饰血管内 皮抑制素； 泳道 2: 20 kDa聚乙二醇修饰的血管内皮抑制素； 泳道 3: 蛋白 分子量标准。
图 9: 20 kDa聚乙二醇修饰具有 SEQ ID NO.2序列的重组人血管内皮抑 制素的 N端后用阳离子柱 SP纯化。 使用阳离子柱 SP吸附蛋白， 再用氯化 钠梯度溶液洗脱， 最后用还原 SDS-PAGE检测纯化效果。 泳道 1 : 纯化前； 泳道 2: 上样穿透的溶液； 泳道 3~10： 分布收集的洗脱液。
图 10: 20 kDa聚乙二醇修饰具有 SEQ ID N0.6序列的重组人血管内皮 抑制素的 N端后用阳离子柱 SP纯化。 使用阳离子柱 SP吸附蛋白， 再用氯 化钠梯度溶液洗脱，最后用还原 SDS-PAGE检测纯化效果。泳道 1 :纯化前; 泳道 2: 上样穿透的溶液； 泳道 3〜8： 分布收集的洗脱液。
图 11: 40 kDa聚乙二醇修饰具有 SEQ ID N0.2序列的重组人血管内皮 抑制素的 N端后用阳离子柱 SP纯化。 使用阳离子柱 SP吸附蛋白， 再用氯 化钠梯度溶液洗脱，最后用还原 SDS-PAGE检测纯化效果。泳道 1:纯化前; 泳道 2: 上样穿透的溶液； 泳道 3〜10： 分布收集的洗脱液。
图 12: 20 kDa聚乙二醇与重组人血管内皮抑制素 N端偶联的产物抑制 血管内皮细胞迁移的活性。 SEQ2: 具有 SEQIDN0.2的血管内皮抑制素； PEG-SEQ2: 20kDa聚乙二醇修饰的具有SEQIDNO.2的血管内皮抑制素； SEQ6: 具有 SEQIDNO.6的血管内皮抑制素； PEG-SEQ6: 20 kDa聚乙二 醇修饰的具有 SEQIDN0.6的血管内皮抑制素。
图 13: 20 kDa聚乙二醇与重组人血管内皮抑制素 N端偶联的产物抑制 小鼠 S180肉瘤的活性。 SEQ2: 具有 SEQIDN0.2的血管内皮抑制素每天 给药； PEG-SEQ2-1: 20 kDa聚乙二醇修饰的具有 SEQ ID N0.2序列的血管 内皮抑制素每天给药； PEG-SEQ 2-2: 20 kDa聚乙二醇修饰的具有 SEQ ID N0.2序列的血管内皮抑制素每两天给药； PEG-SEQ 2-3： 20 kDa聚乙二醇 修饰的具有 SEQIDNO.2序列的血管内皮抑制素每三天给药； SEQ6: 具有 SEQIDN0.6的血管内皮抑制素每天给药； PEG-SEQ6-1: 20kDa聚乙二醇 修饰的具有 SEQ ID N0.6序列的血管内皮抑制素每天给药； PEG-SEQ 6-2： 20 kDa聚乙二醇修饰的具有 SEQ ID N0.6序列的血管内皮抑制素每两天给 药； PEG-SEQ6-3: 20 kDa聚乙二醇修饰的具有 SEQ ID N0.6序列的血管内 皮抑制素每三天给药。
图 14: 20 kDa聚乙二醇修饰具有 SEQ ID N0.6序列的重组人血管内皮 抑制素赖氨酸残基侧链的 ε-氨基后用分子筛纯化。泳道 1:上样；泳道 2〜10： 分布收集的洗脱液。
图 15: 20 kDa聚乙二醇修饰具有 SEQ ID N0.6序列的重组人血管内皮 抑制素赖氨酸残基侧链的 ε-氨基后用阳离子柱 CM纯化。 泳道 1: 上样； 泳 道 2: 穿透； 泳道 3〜8： 分布收集的洗脱液。 发明详述
本发明提供一种具有较长半衰期的血管内皮抑制素产品，其在保持抑制 血管生成的活性的同时， 比未修饰的血管内皮抑制素在代谢上具有更高的稳 定性和更长的体内半衰期。本发明中的血管内皮抑制素优选人血管内皮抑制 素。
本发明的一个具体实施方案涉及一种修饰物与血管内皮抑制素形成的 复合物。 术语 "复合物"在本发明中是指修饰的血管内皮抑制素。 血管内皮 抑制素与修饰物通过共价键相连，或是通过非共价键相互作用形成稳定的复 合物。 因此， 术语 "修饰"既可以是共价键方式的连接， 也可以是非共价键 方式的连接。 修饰物可以是通过化学偶联的方法连接到血管内皮抑制素上 的， 也可以是通过融合表达的方式和血管内皮抑制素连接到一起的。优选的 方案中这种修饰是位点特异性的， 不会影响血管内皮抑制素的生物活性， 例 如与受体的结合。该修饰产物具有抗新生血管生成的活性但相比于未修饰的 血管内皮抑制素在代谢上更稳定， 具有更长的血液半衰期， 可以作为抗新生 血管生成或抗肿瘤的药物。 用于修饰的分子可以是一种或多种生物相容， 能 增加血管内皮抑制素的血液半衰期的高分子聚合物、 蛋白质分子或其片段、 肽链、 或者是其他化学物质。
这里的高分子聚合物，可以有它自己的生物活性，也可以没有生物活性。 合适的聚合物包括， 但不仅限于聚烯醇类化合物、 聚醚类化合物、 聚乙烯吡 咯烷酮、 聚氨基酸、 二乙烯基醚与马来酸酐的共聚物、 N-(2-羟丙基-甲基丙 烯酰胺 (N- (2-Hydroxypropyl)-methacrylamide) 多聚糖类、 聚氧乙基多醇 (polyoxyethylated polyol), 肝素或肝素片段、 聚烃基乙二醇及其衍生物、 聚 烃基乙二醇及其衍生物的共聚物、聚乙烯基乙醚、聚羟乙基天冬酰胺 (a，P-Poly ((2-hydroxyethyl)-DL-aspartamide)) 聚羧酸脂 (polycarboxylates)、氧化乙烯和 甲醛的共聚物 (poly oxyethylene-oxymethylenes) . 聚丙烯酰吗啉 (polyacr loyl morpholines), 氨基化合物与氧化烯烃的共聚物、 聚透明质酸、 聚环氧化物 (polyoxiranes), 乙二酸与丙二酸的共聚物、 聚 1,3-乙二醇次甲基醚、 乙烯 /顺 丁烯二酰肼共聚物、 多聚唾液酸、 环糊精等。 上述的聚烯醇类化合物包括， 但不仅限于聚乙二醇（包括单甲基聚乙二 醇、 单羟基聚乙二醇等）、 聚乙烯醇、 聚丙烯醇、 聚丁烯醇等以及它们的衍 生物。 在一个优选的方案中， 修饰物是聚乙二醇。 一个更优选的方案中， 聚 乙二醇选用单甲基聚乙二醇。
上述的聚醚类化合物包括，但不仅限于聚垸撑二醇 (HO((CH2)xO)nH)、聚 丙二醇、 聚氧化乙烯 (HO((CH2)20)nH)、 聚乙烯醇 ((CH2CHOH)n)等以及它们 的衍生物。
上述的率氨基酸包括， 但不仅限于同种氨基酸的聚合物， 两种或两种以 上的氨基酸的共聚物， 例如： 聚丙氨酸。
上述的多聚糖类包括， 但不仅限于葡聚糖及葡聚糖衍生物如硫酸葡聚 糖、 纤维素及纤维素的衍生物 （包括甲基纤维素和羧甲基纤维素）、 淀粉 及淀粉衍生物、 聚蔗糖等。
这里适合用作修饰载体的蛋白分子优选天然存在的蛋白或是其片段，包 括但不仅限于甲状腺结合蛋白 （thyroxine-binding protein), 转甲状腺蛋白 (transthyretin), 转铁蛋白 （transferring 纤维蛋白原、 免疫球蛋白、 白蛋白 等以及它们的片段。 上述的载体蛋白优选为人源的。 上述蛋白的片段是指任 何比该蛋白小但是保持了载体功能的部分。
血管内皮抑制素与上述修饰物直接或间接共价相连，直接连接指的是血 管内皮抑制素的某一个氨基酸和载体蛋白的某一氨基酸直接相连，可以通过 肽键或二硫键。间接相连指的血管内皮抑制素和载体蛋白通过一定的化学基 团或几个基团的组合连接。
对于血管内皮抑制素曾有文章公开了使用聚乙二醇修饰的方案。牟国营 等 （2005) 和杨胜林等 （2005) 提到采用平均分子量为 6或 10 kDa的 PEG 分子修饰血管内皮抑制素，其修饰物是偶联在血管内皮抑制素赖氨酸残基侧 链 ε-氨基上的， 并且为多个位点修饰的混合产物。 在使用中虽然可以通过分 子筛或是离子交换柱纯化得到某一分子量范围的产物但是产物也存在修饰 位点不均一， 蛋白构象不均一的缺点， 这是由这类修饰试剂的反应特性所决 定的。而且这种对蛋白的多点修饰，会使得某些内部氨基酸的构像发生变化， 造成蛋白活性的降低。对于生物制品药物来说，产物的均一性对于药物毒性， 免疫原型的降低都是十分重要的， 而且在质量控制上， 混合物很难保证药物 每批生产质量均一， 药效稳定。 因此保证药物成分固定， 构像均一， 活性稳 定， 是在设计和生产药物是需要重要考虑和亟待解决的问题。
本发明的一个具体实施方案涉及聚乙二醇修饰的血管内皮抑制素，其特 征为一个血管内皮抑制素分子和一个或多个聚乙二醇分子偶联。偶联的位点 是蛋白的 N端 α-氨基、 赖氨酸残基侧链的 ε-氨基、 半胱氨酸的巯基、 天冬 氨酸残基侧链的羧基、谷氨酸残基侧链的羧基中的一种或是他们的组合。所 有用于偶联的聚乙二醇分子可以是线性的， 也可以是分叉的， 分子量位于 1,000到 100,000道尔顿之间，优选 5,000到 40,000道尔顿。本发明中所述的 聚乙二醇的分子量是指大约的平均的分子量。
聚乙二醇修饰虽然改变药物代谢特性的优势，但是通常这种修饰也会使 得大分子药物的生物活性降低，主要原因是在于不同的修饰位点对药物本身 的结构的改变和对活性部位的屏蔽 [Veronese F.， et al. Drug Discovery Today, 10,21,2005]。 例如用不同的 PEG化修饰试剂对干扰素 β -lb进行修饰后， 不 同修饰手段均导致修饰后的干扰素的活性的降低，最优的方案也仅仅达到原 蛋白活性的 71%，有些修饰方式甚至使得蛋白几乎没有活性（Filpula D., et al. Bioconjugate Chem. 2006, 17, 618-630 )。 所以虽然修饰改变了药代学特征， 但是对药物本身的活性保持却不利， 所以通常在选择修饰方法和修饰试剂 时， 都需要综合考虑这两方面的因素， 使得药物在体内的综合效果最好。 在 本发明的一个优选方案中， 使用蛋白 N 端特异的聚乙二醇修饰试剂 (mPEG-ButyrALD, Nektar)来修饰重组人血管内皮抑制素。 其产物为一个聚 乙二醇与一个重组人血管内皮抑制素相连， 连接位点为蛋白的 N端 a-氨基。 在文献（Filpula D., et al. Bioconjugate Chem. 2006， 17, 618-630 ) 中， 使用相 同的该试剂， 得到的 N端修饰的聚乙二醇干扰素 β -lb的体外活性只有原始 蛋白活性的 70%左右。 在本发明中， 我们惊奇的发现 N端修饰的重组人血 管内皮抑制素其产物在体外细胞学实验中，对内皮细胞的迁移的抑制率是未 修饰蛋白的 2倍或以上， 既蛋白的生物活性有了很大的提高， 这种体外活性 明显增加的现象尚未见报道。而牟国营等（2005 )得到的多修饰的内皮抑制 素的体外活性明显的随着修饰反应的进行而逐渐降低。这说明利用本发明涉 及到反应和纯化方法所的得到的产物比相关报道有更好的体外活性。而另一 方面， 通过对人血管内皮抑制素进行结构分析人们发现， 在人血管内皮抑制 素的 N末端有一个锌离子的结合位点，由野生型人血管内皮抑制素中的第 1， 3， 11， 76位的氨基酸组成， 据文献 (Ding, Y. H, Proc. Natl. Acad. Sci. U.S.A. 95, 10443-10448； Hohenester, E.， EMBO J. 17, 1656-1664； Boehm, T" Biochem. Biophys. Res. Commun. 252, 190-194； Ricard-Blum, S.， J. Biol. Chem. 279, 2927-2936； Tjin Tham Sjin, R. Μ·， Cancer Res. 65(9): 3656-63。）报道， 该结 合位点对人血管内皮抑制素的稳定性和活性至关重要，甚至是人血管内皮抑 制素发挥活性时与受体相结合的部位。 由于 PEG是一个分子量很大的分子， 空间位阻很大， 通常认为对蛋白的化学修饰不宜在蛋白的活性位点进行， 因 为这样对蛋白结构的破坏作用很大， 进而对蛋白的活性会有很大的影响。但 是本发明的这个产物不但没有活性的丧失，反而出乎意料的显示出更高的活 性， 这在现有的知识背景下很难预测。
本发明的一个具体实施方案涉及血管内皮抑制素和蛋白或肽链的偶联， 其特征为血管内皮抑制素与一个或多个蛋白或肽链直接或间接相连， 当与多 个载体蛋白相连时， 所选的载体蛋白之间可以相同也可以是不同的。其中的 载体蛋白优选天然存在的蛋白或是其片段。一个优选的方案中用于修饰的载 体蛋白为人血清白蛋白或其片段。在另一个优选的方案中的载体蛋白为人免 疫球蛋白 IgG的 Fc片段。
本发明的另一个具体实施方案中的修饰物为小分子或是小肽或是其他 化合物，其特征为能使含有内皮抑制素的复合物具有和血液某些成分反应的 能力或是和血液某些成分有较强的亲和力。一种具体化的产物是它具有一个 反应活性的基团， 可以和血液成分的氨基、 羟基、巯基反应形成共价键。 另 一个具体化产物的活性基团是一个马来酰亚胺， 它可以和血液蛋白， 例如白 蛋白中的巯基反应。另一个具体化产物是具有和血液某些成分，例如白蛋白、 免疫球蛋白等有较强的亲和力， 可以与之形成一种更大的复合体。 本发明的另一个具体实施方案中的复合物，是其他小分子或是小肽修饰 的血管内皮抑制素， 可以是血管内皮抑制素被糖基化、磷酸化、酰基化等后 的产物。其中修饰的位点可以是原来蛋白序列中存在的氨基酸， 也可以是通 过突变产生的氨基酸残基。该复合物虽然没有很大的分子量， 但是因为修饰 改变了血管内皮抑制素的性质从而延长了其半衰期。
本发明的另一个具体的实施方案涉及一种含有内皮抑制素的复合物，它 是由血管内皮抑制素和其他分子通过非共价键形成的具有特定组成的复合 物分子， 其中的修饰物可以是蛋白， 小分子等。 该复合物具有抑制血管生成 的活性， 并且在体内有比血管内皮抑制素更长的半衰期。
本发明的另一个具体的实施方案涉及一种含有血管内皮抑制素的缓释 制剂， 它是由血管内皮抑制素或上述的任一复合物和生物相容的物质形成 的。该组合物中的血管内皮抑制素仍然具有生物活性， 但是却可以依靠该载 体改变药物代谢特征达到延长体内保留时间的目的。所述的缓释制剂为但不 仅限于微胶囊、 水凝胶、 微球、 微型渗透泵、 脂质体等。
本发明还提供一种上述含有血管内皮抑制素的复合物或缓释制剂与药 学上可接受的载体形成的药物组合物。本发明还提供一种上述含有血管内皮 抑制素的复合物或缓释制剂和使用说明组成的试剂盒。
本文使用的药学上可接受的载体包括对于以所用剂量和浓度与其接触 的细胞或哺乳动物无毒的药用上可接受的载体、 赋形剂、 或稳定剂。 通常生 理学上可接受的载体是含水的 pH缓冲溶液。 生理学上可接受的载体的例子 包括诸如磷酸盐、 柠檬酸盐、和其他有机酸在内的缓冲液； 包括抗坏血酸在 内的抗氧化剂 ,· 低分子量 (不超过 10个残基)多肽； 诸如血清白蛋白、 明胶、 或免疫球蛋白等蛋白质；诸如聚乙烯吡咯烷酮等亲水性多聚体；诸如甘氨酸、 谷氨酰胺、天冬酰胺、精氨酸或赖氨酸等氨基酸；单糖、二糖和包括葡萄糖、 甘露糖、或糊精等其他糖类； 诸如 EDTA等螯合剂； 诸如甘露醇或山梨醇等 糖醇； 诸如钠等成盐反离子； 和 /或诸如 TWEEN®、 聚乙二醇 (PEG)、 和 PLU OMCS®等非离子表面活性剂。赋形剂优选是无菌的且一般不含不良物 质。 这些组合物可通过常规的熟知灭菌技术进行灭菌。 本发明还涉及到制备上述血管内皮抑制素产品的方法。特别的， 制备聚 乙二醇修饰的血管内皮抑制素的方法， 即将活化的聚乙二醇与血管内皮抑制 素混合， 在适当的溶液、温度、 pH、摩尔比下反应， 并用阳离子柱或是分子 筛纯化偶联产物。 其中反应的 pH优选 pH 3-10之间。
本发明还提供一种上述含有血管内皮抑制素的复合物、缓释制剂、药物 组合物或试剂盒在预防、诊断或治疗肿瘤中的用途。本方法适合的癌症包括 但不仅限于肺癌、 神经内分泌瘤、 结肠癌、 骨癌、 肝癌、 胃癌、 胰腺癌、 口 腔癌、 乳腺癌、 前列腺癌、 淋巴癌、 食道癌、 口腔癌、 鼻咽癌、 宫颈癌、 肉 瘤、 肾癌、 胆癌、 恶性黑色素肿瘤等。
本发明还提供一种上述含有血管内皮抑制素的复合物、药物组合物或缓 释制剂在制备抗肿瘤药物中的用途。
本发明还提供一种上述含有血管内皮抑制素的复合物、缓释制剂、药物 组合物或试剂盒在预防、诊断或治疗其他疾病和制备治疗这些疾病的药物中 的用途，所述疾病的特征为但不限于新生血管异常生成而导致人的组织或器 官病变。
在上述的治疗肿瘤或其他疾病的用途中， 其中的给药方式选自静脉注 射、 静脉滴注、 皮下注射、 肌肉注射、 腹腔注射、 透皮吸收、 皮下包埋、 肝 动脉注射、 口服、 鼻粘膜给药、 口腔粘膜给药、 眼部给药、 直肠给药、 阴道 给药或其他临床给药方式。
本发明还提供一种延长血管内皮抑制素半衰期的方法，所述方法包含将 修饰物与血管内皮抑制素形成复合物的步骤和制备含有血管内皮抑制素的 缓释制剂的步骤。
实验表明，本发明中的一个产品聚乙二醇与重组人血管内皮抑制素 N端 偶联的复合物， 具有抑制血管内皮细胞迁移和小鼠肿瘤生长的活性， 与未修 饰的人血管内皮抑制素相比， 活性明显提高， 并且体内药代学研究表明修饰 产物能有效的减缓药物在体内的代谢， 延长体内半衰期。
本发明中提到的血管内皮抑制素，除了特别说明以外，其来源可以是人， 鼠， 或其他哺乳动物， 其中的优选方案是人血管内皮抑制素。 发明中提到的 血管内皮抑制素， 除特殊说明以外， 包括野生型的血管内皮抑制素， 即体内 自然存在的形式， 或是其具有活性的突变体、 片段、 异构体、 衍生物等或是 它们的组合。血管内皮抑制素的来源可以是但不仅限于动物细胞表达的， 也 可以是从酵母或大肠肝菌中发酵纯化而来的。其中来源于动物细胞表达的野 生型的人血管内皮抑制素的氨基酸序列如 SEQ ID NO.l所示；来源于酵母表 达的重组人血管抑制素具有 SEQ ID N0.1的序列， 或是在此序列的 N端有 10个氨基酸以内的增加或缺失;来源于大肠肝菌表达的重组人血管内皮抑制 素具有 SEQ ID N0.2的序列， 但其 N末端的 Met在表达后有时会被部分删 除。 若此情况发生， N末端的 Met删除后的重组人血管内皮抑制素将具有 SEQ ID NO.l的序列。
血管内皮抑制素的突变体指的是通过氨基酸的取代、缺失、增加而得到 的血管内皮抑制素分子。 血管内皮抑制素的片段是指序列属于 SEQ ID NO.l 或 SEQ ID N0.2的任何更小的部分，可以是但不限于通过酶切得到的， 或是 基因工程表达的，或是通过多肽合成的方法得到的。例如具有 SEQ ID N0.3、 SEQ ID N0.4、 SEQ ID NO.5序列的片段曾被报道具有抑制小鼠实体瘤的活 性 （Cattaneo MG et al. Experimental Cell Research 283 (2003) 230-236； Tjin Tham Sjin RM et al. Cancer Research (2005) 65(9) 3656-3663 )。 血管内皮抑制 素的异构体是指具有和野生型血管内皮抑制素相同的氨基酸序列或分子式， 但是却有不一样的构象，包括蛋白二级或三级结构的不同或是局部氨基酸旋 光性的改变， 异构体的来源可以是天然存在的突变或是通过人为设计而得到 的。血管内皮抑制素的衍生物是指在野生型血管内皮抑制素的基础上进行修 饰的产物， 其中的修饰是指在蛋白上共价连接一个或多个其他小分子， 例如 磷酸分子、 糖分子等， 或是 20个氨基酸以内的小肽链， 连接位点可以是蛋 白内的任意一个氨基酸。在一个优选的方案中， 可以是 N端附加一段 9个氨 基酸组成的含有 His-tag的肽链 MGGSHHHHH的人血管内皮抑制素， 其具 有 SEQ ID N0.6的序列 （Luo Yongzhang et al. US2004110671), 当由大肠杆 龠表达时其 N末端的 Met在表达后有时会被部分删除。 若此情况发生， N 末端的 Met删除后的重组人血管内皮抑制素衍生物将具有 SEQ ID N0.7的序 列。 血管内皮抑制素具有活性的突变体、 片段、 异构体、 衍生物的组合是指 同时具有两种或两种以上上述特征的变化的产物，例如但不仅限于片段的突 变体、 突变体的修饰产物等。
实施例 1、 20 kDa聚乙二醇与重组人血管内皮抑制素 N端的偶联
本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID NO.2和 SEQ ID N0.6序列的重组人血管内皮抑制素。具体为将重组人血管内皮抑制素 (北 京普罗吉生物科技发展有限公司） 透析到 pH 5.5的 30 mM醋酸钠溶液中。 用紫外分光光度计 （Agilent Technologies) 在 280 nm波长下测量蛋白浓度， 然后将浓度调节到 2 mg/ml。 与 20 kDa的特异修饰蛋白 N端的聚乙二醇 (mPEG-ButyrALD 20 kDa, Nektar)偶联时， 取 20 ml上述蛋白溶液 （含蛋白 40 mg), 加入 20 kDa聚乙二醇固体 80 mg， 并室温搅拌， 直到完全溶解， 使 得聚乙二醇与血管内皮抑制素的摩尔比为 2:1。 加入还原剂 CH3BNNa (Sigma) , 浓度为 20 mM, 并最后调节溶液的 pH值为 5。 室温静置 10小 时以后大于 80% 的血管内皮抑制素被单一聚乙二醇修饰， 即一个聚乙二醇 与一个血管内皮抑制素分子偶联，并且偶联的位点是血管内皮抑制素 N端的 α-氨基， 少量血管内皮抑制素会被非特异性的多位点修饰。 这时反应液可直 接用于上柱纯化。 反应前后溶液电泳结果如附图 7和 8所示。 实施例 2、 20 kDa聚乙二醇修饰重组人血管内皮抑制素 N端后用阳离子柱 SP纯化
本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID N0.2和 SEQ ID N0.6序列的重组人血管内皮抑制素。具体实施方式为将 20 kDa聚乙二醇 修饰的血管内皮抑制素用 SP层析柱（Amersham)纯化。 将反应后的混合液 调节 pH为 6。层析柱用含有 10 mM磷酸盐， pH值为 6.0的平衡缓冲液平衡 后上样。 上样后再用含有 10 mM磷酸盐， 1M氯化钠， pH 6.0的洗脱缓冲 液梯度洗脱， 未反应的聚乙二醇由于带电荷极少， 所以在穿透和冲洗的过程 中出现， 洗脱出峰的顺序为多修饰的血管内皮抑制素、 单修饰的血管内皮抑 制素、未修饰的血管内皮抑制素。根据 280nm的紫外检测可以收集不同的分 离组分。 纯化结果如附图 9和 10所示。 实施例 3、 40 kDa聚乙二醇与重组人血管内皮抑制素 N端的偶联
本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID N0.2和 SEQ ID N0.6序列的重组人血管内皮抑制素。 具体实施方式为将重组人血管内皮 抑制素透析到 pH 5.5的 30 mM醋酸钠溶液中。用紫外分光光度计在 280 nm 波长下测量蛋白浓度， 然后将浓度调节到 2.5 mg/ml。 与 40 kDa的特异修饰 蛋白 N端的聚乙二醇 (mPEG-ButyrALD 40 kDa, Nektar)偶联时， 取 20 ml上 述蛋白溶液（含蛋白 40 mg)， 加入 40 kDa聚乙二醇固体 160 mg， 并室温搅 拌， 直到完全溶解， 使得聚乙二醇与血管内皮抑制素的摩尔比为 2:1。 加入 还原剂 C¾BNNa (Sigma) ,浓度为 20 mM， 并最后调节溶液的 pH值为 5。 室温静置 10小时以后大于 80% 的血管内皮抑制素被单一聚乙二醇修饰，即 一个聚乙二醇与一个血管内皮抑制素分子偶联，并且偶联的位点是蛋白 N端 的 a-氨基， 少量血管内皮抑制素会被非特异性的多位点修饰。这时反应液可 直接用于上柱纯化。 实施例 4、 40 kDa聚乙二醇修饰重组人血管内皮抑制素 N端后用阳离子柱 SP纯化
本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID N0.2和 SEQ ID N0.6序列的重组人血管内皮抑制素。具体实施方式为将 40 kDa聚乙二醇 血管内皮抑制素偶联物用 SP层析柱（Amersham)纯化。 将反应后的混合液 调节 pH为 6。层析柱用含有 10 mM磷酸盐， pH值为 6.0的平衡缓冲液平衡 后上样。 上样后再用含有 10 mM磷酸盐， 1M氯化钠， pH 6.0的洗脱缓冲 液梯度洗脱， 未反应的聚乙二醇由于带电荷极少， 所以在穿透和冲洗的过程 中出现， 洗脱出峰的顺序为多修饰的血管内皮抑制素、 单修饰的血管内皮抑 制素、未修饰的血管内皮抑制素。根据 280nm的紫外检测可以收集不同的分 离组分。 具有 SEQ ID N0.2序列的重组人血管内皮抑制素纯化结果如附图 11所示， 具有 SEQ ID N0.6序列的纯化结果与 SEQ ID N0.2序列的纯化结 果类似， 在此省略。 实施例 5、 20 kDa聚乙二醇与重组人血管内皮抑制素 N端偶联的产物在血液 中的长效效应
本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID N0.2 和 SEQ ID N0.6序列的重组人血管内皮抑制素。测量聚乙二醇修饰和未修饰的 重组人血管内皮抑制素在小鼠体内的半衰期来检验聚乙二醇修饰后的长效 效益。选用 6只 25g左右体重的健康昆明种小鼠（维通利华实验动物中心）， 分为两组， 每组 3只， 分别尾静脉注射未修饰的重组人血管内皮抑制素（北 京普罗吉生物科技发展有限公司）和 20 kDa聚乙二醇与重组人血管内皮抑 制素 N端偶联的产物， 剂量为 15mg/kg体重。 按照 2、 10、 30分钟、 1、 2、 4、 8、 16、 24、 36、 48、 72小时的时间间隔尾静脉取血。 收集血浆， 用夹心 法 ELISA分别测血管内皮抑制素和聚乙二醇修饰的血管内皮抑制素的浓度。 体内药代动力学显示 20 kD聚乙二醇修饰后，具有 SEQ ID N0.2序列的重组 人血管内皮抑制素在小鼠体内的半衰期从平均 4.8小时增加到平均 16小时。 具有 SEQ ID N0.6序列的重组人血管内皮抑制素经修饰以后在小鼠体内的 半衰期从平均 7.5小时增加到平均 16小时这表明偶联高分子量的聚乙二醇能 有效的增加重组人血管内皮抑制素的体内代谢的半衰期， 达到长效的目的。 实施例 6、 20 kDa聚乙二醇与重组人血管内皮抑制素 N端偶联的产物抑制血 管内皮细胞迀移的活性
本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID N0.2和 SEQ ID N0.6序列的重组人血管内皮抑制素。 具体实施方案为将人血管内皮细胞 (HMEC)在含 10%血清的 DMEM培养基（Hyclone)中培养至对数生长期。 然后饥饿细胞 12小时。 将细胞用胰酶（Sigma)消化以后， 加入测细胞迁移 的皿中， 每孔细胞数 10000个。迁移条件为 DMEM培养基、 1%血清、 10ng/ml bFGF (Sigma) 和双抗（链霉素和氨苄青霉素各自 10 g/ml, Sigma) 0 对于 加药组分别加入血管内皮抑制素和修饰后的血管内皮抑制素， 浓度为 30 g/ml。 37°C培养 8小时以后， 取出小皿， 用 1%戊二醛固定细胞， 刮掉膜上 层的未迁移细胞， 用苏木精对下层细胞染色。最后在显微镜（Olympus)下， 取相同大小视野数细胞数。同一块皿中选取三个不同视野，最后算出抑制率。 结果显示具有 SEQ ID NO.2序列的重组人血管内皮抑制素对细胞迁移的抑 制率为 42%， 修饰后对细胞迁移的抑制率达到 90%。 具有 SEQ ID N0.6序 列的重组人血管内皮抑制素对细胞迁移的抑制率为 45。/。，修饰后对细胞迀移 的抑制率达到 88%。以上结果表明经过聚乙二醇修饰后的血管内皮抑制素不 仅保持而且还大大增强了生物活性。 实验结果如图 12所示。 实施例 7、 20 kDa聚乙二醇与重组人血管内皮抑制素 N端偶联的产物在小鼠 肿瘤模型治疗中的活性
本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID NO.2 和 SEQ ID NO.6序列的重组人血管内皮抑制素。 实验观察了 20 kDa聚乙二醇 修饰的血管内皮抑制素对小鼠 S180肿瘤的体内抑制活性。 实验材料选用 20 g体重的昆明小鼠， 分别腋下接入 S180肉瘤细胞（中国细胞库 QK10952), 每只接入细胞数 2 X 106个。第二天随机分组， 每组 8只， 分别设定阴性对照 组（生理盐水）、 阳性对照组（血管内皮抑制素 1.5 mg/kg体重， 每天给药）、 三组给药组（聚乙二醇修饰的血管内皮抑制素 1.5 mg/kg体重， 分别为每天， 每两天， 每三天给药）。 分组后开始给药， 给药方式为尾静脉注射。 给药周 期为 10天，第 11天处死小鼠，称瘤重， 以抑瘤率来评价药效， 计算方法为： 抑瘤率 = (阴性对照组瘤重-给药组瘤重） /阴性对照组瘤重 X 100%。 对于具 有 SEQ ID N0.2序列的重组人血管内皮抑制素， 实验结果显示： 阳性对照组 的抑瘤率为 34%， 给药组每天， 每两天，每三天给药的抑瘤率分别为： 47%， 53%, 40% 对于具有 SEQ ID NO.6序列的 N端附加氨基酸的重组人血管内 皮抑制素， 实验结果显示： 阳性对照组的抑瘤率为 40%， 给药组每天， 每两 天， 每三天给药的抑瘤率分别为： 45%， 57%, 50% 表明修饰后的重组人 血管内皮抑制素具有体内的抑瘤活性， 而且由于其体内代谢速度减慢， 使得 药效优于未修饰的重组人血管内皮抑制素， 并且在延长给药间隔的情况下仍 能保持药效优于未修饰的重组人血管内皮抑制素。 实验结果如图 13所示。 实施例 8、 20 kDa聚乙二醇修饰重组人血管内皮抑制素赖氨酸残基侧链的 ε- 氨基
本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID NO.2和 SEQ ID N0.6序列的重组人血管内皮抑制素。 具体实施方式为将重组人血管内皮 抑制素透析到 pH 7.8的 30 niM磷酸缓冲液中。用紫外分光光度计在 280 mn 波长下测量蛋白浓度， 然后将浓度调节到 2 mg/ml。 加入 20kDa的修饰赖氨 酸残基侧链 ε-氨基的聚乙二醇 (mPEG-SPA 20 kDa, Nektar)分子， 使得聚乙二 醇分子与蛋白的摩尔比分别为 8: 1。室温放置。重组人血管内皮抑制素能被 较快的修饰， 30 分钟以后 80%的血管内皮抑制剂能被修饰， 随着反应时间 增长， 修饰物产量增多， 但反应速度在 1小时以后减慢。 反应产物主要有单 分子聚乙二醇修饰和多分子聚乙二醇修饰的血管内皮抑制素。这时反应液可 直接用于上柱纯化。 实施例 9、 20 kDa聚乙二醇修饰重组人血管内皮抑制素赖氨酸残基侧链的 ε- 氨基后用分子筛纯化
本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID N0.2和 SEQ ID N0.6序列的重组人血管内皮抑制素。具体实施方式为将 20 kDa聚乙二醇 血管内皮抑制素偶联物用分子筛纯化。将反应后的混合液调节 pH为 7.4。层 析柱用含有 20 mM磷酸盐， 100 mM氯化钠， pH值为 7.4的平衡缓冲液平 衡后上样。 根据 280nm的紫外捡测可以收集不同的分离组分。 具有 SEQ ID N0.6序列的血管内皮抑制素的修饰产物的纯化结果如附图 14所示。 实施例 10、20 kDa聚乙二醇修饰重组人血管内皮抑制素赖氨酸残基侧链的 ε- 氨基后用离子柱纯化 本实施例涉及的重组人血管内皮抑制素包括具有 SEQ ID N0.2和 SEQ ID N0.6序列的重组人血管内皮抑制素。具体实施方式为将 20 kDa聚乙二醇 血管内皮抑制素偶联物用 CM层析柱纯化。将反应后的混合液调节 pH为 6.5。 层析柱用含有 10 mM磷酸盐， pH值为 6.5的平衡缓冲液平衡后上样。 上样 后再用含有 10 mM磷酸盐， 1M氯化钠， pH 6.5的洗脱缓冲液梯度洗脱。 未反应的聚乙二醇由于带电荷极少， 所以在穿透和冲洗的过程中出现， 洗脱 出峰的顺序为多修饰的血管内皮抑制素， 单修饰的血管内皮抑制素， 未修饰 的血管内皮抑制素。根据 280nm的紫外检测可以收集不同的分离组分。具有 SEQ ID N0.6序列的血管内皮抑制素的修饰产物的纯化结果如附图 15所示。 实施例 11、 20 kDa聚乙二醇与重组人血管内皮抑制素 N端偶联的产物在小 鼠肿瘤模型治疗中的活性
本实施例涉及的重组人血管内皮抑制素具有 SEQ ID N0.6序列的重组 人血管内皮抑制素。实验观察了 20 kDa聚乙二醇（mPEG-ButyrALD 20 kDa, Nektar) 修饰的血管内皮抑制素增加给药间隔时间的条件下对小鼠 S180 肿 瘤的体内抑制活性。实验材料选用 20 g体重的昆明小鼠，分别腋下接入 S180 肉瘤细胞， 每只接入细胞数 2 X 106个。第二天随机分组， 每组 8只， 分别设 定阴性对照组 （生理盐水）、 阳性对照组 （血管内皮抑制素 1.5 mg/kg体重， 每 5天给药）、 四组给药组 （聚乙二醇修饰的血管内皮抑制素 1.5 mg/kg体 重， 分别为每 5天， 每 7天， 每 10天， 每 14天给药）。 接瘤后次日开始给 药， 给药方式为尾静脉注射。给药周期为 14天， 第 15天处死小鼠，称瘤重， 以抑瘤率来评价药效，计算方法为：抑瘤率- (阴性对照组瘤重-给药组瘤重） /阴性对照组瘤重 X 100%。对于具有 SEQ ID NO.6序列的 N端附加氨基酸的 重组人血管内皮抑制素， 实验结果显示： 阳性对照组的抑瘤 为 34%， 给药 组每 5天， 每 7天， 每 10天， 每 14天给药的抑瘤率分别为： 56.4%， 37.1%, 31.8%， 0%。 表明修饰后的重组人血管内皮抑制素在该剂量下延长时间到每 10天给药仍然具有一定的体内抑瘤活性。

Claims

权 利 要 求 书
1.一种修饰物与血管内皮抑制素形成的复合物，其中所述修饰物能够延 长血管内皮抑制素的体内半衰期。
2.权利要求 1的复合物，所述修饰物与血管内皮抑制素通过共价键连接。
3.权利要求 2的复合物， 所述修饰物选自高分子聚合物、 蛋白质分子或 其片段、 肽链、 小分子或其他化学物质。
4.权利要求 3的复合物， 所述高分子聚合物选自聚烯醇类化合物、 聚醚 类化合物、 聚乙烯吡咯烷酮、 聚氨基酸、 二乙烯基醚与马来酸酐的共聚物、 N-(2-羟丙基) -甲基丙烯酰胺 (N- (2-Hydroxypropyl)-methacrylamide) 葡聚糖及 葡聚糖衍生物如硫酸葡聚糖、 纤维素及纤维素的衍生物（包括甲基纤维素和 羧甲基纤维素）、淀粉及淀粉衍生物、聚蔗糖、聚氧乙基多醇 (polyoxyethylated polyol), 肝素或肝素片段、 聚烃基乙二醇及其衍生物、 聚烃基乙二醇和其衍 生物的共聚物、 聚乙烯基乙醚、 聚羟乙基天冬酰胺（a,P-Poly ((2-hydroxyet yl)-DL-aspartamide)) 聚羧酸脂 (polycarboxylates)、氧化乙烯和 甲醛的共聚物 (poly oxyethylene-oxymethylenes) 聚丙烯酰吗啉 (polyacryloyl morpholines), 氨基化合物与氧化烯烃的共聚物、 聚透明质酸、 聚环氧化物 (polyoxiranes), 乙二酸与丙二酸的共聚物、 聚 1,3-乙二醇次甲基醚、 乙烯和 顺丁烯二酰肼的共聚物、 多聚唾液酸、 环糊精或其他药学上可接受的高分子 聚合物。
5.权利要求 4 的复合物， 所述聚醚类化合物选自聚烷撑二醇 (HO((CH2)xO)nH)、 聚丙二醇、 聚氧化乙烯 (HO((CH2)20)nH)、 聚乙烯醇 ((CH2CHOH)n)或它们的衍生物。
6.权利要求 4的复合物， 所述聚烯醇类化合物选自聚乙二醇（包括单甲 基聚乙二醇、 单羟基聚乙二醇)、 聚乙烯醇、 聚丙烯醇、 聚丁烯醇或它们的 衍生物。 '
7.权利要求 6的复合物， 所述聚烯醇类化合物为聚乙二醇。
8.权利要求 7的复合物， 所述聚乙二醇是单甲基聚乙二醇。
9.权利要求 7或 8的复合物，其特征为一个血管内皮抑制素分子和一个 或多个聚乙二醇分子偶联。
10. 权利要求 7-9任一的复合物， 血管内皮抑制素与聚乙二醇偶联的 位点是血管内皮抑制素的 N端 a-氨基、 赖氨酸残基侧链的 ε-氨基、 半胱氨 酸残基侧链的巯基、天冬氨酸残基侧链的羧基、谷氨酸残基侧链的羧基中的 一种或是他们的组合。 .
11. 权利要求 7-10任一的复合物， 其特征为一个血管内皮抑制素分子 与一个聚乙二醇分子偶联， 偶联位点为血管内皮抑制素 Ν端的 a-氨基。
12. 权利要求 7-10任一的复合物， 其特征为血管内皮抑制素分子与一 个或多个聚乙二醇分子偶联， 偶联的位点是血管内皮抑制素 Ν端 a-氨基或 野生型人血管内皮抑制素 75、 95、 106、 117、 183位的赖氨酸残基侧链上的 S-氨基或其组合。
13. 权利要求 7-10任一的复合物， 其特征为血管内皮抑制素分子与一 个或多个聚乙二醇分子偶联，偶联方法为在血管内皮抑制素分子内部或其 Ν 末端或 C末端附加半胱氨酸或是含有半胱氨酸的肽链，使得偶联位点为附加 的半胱氨酸残基侧链上的巯基。
14. 权利要求 7-10任一的复合物， 其特征为血管内皮抑制素分子与一 个或多个聚乙二醇分子偶联，偶联的位点是血管内皮抑制素天冬氨酸或谷氨 酸残基侧链上的羧基。
15. 权利要求 7-14任一的复合物， 所述聚乙二醇分子是线性的或是分 叉的。
16. 权利要求 7-14任一的复合物， 所述聚乙二醇分子的平均分子量位 于 1,000到 100,000道尔顿之间， 优选 5,000到 40,000道尔顿之间。
17. 权利要求 3的复合物， 所述蛋白选自白蛋白、 免疫球蛋白、 甲状 腺结合蛋白、 转甲状腺蛋白、 转铁蛋白、 纤维蛋白原或它们的片段。
18. 权利要求 17的复合物，所述复合物是白蛋白偶联的血管内皮抑制 素， 其特征为一个血管内皮抑制素分子和一个或多个白蛋白偶联， 其中的白 蛋白优选人血清白蛋白或其片段。
19. 权利要求 17的复合物， 所述复合物是免疫球蛋白 Fc片段偶联的 血管内皮抑制素，其特征为一个血管内皮抑制素分子和一个或多个免疫球蛋 白 Fc片段偶联，其中的 Fc片段优选人免疫球蛋白 IgG中的 Fc区域的片段。
20. 权利要求 3 中的复合物， 所述复合物是小分子或是小肽或是其他 化合物修饰的血管内皮抑制素，其特征是该复合物具有同体内其他分子或成 分反应或结合的活性，使得该聚合物可以在体内和其他成分形成更大的复合 物。 '
21. 权利要求 20中的复合物， 所述复合物带有具有反应活性的基团， 可以和血液成分的氨基、 羟基、 巯基反应形成共价键。
22. 权利要求 21中的复合物，所述具有反应活性的基团是马来酰亚胺, 它可以和血液成分， 例如白蛋白中的巯基反应。
23. 权利要求 20中的复合物， 和血液某些成分， 例如白蛋白、 免疫球 蛋白有较强的亲和力， 能相互形成更大的复合物。
24. 权利要求 3 中的复合物， 所述复合物是其他分子或是小肽修饰的 血管内皮抑制素， 例如血管内皮抑制素被糖基化、 磷酸化或酰基化的产物， 其中修饰的位点是原来蛋白序列中存在的氨基酸残基，或是通过突变产生的 氨基酸残基。
25. 权利要求 1 的复合物， 所述修饰物与血管内皮抑制素通过非共价 键相互作用， 其中的修饰物可以是蛋白、 小分子或其他化学物质。
26. 血管内皮抑制素或权利要求 1-25任一的复合物和生物相容的物质 形成的缓释制剂。
27. 权利要求 26的缓释制剂， 所述的缓释制剂选自微胶囊、 水凝胶、 微球、 微型渗透泵或脂质体。
28. 权利要求 1-25任一中的复合物以及权利要求 26或 27的缓释制剂， 其中血管内皮抑制素是人、 鼠、 或其他哺乳动物来源的。
29. 权利要求 1-25任一中的复合物以及权利要求 26或 27的缓释制剂， 其中血管内皮抑制素是野生型的人血管内皮抑制素， 其天然形式具有 SEQ ID NO.1所示的序列。
30. 权利要求 1-25任一中的复合物以及权利要求 26或 27的缓释制剂， 其中所述血管内皮抑制素当由大肠肝菌表达时，其野生型的人血管内皮抑制 素具有 SEQ ID N0.2或者 SEQ ID NO.l的序列。
31. 权利要求 1-25任一中的复合物以及权利要求 26或 27的缓释制剂， 其中血管内皮抑制素可以是血管内皮抑制素的具有活性的片段、突变体、衍 生物、 异构体或是它们^组合。
32. 权利要求 31中的复合物或缓释制剂，其中血管内皮抑制素片段是 具有 SEQ ID N0.3、 SEQ ID N0.4、 或 SEQ ID N0.5序列的肽链。
33. 权利要求 31中的复合物或缓释制剂，其中血管内皮抑制素衍生物 是在野生型血管内皮抑制素的 N端或 C端附加一段长度为 1-15个氨基酸的 肽链形成的。
34. 权利要求 33中的复合物或缓释制剂，其中血管内皮抑制素衍生物 是在野生型人血管内皮抑制素的 N端附加一段 9个氨基酸组成的含有 His-tag 的肽链 MGGSHHHHH,该衍生物具有 SEQ ID N0.6或者 SEQ ID N0.7的序 列。
35. 权利要求 1-34中任一的含有血管内皮抑制素的复合物或缓释制剂 与药学上可接受的载体形成的药物组合物。
36. 权利要求 35的药物组合物，其中的药学上可接受的载体为含水的 pH缓冲溶液， 包括磷酸盐、 柠檬酸盐、 或其他有机酸的缓冲液、 抗坏血酸 或其他抗氧化剂、低分子量 (不超过 10个残基)多肽、血清白蛋白、 明胶、免 疫球蛋白或其他蛋白质、 聚乙烯吡咯烷酮或其他亲水性多聚体、 甘氨酸、 谷 氨酰胺、 天冬酰胺、 精氨酸、 赖氨酸或其他氨基酸、 单糖、 二糖、 葡萄糖、 甘露糖、 糊精或其他糖类、 EDTA或其他螯合剂、 甘露醇、 山梨醇或其他糖 醇、 钠离子或其他成盐反离子、 TWEEN®、 聚乙二醇 (PEG)、 PLURONICS® 或其他非离子表面活性剂。
37. 一种试剂盒， 其包含权利要求 1-36的含有血管内皮抑制素的复合 物、 缓释制剂或药物组合物和使用说明。
38. —种试剂盒， 其包含权利要求 7-16的聚乙二醇修饰的血管内皮抑 制素和使用说明。
39. 制备权利要求 7-16中的含有血管内皮抑制素的复合物的方法， 其 特征是用活化的聚乙二醇与血管内皮抑制素混合，在适当的溶液、温度、 pH、 摩尔比下反应。
40. 权利要求 39的方法， 其中 pH为 pH 3-10。
41. 制备权利要求 7-16中的含有血管内皮抑制素的复合物的方法， 其 特征是将偶联产物用阳离子柱纯化。
42. 制备权利要求 7-16中的含有血管内皮抑制素的复合物的方法， 其 特征是将偶联产物用分子筛纯化。
43. 权利要求 1-38的含有血管内皮抑制素的复合物、 缓释制剂、 药物 组合物或试剂盒在预防、 诊断或治疗肿瘤中的用途。
44. 权利要求 43的用途，所述肿瘤选自肺癌、神经内分泌瘤、结肠癌、 骨癌、肝癌、 胃癌、胰腺癌、 口腔癌、乳腺癌、前列腺癌、淋巴癌、 食道癌、 口腔癌、鼻咽癌、 宫颈癌、 肉瘤、 肾癌、胆癌、恶性黑色素肿瘤或其他肿瘤。
45. 权利要求 1-38的含有血管内皮抑制素的复合物、 缓释制剂、 药物 组合物或试剂盒在预防、诊断或治疗其他疾病中的用途， 所述疾病的特征为 新生血管异常生成而导致人的组织或器官病变。
46. 权利要求 43-45 的用途， 其中的给药方式选自静脉注射、 静脉滴 注、 皮下注射、 肌肉注射、 腹腔注射、 皮下包埋、 透皮吸收、 肝动脉注射、 口服、 鼻粘膜给药、 口腔粘膜给药、 眼部给药、 直肠给药、 阴道给药或其他 临床给药方式。
47. 权利要求 1-36的含有血管内皮抑制素的复合物、 缓释制剂或药物 组合物在制备抗肿瘤药物中的用途。
48. 权利要求 1-36的含有血管内皮抑制素的复合物、 缓释制剂或药物 组合物在制备治疗其他疾病的药物中的用途，所述疾病的特征为新生血管异 常生成而导致人的组织或器官病变。
49. 一种延长血管内皮抑制素半衰期的方法， 所述方法包含将能够延 长血管内皮抑制素的体内半衰期的修饰物与血管内皮抑制素形成复合物的 步骤。
50. —种延长血管内皮抑制素半衰期的方法， 所述方法包含将血管内 皮抑制素或含有血管内皮抑制素的复合物与生物相容的物质形成缓释制剂 的步骤。
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US8802824B2 (en) * 2007-09-05 2014-08-12 Jiangsu Simcere Pharmaceutical R&D Co., Ltd. Modified recombinant human endostatin and uses thereof
CN109963597A (zh) * 2016-11-10 2019-07-02 北京普罗吉生物科技发展有限公司 聚乙二醇化血管内皮抑制素类似物及其应用
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