WO2008083615A1 - Complexes comprenant de l'angiostatine et ses fragments, leurs procédés de préparation et leurs utilisations - Google Patents

Complexes comprenant de l'angiostatine et ses fragments, leurs procédés de préparation et leurs utilisations Download PDF

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Publication number
WO2008083615A1
WO2008083615A1 PCT/CN2008/000067 CN2008000067W WO2008083615A1 WO 2008083615 A1 WO2008083615 A1 WO 2008083615A1 CN 2008000067 W CN2008000067 W CN 2008000067W WO 2008083615 A1 WO2008083615 A1 WO 2008083615A1
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Prior art keywords
angiostatin
fragment
complex
polyethylene glycol
cancer
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PCT/CN2008/000067
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English (en)
French (fr)
Inventor
Yongzhang Luo
Guodong Chang
Shuling Yang
Lei Gao
Yan Fu
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Protgen Ltd.
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Application filed by Protgen Ltd. filed Critical Protgen Ltd.
Priority to EP08700623.5A priority Critical patent/EP2113517B1/en
Priority to US12/522,661 priority patent/US20100184661A1/en
Priority to AU2008204647A priority patent/AU2008204647A1/en
Priority to JP2009545050A priority patent/JP2010515694A/ja
Priority to CA2675231A priority patent/CA2675231C/en
Publication of WO2008083615A1 publication Critical patent/WO2008083615A1/zh
Priority to US13/488,932 priority patent/US20140056966A1/en

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    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

  • the present invention relates to a complex comprising a vasopressin or a fragment thereof, preferably a fragment K1-3, and a sustained release preparation and a process for the preparation thereof.
  • the complexes of the present invention have no toxic side effects, higher biological activity and better metabolic stability.
  • the present invention also provides a pharmaceutical composition comprising such a complex and a sustained release preparation, and a kit containing the same.
  • the present invention also provides a method of preventing, diagnosing, treating a tumor, preparing an antitumor drug, and anti-angiogenic disease using the complex, the sustained release preparation, the pharmaceutical combination and the kit of the present invention. Background technique
  • Cancer is a common and frequently-occurring disease in the world today, and it is the number one killer of human life. Therefore, the development and development of anticancer drugs has been a hot topic at home and abroad. At present, most of the anticancer drugs commonly used in clinical practice are traditional chemotherapy drugs. The long-term use is easy to cause drug resistance and has great toxic and side effects on the human body, and is greatly limited in clinical application.
  • Neovascularization refers to the creation of new capillaries on existing blood vessels. Therefore, blocking neovascularization of the tumor may prevent tumor growth and metastasis.
  • the use of neovascular endothelial cells in neoplasms as a target for cancer treatment provides a treatment for tumors, namely anti-angiogenic therapy (Folkman J. N EngU Med 1971; 285: 1182 -1186).
  • Angiostatin is an endogenous angiogenesis inhibitor extracted from the serum and urine of Lewis lung cancer-bearing mice in 1994 by O'Reilly et al. It is a sequence starting from the 98th amino acid residue of the mouse Plasminogen (starting amino acid residue Met as the first amino acid) (O'Reilly MS, Holmgren L, Shing Y, et al. Cell, 1994, 79: 315-328), which includes at least the first four of the five Kringles structures of plasminogen, each of which consists of approximately 80 amino acid residues (Lerch PG, Riskli EE, Lergier W, et al.
  • K1, ⁇ 2, ⁇ 3, ⁇ 5, ⁇ 1-3, ⁇ 2-3, K1-5, etc. of recombinant human plasminogen can effectively inhibit the proliferation of bovine capillary endothelial cells (BCE) cells.
  • BCE bovine capillary endothelial cells
  • K4 has almost no such activity.
  • the activity of K1-3 to inhibit cell proliferation was significantly stronger than that of the originally found angiostatin Kl-4 (Cao Y, Ji RW, Davidson D, et al. J Biol Chem, 1996, 271(46): 29461-29467.).
  • Kl-3 can inhibit the growth and metastasis of Lewis lung cancer in mice, and also inhibit the growth of various primary tumors such as Lewis lung cancer primary tumor and mouse hemangioendothelioma.
  • the mechanism by which angiostatin acts is that it inhibits the proliferation of vascular endothelial cells, thereby inhibiting the formation of neovascularization in tumor tissues, thereby cutting off the transport pathway that provides nutrients and oxygen to tumor tissues, and finally starving the tumor.
  • angiostatin is a fragment of endogenous plasminogen that acts specifically on proliferating vascular endothelial cells and therefore does not generally cause an immune response. Long-term injection does not cause weight loss or activity.
  • angiostatin has achieved remarkable curative effect on the treatment of tumor cells and is expected to become the main anticancer drug in the 21st century.
  • polypeptides and proteinaceous drugs including angiostatin have advantages such as less toxic side effects than chemical drugs, and no drug resistance, and in order to increase the bioavailability of protein drugs and reduce their In vivo degradation is usually administered intravenously, but for proteins with small molecular weight, the in vivo half-life of intravenous administration is very short. Protein degradation in the body is only one of the reasons. Another important aspect is that small proteins can be quickly eliminated by renal filtration. Studies have found that proteins with a hydraulic radius in excess of albumin or a molecular weight greater than about 66,000 daltons (66 kDa) are generally stable in the circulatory system. However, small molecules are quickly eliminated by glomerular filtration.
  • Angiostatin which is a protein drug, also has a shortcoming of a short half-life and a high clearance rate in the body.
  • the object of the present invention is to improve the in vivo generation characteristics of the protein, resulting in higher stability and longer in vivo half-life for better therapeutic effects.
  • High molecular weight polymers are commonly used methods for altering and controlling the kinetic properties of drugs such as half-life, immunological characteristics, and toxicological properties. It 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.
  • the PEG molecule is amphiphilic and can be dissolved in 7 or dissolved in most organic solvents. It is non-toxic, non-immunogenic, and highly soluble in aqueous solution. It is included in the US FDA and China 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, the elimination efficiency of the kidney can be greatly reduced. This method 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;
  • PEG modification uses an amino group as a reaction site, which mainly includes 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 lysine residue side chain are also often produced because the reaction site is not specific to produce a modified isomer.
  • the isoelectric point of the ⁇ -amino group of the side chain ⁇ -amino and lysine residues Differences, and newly developed PEG modification reagents only for the N-terminus of the protein the modification sites are consistent, and the modified product composition is uniform.
  • Another protein site that can be used for PEG modification is the thiol group of cysteine. Since the number of thiol groups in the protein is usually less than the amino group, such modified sites are more specific. Using genetic engineering techniques, it is possible to introduce cysteine as a specific modification site in any part of the protein. However, the introduction of cysteine as a modification site has certain limitations, because for some proteins with cysteine itself, mismatch or irreversibility of disulfide bonds may occur.
  • the carboxyl group of the protein is also a commonly used modification site (Veronese FM et al. Drug Discov, Today. 2005Nov
  • PEG modification technology has been successfully applied to many protein drugs, including: PEG-asparaginase (Graham ML Adv. Drug Deliv. Rev. 55, 1293- 1302 Avramis Vassilios I. et al. W09939732 and
  • polystyrene resin In addition to PEG, other polymer modifiers commonly used in the art are dextran, sucrose, starch, polyalanine, copolymers of oxalic acid and malonic acid, carboxymethyl cellulose, polyvinylpyrrolidone, poly 1,3-ethylene glycol methine ether, a copolymer of ethylene and maleic hydrazide, polysialic acid, cyclodextrin, and the like.
  • the blood protein or a fragment thereof is used as a carrier, and is coupled or fused with a pharmaceutical protein to thereby increase the molecular weight of the protein, and is also a 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 applied to a Notch l receptor protein (Kitajewsky Jan et al. WO2005-111072), Erythropoietin (EPO) (Gillies Stephen D et al. WO2005- 063808), human growth hormone (Kim Young Min et al. WO2005047337) and other proteins.
  • Plasma albumin is also a commonly used coupling carrier and can be applied to antibiotics, anti-inflammatory, anti-oxidant and other proteins (Ezrin Alan M et al. WO0117568, Otagiri Masaki et al.
  • Another method for extending the in vivo half-life of a protein is to prepare 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. Pharai. 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)).
  • Liposomes are ultramicroscopic particles in the form of a hollow sphere with a two-layer membrane structure.
  • 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 lumen 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 the invention
  • Kl refers to a fragment of Plasminogen, wherein the sequence of human plasminogen is shown in SEQ ID NO: 7. (See U.S. Patent Nos. 7, 157, 556 and WO 02/26782 A2) - where "K1-3” means “Kringlesl-3".
  • PEG refers to polyethylene glycol, preferably methylpolyethylene glycol (mPEG).
  • the inventors have surprisingly discovered a complex formed by a modification with angiostatin or a fragment thereof, which complex has an unmodified angiostatin Or a fragment thereof having a longer in vivo half-life, the modification being selected from the group consisting of a high molecular polymer, a protein molecule or a fragment thereof, a peptide chain, a small molecule, or any other form of chemical.
  • the complex not only maintains the original activity of inhibiting angiogenesis, but also has higher biological activity and longer half-life in vivo, and has no toxic and side effects on cells and animals, and does not cause drug resistance for long-term use. Sexual side effects.
  • the angiostatin of the invention is of human, murine or other mammalian origin. More preferably, the angiostatin is human angiostatin.
  • the angiostatin of the invention is a recombinant angiostatin. More preferably, the recombinant angiostatin is recombinant human angiostatin.
  • the fragment of angiostatin of the invention is selected from the group consisting of angiostatin K1, K2, K3, K5, Kl-2, Kl-3, Kl-5, ⁇ 2-3, ⁇ 2-5 and ⁇ 3 -5. More preferably, the angiostatin fragment is angiostatin K1 having the sequence of SEQ ID NO: 3, angiostatin K2 having the sequence of SEQ ID NO: 4 or having the sequence of SEQ ID NO: Angiostatin K3.
  • the fragment of angiostatin of the present invention is angiostatin K1-3 having the sequence of SEQ ID NO: 1, and when the human angiostatin is a recombinant human vascular suppressor expressed by Escherichia coli In the case of K1-3, it has the sequence of SEQ ID NO: 2, wherein the N-terminal Met is optionally deleted in the expression of E. coli.
  • the angiostatin or fragment thereof for use in the complex of the invention further comprises an active mutant, derivative, isomer or combination thereof of angiostatin or a fragment thereof.
  • the derivative of angiostatin or a fragment thereof is added to the N-terminus or C-terminus of angiostatin or a fragment thereof for a length of 1-15 ammonia.
  • the peptide chain of the base acid is formed.
  • the derivative of angiostatin or a fragment thereof is a His-tag-containing peptide chain MMHHHHH comprising a 7 amino acid sequence at the N-terminus of human angiostatin, the derivative having SEQ ID NO: The sequence of 6, and its N-terminal Met is optionally deleted when expressed by E. coli.
  • the derivative of angiostatin or a fragment thereof is a peptide chain having a sequence of MGGSHHHHH or MGGSHHHHHH attached to the N-terminus of human angiostatin, and its N-terminus when expressed by Escherichia coli Met is optionally removed.
  • the modification in the complex is linked to angiostatin or a fragment thereof by a covalent bond. In another embodiment of the invention, the modification in the complex is linked to angiostatin or a fragment thereof by a non-covalent bond. Preferably, the modification may be linked to angiostatin or a fragment thereof by chemical coupling, or may be linked to angiostatin or a fragment thereof by fusion expression.
  • the polymer of the present invention may or may not be biologically active.
  • 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 Acrylamide, dextran and dextran derivatives such as dextran sulfate, cellulose and cellulose derivatives (including methyl cellulose and carboxymethyl cellulose), starch and starch derivatives, polysucrose, poly Oxyethyl alcohol, heparin or heparin fragment, polyalkyl glycol and its derivatives, copolymer of polyalkyl glycol and its derivatives, polyvinyl ether, polyhydroxyethyl asparagine
  • polyoxyethylene-oxymethylenes ⁇ polyacry morpholines, copolymers of amino compounds with alkylene oxides, polyhyaluronic acid, polyoxiranes, copolymers of oxalic acid and malonic acid , poly1,3-ethylene glycol methine ether, a copolymer of ethylene and maleic hydrazide, polysialic acid, cyclodextrin or other pharmaceutically acceptable high molecular polymer.
  • the polyether compounds of the present invention include, but are not limited to, polyalkylene glycols (HO((CH 2 )xO)neigH), polypropylene glycol, polyethylene oxide (HO((CH 2 ) 2 0) n H), polyvinyl alcohol (XCH 2 CHOH) n ) or their derivatives
  • the polyenol compounds include, but are not limited to, polyethylene glycol (including monomethyl polyethylene glycol, monohydroxy polyethylene glycol, etc.), polyvinyl alcohol, polypropylene alcohol, polybutenol or derivatives thereof.
  • the modification is polyethylene glycol.
  • the polyethylene glycol is monomethylpolyethyl alcohol.
  • the polyethylene glycol molecules used in the present invention are linear or bifurcated.
  • the polyethylene glycol molecules have an average molecular weight between 1,000 and 100,000 Daltons. More preferably, the polyethylene glycol molecules have an average molecular weight between 5,000 and 40,000 Daltons. Most preferably, the polyethylene glycol is a monomethyl polyethylene glycol having a molecular weight of 20 kDa.
  • an angiostatin molecule or fragment thereof is conjugated to one or more polyethylene glycol molecules.
  • the modification is site specific and does not affect the biological activity of angiostatin or a fragment thereof.
  • the modified product has anti-angiogenic activity and is more metabolically stable than the unmodified angiostatin or a fragment thereof, has a longer blood half-life, and can be used as an anti-angiogenic disease or anti-tumor drug. .
  • the site at which the angiostatin molecule or fragment thereof is coupled to the polyethylene glycol molecule is selected from the group consisting of an N-terminal ⁇ -amino group of angiostatin or a fragment thereof, and an ⁇ -amino group of a side chain of a lysine residue, One or more of a thiol group of a side chain of a cysteine residue, a carboxyl group of a side chain of an aspartic acid residue, and a carboxyl group of a side chain of a glutamic acid residue.
  • the site of the angiostatin molecule or a fragment thereof conjugated to the polyethylene glycol molecule is selected from the group consisting of angiostatin or a fragment thereof, a-amino group or the sequence shown in SEQ ID NO: 1
  • an angiostatin molecule or a fragment thereof is conjugated to a polyethylene glycol molecule, and the coupling site is an ⁇ -amino group at the terminal end of angiostatin or a fragment thereof.
  • a fragment K1-3 having the recombinant human angiostatin molecule of SEQ ID NO: 2 is conjugated to a polyethylene glycol molecule, and the coupling site is an angiostatin fragment.
  • the present invention specifically modifies the ⁇ -amino group of the K1-3 N-terminus using PEG, the product of which has biological activity of inhibiting proliferation and migration of endothelial cells, inhibiting tumor cell proliferation, and inhibiting tumor growth in mice.
  • an angiostatin molecule or fragment thereof is conjugated to one or more polyethylene glycol molecules, either in the angiostatin molecule or a fragment thereof or at the N-terminus or C thereof. Cysteine or a cysteine-containing peptide chain is added at the end such that the coupling site is a thiol group on the side chain of the additional cysteine residue.
  • Suitable polyamino acids according to the invention include, but are not limited to, polymers of the same amino acid, copolymers of two or more amino acids, for example: polyalanine.
  • Protein molecules suitable for use as a modified vector are preferably naturally occurring proteins or fragments thereof according to the invention, including but not limited to thyroid binding proteins, transthyretin, transferrin, fibrinogen, immunoglobulin, albumin, etc. and Fragment of.
  • the above carrier proteins are 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 vector.
  • the complex of the invention is an albumin-conjugated angiostatin or a fragment thereof, characterized in that an angiostatin molecule or fragment thereof is coupled to one or more albumin, the coupling
  • the substance may be obtained by chemical modification, or obtained by fusion expression, or obtained by other methods, wherein the albumin is preferably human serum albumin or a fragment thereof.
  • the complex of the invention is an immunoglobulin Fc fragment-conjugated angiostatin or a fragment thereof, characterized by an angiostatin molecule or fragment thereof and one or more immunoglobulin Fc
  • the fragment is coupled, and the conjugate may be obtained by chemical modification, 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.
  • Modifications of the invention also include small molecules or small peptides or other compounds.
  • the complex of the invention is a small molecule or a small peptide or other compound modification Angiostatin or a fragment thereof, characterized in that the complex has an activity of reacting or binding with other molecules or components in the body, such that the polymer can form a larger complex in vivo and other components, wherein the activity is It can be produced by reacting with an amino group, a hydroxyl group or a thiol group of a blood component to form a covalently bonded reactive group.
  • the reactive group is a maleimide, which can be combined with a blood component such as a thiol group in albumin. reaction.
  • the complex of the invention is a product of glycosylation, phosphorylation or acylation of angiostatin or a fragment thereof, wherein the site of modification is an amino acid residue present in the original protein sequence, or It is an amino acid residue produced by mutation.
  • the complex does not have a large molecular weight, but because the modification changes the nature of K1-3, it extends its half-life.
  • the angiostatin or a fragment thereof of the present invention is directly or indirectly covalently linked to the above-mentioned modification, and the direct linkage means that an amino acid of angiostatin or a fragment thereof is directly linked to an amino acid of a carrier protein, and may be through a peptide bond or Disulfide bond.
  • An indirect linker refers to angiostatin or a fragment thereof and a carrier protein linked by a chemical group or a combination of several groups.
  • sustained release preparation which is formed from angiostatin or a fragment thereof, preferably fragment Kl-3, or any of the above complexes and a biocompatible substance.
  • Angiostatin or a fragment thereof in the composition is still biologically active, but can rely on the carrier to alter the metabolic profile of the drug for the purpose of prolonging retention time in the body.
  • the sustained release preparations include, but are not limited to, microcapsules, hydrogels, microspheres, micro osmotic pumps, liposomes, and the like.
  • a pharmaceutical composition comprising the above complex or sustained release preparation and a pharmaceutically acceptable carrier, the complex comprising a modification and angiostatin or a fragment thereof
  • the angiostatin fragment K1-3 is formed.
  • the pharmaceutically acceptable carrier for use in the present invention comprises 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.
  • the physiologically acceptable carrier is an aqueous pH buffer solution.
  • physiologically acceptable carrier Substances include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (up to 10 residues) polypeptides; such as serum albumin, gelatin, or immunoglobulins Proteins such as proteins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and including glucose, mannose, or Other sugars such as dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfaces such as TWEEN®, polyethylene glycol, and PLURONICS® Active agent.
  • the excipients are preferably sterile and generally free of undesirable materials. These compositions can be sterilized by conventional sterilization techniques.
  • kits comprising the above complex, sustained release preparation or pharmaceutical composition and instructions for use, wherein the complex is formed from a modification with angiostatin or a fragment thereof, preferably The ground is a polyethylene glycol modified angiostatin fragment Kl-3.
  • the invention further relates to a method of preparing a complex of the invention consisting of a modification with angiostatin or a fragment thereof, preferably fragment K1-3. More particularly, a method for preparing a polyethylene glycol (PEG)-modified angiostatin or a fragment thereof, that is, an activated PEG is mixed with an angiostatin or a fragment thereof, and reacted at an appropriate solution, temperature, pH, molar ratio, and The coupled product is optionally purified using a cation column or molecular sieve.
  • the pH of the reaction is preferably between pH 3 and 10, more preferably between pH 5 and 7, and the polyethylene glycol to angiostatin molar ratio is from 1:1 to 10:1.
  • the present invention also provides the use of the above complex, sustained release preparation, pharmaceutical composition or kit for preventing, diagnosing or treating a tumor, wherein the complex is formed by a modification with angiostatin or a fragment thereof, Preferred is a polyethylene glycol modified angiostatin fragment Kl-3.
  • Suitable cancers for the present 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 a use of the above-mentioned complex, pharmaceutical composition or sustained release preparation containing angiostatin or a fragment thereof, preferably K1-3, in the preparation of an antitumor drug, wherein
  • the medicament is suitable for intravenous injection, intravenous drip, subcutaneous injection, intramuscular injection, intraperitoneal injection, subcutaneous embedding, transdermal absorption, hepatic artery injection, oral administration, nasal mucosal administration, oral mucosal administration, ocular administration,
  • the administration time is from daily to every 21 days, preferably from daily to weekly.
  • the present invention also provides the use of the above complex, sustained release preparation, pharmaceutical composition or kit for preventing, diagnosing or treating a medicament for non-tumor diseases, wherein the complex consists of a modification with angiostatin or Its fragment is formed, preferably a polyethylene glycol modified angiostatin fragment Kl-3.
  • the non-neoplastic disease is characterized by abnormal neovascularization resulting in human tissue or organ lesions, and the medicament is suitable for intravenous injection, intravenous drip, subcutaneous injection, intramuscular injection, intraperitoneal injection, subcutaneous embedding, transdermal absorption, Hepatic artery injection, oral, nasal mucosal administration, oral mucosal administration, ocular administration, rectal administration, vaginal administration or other clinical administration methods, administration time is from daily to every 21 days, preferably daily to Dosing weekly.
  • the present invention also provides a method of prolonging angiostatin or a fragment thereof, preferably extending a half-life of a fragment K1-3, the method comprising the step of forming a complex with an angiostatin or a fragment thereof, and optionally comprising preparing A step of a sustained release preparation formed by the above complex and a biocompatible substance.
  • the complex of PEG and Kl-3 N-terminally coupled in a preferred embodiment of the present invention has an activity of inhibiting vascular endothelial cell migration and tumor growth in mice, and the activity is remarkably improved as compared with K1-3, and In vivo pharmacokinetic studies have shown that modified K1-3 can effectively slow the metabolism of K1-3 in the body and prolong the metabolism time in the body.
  • Angiostatin or a fragment thereof as referred to in the present invention includes wild type angiostatin or a fragment thereof, that is, a naturally occurring form in vivo or an active mutant, fragment, isomer thereof, derivative thereof, unless otherwise specified. Things or the like or a combination thereof.
  • the source of angiostatin or a fragment thereof may be, but is not limited to, expressed by animal cells, or may be fermentatively purified from yeast or E. coli.
  • the human angiostatin fragment K1-3 derived from animal cell or yeast cell expression has the amino acid sequence shown in SEQ ID NO: 1, or has an increase within 10 amino acids at the N-terminus and/or C-terminus of the sequence. Or a deletion sequence derived from E. coli expressing K1-3 having the sequence of SEQ ID NO: 2, but the N-terminal Met is expressed after Sometimes it will be deleted.
  • the mutant of K1-3 refers to Kl-3 which is obtained by substitution, deletion, and addition of an amino acid.
  • the fragment thereof refers to any smaller part of the sequence belonging to SEQ ID ⁇ : 1 or SEQ ID NO: 2, which can be It is, but not limited to, obtained by enzymatic cleavage, or expressed by genetic engineering, or by a method of polypeptide synthesis.
  • the mutant has 60% amino acid sequence homology to SEQ ID NC, or has 70% amino acid sequence homology, more desirably 80% to 90% homology, especially 95 % sequence homology.
  • the fragment refers to a sequence comprising a portion of SEQ ID NO: 1, such as the sequence having SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5.
  • a fragment having the sequence of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 has been reported to have an activity of inhibiting endothelial cell proliferation (Cattaneo MG et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY. Vol. 271, No. 46, Issue of November 15, pp. 29461 - 29467, 1996).
  • the isomer of Kl-3 refers to the same amino acid sequence or molecular formula as SEQ ID NO: 1, but has a different conformation, including a difference in the secondary or tertiary structure of the protein or a change in the optical rotation of the local amino acid.
  • the source of the construct can be a naturally occurring mutation or obtained by artificial design.
  • the derivative of K1-3 refers to a product modified based on SEQ ID NOrl, wherein the modification refers to covalently linking one or more other small molecules such as a phosphate molecule, a sugar molecule, etc., or 30 to the protein.
  • a small peptide chain within one amino acid, and the attachment site may be any amino acid in the protein.
  • the combination of mutants, fragments, isomers and derivatives in which K1-3 is active means a product having two or more of the above characteristic changes at the same time, but is not limited to mutants of the fragments, modified products of the mutants, and the like.
  • Figure 1 shows the amino acid sequence of human angiostatin fragment K1-3 SEQ ID ⁇ : 1.
  • Figure 2 shows the amino acid sequence of human angiostatin fragment K1-3 expressed by E. coli SEQ ID NO: 2. Here the M-term at the N-terminus is sometimes deleted after expression.
  • Figure 3 shows the amino acid sequence of human angiostatin k 1 SEQ ID NO: 3.
  • Figure 4 shows the amino acid sequence of human angiostatin k 2 SEQ ID NO:4.
  • Figure 5 shows the amino acid sequence of human angiostatin k 3 SEQ ID NO: 5.
  • Figure 6 Amino acid sequence of K1-3 showing an active N-terminal additional amino acid SEQ ID NO: 6. The Met at the N-terminus is sometimes deleted after expression.
  • Figure 7 Coupling of PEG to the N-terminus of K1-3 with the sequence of SEQ ID NO: 2.
  • the reaction conditions were 4 ° C, 10 h.
  • the solution was detected by SDS-PAGE, lane 1: protein molecular weight standard; lane 2: K1-3; lanes 3 to 8 were PEG and K1-3 molar ratios of 5:1, respectively, pH 5.0, 5.5, 6.0 K1-3 after modification of conditions 6.5, 7.0, 8.0; Lanes 9 to 12 are pH 5.5, 20 kDa, respectively.
  • the molar ratio of PEG to K1-3 is 1:1, 2:1, 5:1, 10:1 Conditionally modified K1 -3.
  • Figure 8 Coupling of PEG to the N-terminus of K1-3 with the sequence of SEQ ID NO: 2.
  • the reaction conditions were room temperature, 10 h. After the reaction, the solution was detected by SDS-PAGE, lane 1: protein molecular weight standard; lane 2: K1-3; lanes 3 to 8 were PEG and K1-3, respectively, the molar ratio was 5:1, and the pH was 5.0, 5.5, 6.0, respectively. , 6.5, 7.0, 8.0 modified K1-3; lanes 9 ⁇ 12 are pH5.5, PEG and K1-3 molar ratio are 1:1, 2:1, 5:1, 10:1 conditional modification After Kl-3.
  • Figure 9 Coupling of PEG to the ⁇ end of K1-3 having the sequence of SEQ ID NO: 2.
  • the solution before and after the reaction was detected by SDS-PAGE, lane 1 : protein molecular weight standard; lane 2: K1-3; lane 3: PEG-modified Kl-3.
  • Figure 10 PEG-modified N-terminal coupling of K1-3 having the sequence of SEQ ID NO: 2, 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 purified by SDS-PAGE.
  • Lane 1 Protein Molecular Weight Standard
  • Lane 2 Before Purification
  • Lane 3 Sample Penetrating Solution
  • Lane 4-13 Distribute the collected eluate.
  • Figure 11 Results of in vivo metabolism assays of products of PEG coupled to the N-terminus of Kl-3.
  • K1-3 Kl-3 having the sequence of SEQ ID NO: 2;
  • PEG-K1-3 PEG modified with SEQ IDNO: Kl-3 of the 2 sequence.
  • FIG. 12 Activity of PEG coupled to the K1-3 N-terminus to inhibit migration of human umbilical vein endothelial cells (HUVEC).
  • K1-3 Kl-3 having the sequence of SEQ ID NO: 2;
  • PEG-Kl-3 PEG-modified Kl-3 having the sequence of SEQ ID NO: 2.
  • Figure 13 The product of PEG coupled to the K1-3 N-terminus inhibits the self-repairing activity of human umbilical vein endothelial cells (HUVEC) lesions.
  • K1-3 Kl-3 having the sequence of SEQ ID NO: 2;
  • PEG-K1-3 PEG-modified Kl-3 having the sequence of SEQ ID NO: 2.
  • FIG. 14 Activity of PEG coupled to the K1-3 N-terminus to inhibit proliferation of human umbilical vein endothelial cells (HUVEC).
  • K1-3 Kl-3 having the sequence of SEQ ID NO: 2;
  • PEG-Kl-3 PEG-modified Kl-3 having the sequence of SEQ ID NO: 2.
  • FIG. 15 Activity of PEG coupled to the K1-3 N-terminus to inhibit proliferation of human microvascular endothelial cells (HMEC).
  • K1-3 Kl-3 having the sequence of SEQ ID NO: 2;
  • PEG-Kl-3 PEG-modified Kl-3 having the sequence of SEQ ID NO: 2.
  • FIG. 16 Activity of PEG coupled to the K1-3 N-terminus to inhibit tumor cell (Hela) proliferation.
  • K1-3 Kl-3 having the sequence of SEQ ID NO: 2;
  • PEG-K1-3 PEG-modified Kl-3 having the sequence of SEQ ID NO: 2.
  • Figure 17 The product of PEG coupled to the Kl-3 terminus inhibits the activity of mouse melanoma (B16).
  • K1-3-4.5-1 Kl-3 having the sequence of SEQ ID NO: 2, 4.5 mg/kg body weight, administered daily;
  • PEG-K1-3-1.5-1 PEG-modified Kl-3 having the sequence of SEQ ID NO: 2 1.5 mg/kg body weight, administered daily;
  • PEG-Kl-3-4.5-1 PEG-modified Kl-3 with SEQ ID NO: 2 sequence, 4.5 mg/kg body weight, administered daily;
  • PEG-Kl- 3-4.5-3 PEG-modified K1-3 with SEQ ID NO: 2 sequence, 4.5 mg/kg body weight, administered every 3 days;
  • PEG-K1-3-4.5-6 PEG modified with SEQ ID NO: 2
  • PEG-K1 -3-4.5-9 PEG-modified Kl-3 having the sequence of SEQ ID NO: 2, 4.5 mg/
  • Figure 18 The product of PEG coupled to the N-terminus of Kl-3 inhibits the activity of mouse liver cancer (H22).
  • K1-3- 4.5-1 K1-3 with a sequence of SEQ ID NO: 2, 4.5 mg/kg body weight, Administered daily;
  • PEG-K1-3-1.5-1 PEG-modified Kl-3 with the sequence of SEQ ID NO: 2, 1.5 mg/kg body weight, administered daily;
  • PEG- K1-3-4.5-1 PEG Modified Kl-3 having the sequence of SEQ ID NO: 2, 4.5 mg/kg body weight, administered daily;
  • PEG-Kl-3-4.5-3 PEG modified with 8 £( ⁇ 10 ⁇ [0: 2 sequence 1 ⁇ 1-3, 4.5 mg/kg body weight, administered twice a week;
  • PEG-Kl-3-4.5-7 PEG-modified Kl-3 with a sequence of SEQ ID NO: 2, 4.5 mg/kg body weight, Dosing once a week.
  • polyethylene glycol (PEG) used in the following examples was 082M0P01 MPEG-BUTY ALD-20K, NETKTARTM, Lot#: PT-03G-05.
  • PEG polyethylene glycol
  • Kl-3 (purchased from Prometheus, China) referred to in this example is Kl-3 having the sequence of SEQ ID NO: 2.
  • Kl-3 was dialyzed into a solution of 30 mM sodium acetate (purchased from Beijing Chemical Laboratory, China) of pH 5.5. The protein concentration was measured at 280 nm using an ultraviolet spectrophotometer (8453, Agilent), and then the concentration was adjusted to 1 mg/ml.
  • Kl-3 is coupled to the PEG of the N-terminus of the specific modified protein. In the first case, at a pH of 5.5, 10 ml of the above-mentioned dialysis solution containing K1-3 (containing protein 10 mg) was taken, and the molar ratio of PEG to K1-3 was 1:1, 2:1, respectively.
  • the K1-3 was observed to be modified by a single PEG.
  • the coupled site is the a-amino group at the N-terminus of K1-3, and a small amount of K1-3 is modified by a non-specific multi-site. This will find the best conditions for the reaction of PEG with K1-3.
  • the results of solution electrophoresis before and after the reaction are shown in Figure 7-8.
  • K1-3 according to the present embodiment is K1-3 having the sequence of SEQ ID NO: 2.
  • K1-3 was 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 1 mg/ml.
  • When conjugated to the N-terminal PEG of the specific modified protein take 20 ⁇ 1 of the above 1-3 dialyzed solution (containing protein 20 mg), add PEG solid 100 mg, and stir at room temperature until completely dissolved, so that PEG and K1 The molar ratio of -3 is 5:1.
  • the reducing agent C3 ⁇ 4BNNa was added at a concentration of 20 mM, and finally the pH of the solution was adjusted to 5.5.
  • K1-3 involved in the present example is Kl-3 having the sequence of SEQ ID NO: 2.
  • the PEG-modified Kl-3 was purified using an SP column (Amersham). The pH of the mixture after the reaction was adjusted to 5. The column was equilibrated with equilibration buffer containing 20 mM sodium acetate, pH 5.0 and loaded. After loading, it was eluted with a gradient of elution buffer containing 20 mM sodium acetate, 1 M sodium chloride, pH 5.0. Unreacted polyethylene glycol appeared in the process of penetration and rinsing due to the little charge. , the order of eluting peaks is multi-modification Kl-3, single modification Kl-3, Kl-3.
  • K1-3 involved in the present example is Kl-3 having the sequence of SEQ ID NO: 2.
  • the metabolic rate of ⁇ 1-3 and PEG-modified K1-3 in Wistar rats was determined to examine the long-term benefit of PEG-modified products in the blood. Two healthy rats weighing about 200 g were used, and K1-3 and PEG-modified K1-3 N-terminal coupling products were injected into the tail vein, respectively, 4.5 mg/kg body weight.
  • In vivo pharmacokinetics showed that the half-life of PEG-modified K1-3 in rats was 81.5 h, while the half-life of K1-3 in rats was only 12.2 h, indicating that the coupling of high molecular weight PEG can effectively increase.
  • the product of PEG coupled to the Kl-3 N-terminus inhibits the migration of human umbilical vein endothelial cells (HUVEC).
  • K1 -3 involved in the present example is Kl-3 having the sequence of SEQ ID NO: 2.
  • HUVEC Progis, Beijing
  • M199 medium Hyclone
  • fetal bovine serum Hyclone
  • the migration conditions were M199 medium containing 5% fetal bovine serum.
  • K1-3 and PEG-modified K1-3 were added to the drug-added groups at concentrations of 0.004 g/ml, 0.04 g/ml, 0.4 g/ml, and 4 g/ml, respectively. 37.
  • the cells were fixed with 1% glutaraldehyde (Beijing Chemical Plant, China), and the unmigrated cells in the upper layer of the membrane were scraped off, and stained with hematoxylin and eosin (Beijing Chemical Reagent Co., Ltd., China). Under the microscope, take three fields of the same size, calculate the number of cells, and finally calculate the inhibition rate.
  • the results show In the range of 0.04-4 g/ml, the inhibition of cell migration was enhanced as the concentration of the drug was increased, and the results showed that the inhibition rate of cell migration of K1-3 having the sequence of SEQ ID NO: 2 was 43%, respectively.
  • the K1-3 involved in this example is the effect of K1-3 having the sequence of SEQ ID NO: 2 on the repair of HUVEC damage.
  • HUVEC was seeded in a 12-well plate (Coming) at a density of 2 ⁇ 10 5 /ml, and cultured in a logarithmic growth phase in a M199 complete medium containing 20% fetal calf serum. Then starve for 12 h, gently cross a line in the well and wash the suspended cells.
  • the test was set as a negative control group (PBS), and the positive control group (K1-3 concentration was 0.04 g/ml, 0.4, respectively).
  • the administration group (concentration of PEG-modified K1-3 was: 0.04 g/ml, 0.4 g/ml, 4 ⁇ / ⁇ 1). After incubation at 37 ° C for 16 h, the amount of cell migration was measured. The efficacy is calculated by inhibition of cell migration. The results showed that in the range of 0.04-4 g/ml, the inhibition of cell migration increased with the increase of the concentration of administration, and the inhibition rates were 0%, 20%, 25%, respectively; inhibition of PEG-modified K1-3 The rates are 35%, 40%, and 105%, respectively. The results showed that K1-3 modified by PEG not only maintained and greatly enhanced its biological activity, but also died at the concentration of 4 g/ml with shrinkage of some cells. The result is shown in FIG. Example 7. The product of PEG coupled to the N-terminal of Kl-3 inhibits the proliferation of human umbilical vein endothelial cells (HUVEC).
  • HAVEC human umbilical vein endothelial cells
  • K1-3 involved in the present example is Kl-3 having the sequence of SEQ ID NO: 2.
  • HUVECs that were starved overnight were inoculated at a density of 2 ⁇ 10 4 /ml in 96-well plates (Coming), and cultured in M199 containing 20% fetal calf serum to logarithmic growth phase. Then hungry 12 ho Different concentrations of K'l-3 and PEG-modified Kl-3 were added according to the experimental design.
  • the experiment was set as negative control group (PBS), positive control group (K3 concentration was 0.04 g/ml, 0.44 g/ml, respectively), and the concentration of PEG-modified Kl-3 was 0.04 ⁇ . ⁇ 0.4 ⁇ 4 ⁇ ).
  • Example 8 The product of PEG coupled to the N-terminal of Kl-3 inhibits the proliferation of human microvascular endothelial cells (HMEC).
  • HMEC human microvascular endothelial cells
  • K1-3 involved in the present example is Kl-3 having the sequence of SEQ ID NO: 2.
  • the cells were seeded in 96-well plates at a density of 2 ⁇ 10 4 /ml using starvation overnight HMEC (ATCC# CRL 10636, USA) and cultured in logarithmic growth phase in DMEM containing 10% serum. Then starved for 12 h.
  • Different concentrations of unmodified and modified Kl-3 were added in a pre-test design.
  • the experiment set negative control group (PBS), positive control group (K3 concentration was 0.04 ⁇ 0.4 ⁇ 4 ⁇ ⁇ / ⁇ 1), and the concentration of PEG-modified Kl-3 was 0.04. g/ml, 0.4 g/ml, 4 g/ml).
  • K1-3 involved in the present example is Kl-3 having the sequence of SEQ ID NO: 2.
  • Hela cells ATCC# CCL-2, USA
  • DMEM fetal calf serum
  • Different concentrations of K1-3 and PEG-modified Kl-3 were added according to the experimental design.
  • the experiment set negative control group (PBS) and positive control group (K1-3 concentration was 0.4 g/ml, 4 g/ml, respectively).
  • K1-3 involved in the present example is Kl-3 having the sequence of SEQ ID NO: 2.
  • the in vivo inhibitory activity of PEG-modified K1-3 on mouse B16 tumor was observed experimentally.
  • the experimental material was selected from 20 g of C57B/L mice (Vitronius Experimental Animal Center), and the back was connected to I x10 6 melanoma cells (ATCC # CRL-6475 TM , USA).
  • the tumors were grouped into groups, 8 rats in each group, respectively, a negative control group (20 ⁇ sodium acetate solution), a positive control group (K1-3 4.5 mg/kg body weight, administered daily), and a drug-administered group (PEG-modified K1- 3 1.5 mg/kg body weight, administered daily; PEG-modified K1-3 4.5 mg/kg body weight, administered every 1, 3, 6, or 9 days).
  • the tumors were grouped up to a length of 1 cm 3 and were administered after grouping.
  • the mode of administration was subcutaneous injection. The administration period was 10 days, and the mice were sacrificed on the 11th day, and the tumor weight was weighed, and the drug effect was evaluated by the tumor inhibition rate.
  • the experimental results showed that: the positive control group had a tumor inhibition rate of 45%, the administration group: 1.5 mg/kg body weight, and the tumor-administered group had tumor inhibition. The rate was 28%; the tumor inhibition rate of the 4.5 mg/kg body weight of the 1, 3, 6, and 9 day administration groups was 60%, 34%, 25%, and 24%, respectively. It is indicated that the same dose of PEG-modified K1-3 has higher antitumor activity than K1-3 in vivo; and the lower dose of PEG-modified K1-3 also has a certain anti-tumor effect. The result is shown in Fig. 17.
  • Example 11 Activity of a product of PEG coupled to the K1-3 N-terminus in the early treatment of a mouse liver cancer (H22) model.
  • K1-3 is K1-3 having the sequence of SEQ ID NO: 2.
  • the in vivo inhibitory activity of PEG-modified K1-3 on mouse liver cancer was observed experimentally.
  • the experimental materials were selected from 20 gram body weight of Babl/c mice (Viton Lihua Experimental Animal Center), and the back was connected with lxlO 6 liver cancer cells.
  • the second day of the tumor was administered in groups.
  • the mode of administration is subcutaneous injection.
  • the drug administration period was 3 weeks, and the mice were sacrificed on the 22nd day, and the tumor weight was weighed, and the drug efficacy was evaluated by the tumor inhibition rate.
  • the experimental results showed that: the positive control group had a tumor inhibition rate of 51%, the administration group: 1.5 mg/kg body weight, and the tumor inhibition rate per day was 54%; The tumor inhibition rate of 4.5 mg/kg body weight per day, twice a week, and once a week was 60%, 84%, and 50%, respectively, indicating the same dose of K1-3, which was higher in vivo than K1-3.
  • K1-3 involved in the present example is Kl-3 having the sequence of SEQ ID NO: 2.
  • NCI-H226 cells human lung adenocarcinoma cell line, obtained from ATCC, accession number CRL-5826
  • mice Institute of Laboratory Animals, Chinese Academy of Medical Sciences
  • the treatment is as shown in Table 1. Animal weight and tumor size were monitored twice a week. Animals were sacrificed on day 28 of treatment and tumors were collected and photographed.
  • Table 1 Animal grouping and treatment plan
  • Taxol was purchased from Peking Union Pharmaceutical Co., Ltd., batch number 060408.
  • the experimental results showed: as shown in Figure 19A, animals treated with 4.5 mg/kg PEG-K1-3 on the 22nd and 25th day after treatment (Group 2) There was a significant difference in body weight compared with the control group (Group 1) (the body weight of the animals in Group 2 was significantly higher than that in Group 1, univariate ANOVA, *p ⁇ 0.05).
  • animals treated with 20 mg/kg Taxol Group 3 showed significant differences in body weight compared to control animals (Group 3 animals weighed significantly less than Group 1, # p ⁇ 0.05).
  • the animals treated with 4.5 mg/kg PEG-K1-3 had a significantly smaller tumor volume than the control group (p ⁇ 0.05).
  • the tumor volume of the third group of animals on days 11, 15, and 18 was significantly different from that of the control group.
  • the T/C ratios of the tumors of Group 2 (4.5 mg/kg PEG-K1-3 ivx 21 days) were 73%, 63%, 62%, and 66%, respectively.
  • the third group (20 mg/kg Taxol) was 69%, 39%, 28% and 18%, respectively.
  • mice treated with 4.5 mg/kg PEG-K1-3 (group 2) and 20 mg/kg were compared with the control group (group 1) as compared with the control group (group 1).
  • the tumor inhibition rates of Taxol-treated animals (Group 3) were 17.18% and 84.43%, respectively.

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Description

包含血管抑素或其片段的复合物、 其制备方法及应用 技术领域
本发明涉及含有血管抑素或其片段， 优选片段 K1-3的复合物和 缓释制剂及其制备方法。本发明涉及的复合物无毒副作用、具有更高 的生物活性以及更好的代谢稳定性。本发明还提供含有这种复合物和 缓释制剂的药物组合物以及含有这种复合物的试剂盒。本发明还提供 使用本发明中的复合物、缓释制剂、药物组合和试剂盒来预防、诊断、 治疗肿瘤、 制备抗肿瘤药物以及抗新生血管疾病的方法。 背景技术
癌症是当今世界的常见病以及多发病，是威胁人类生命的头号杀 手。因此抗癌药物的开发以及研制一直是国内外的热门课题。 目前临 床上常用的抗癌药物多为传统的化疗药物，长期使用易导致耐药性以 及对人体也有很大的毒副作用， 在临床应用上受到很大的'限制。
1971年， Folkman提出肿瘤的生长和迁移依赖于新生血管生成理 论， 新生血管生成是指在原有血管上生成新的毛细血管。 因此， 阻断 胂瘤新生血管生成就可能阻止肿瘤的生长和转移。将胂瘤中的新生血 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， 即抗血管生成治疗 (Folkman J. N EngU Med 1971;285： 1182 -1186)。
血管抑素（Angiostatin)是 1994年 O'Reilly等在 Lewis肺癌荷瘤 小鼠的血清和尿液中提取的内源性血管生成抑制剂。它是小鼠血纤溶 酶原（Plasminogen) N端从第 98位氨基酸残基（以起始氨基酸残基 Met为第一位氨基酸）起始的一段序列 （O'Reilly MS, Holmgren L, Shing Y, et al. Cell, 1994， 79: 315-328) ， 它至少包括血纤溶酶原 5个 Kringles结构中的前 4个，每个 ringle结构由 80个左右氨基酸残基 组成（Lerch P G， Riskli E E, Lergier W, et al. Eur J Biochem, 1980, 107: 研究发现重组人纤溶酶原的 Kl、 Κ2、 Κ3、 Κ5、 Κ1-3、 Κ2-3、 K1-5等都能有效抑制牛毛细血管内皮细胞（bovine capillary endothelial cell, BCE) 细胞增殖， 而 K4几乎无此活性。 K1-3抑制细 胞增殖的活性明显强于最初发现的血管抑素 Kl-4 (Cao Y， Ji RW, Davidson D, et al. J Biol Chem, 1996， 271(46): 29461-29467.)。研究进 一步发现 Kl-3能抑制小鼠 Lewis肺癌生长及转移作用， 也能抑制小 鼠 Lewis肺癌原发瘤及小鼠血管内皮瘤等在内的多种原发瘤的生长。 血管抑素发挥活性的机理在于它通过抑制血管内皮细胞的增殖，从而 抑制了肿瘤组织新生血管的生成，从而切断提供肿瘤组织营养和氧气 的运输通路， 最后饿死肿瘤。 O'Reilly指出， 血管抑素是内源性纤溶 酶原内部的一个片断，它特异性的作用于增生的血管内皮细胞， 因此 一般不会引起免疫反应， 长期注射也不出现体重减轻、活动减少、食 欲下降及条件致病菌的感染等副作用 （ Sim BKL, O'Reilly MS, et al. Cancer Res, 1997, 57: 1329-1334.) ， 同时也不会影响肝、 肾的功能 (Kirsch M, Strasser J, Allende R， et al. J Biol Chem, 1996, 271(46):
29461-29467)。 相比之下， 目前市场上大部分的化疗药物， 主要是针 对肿瘤细胞，但是肿瘤细胞具有遗传的不稳定性以及高的突变率，故 而极易产生耐药性， 影响药效。鉴于以上原因， 血管抑素在肿瘤细胞 的治疗上已经取得很明显的疗效，有望成为二十一世纪的主打抗癌药 物。
然而，尽管包括血管抑素在内的多肽和蛋白质类药物相比于化学 药物具有毒副作用小， 以及不产生耐药性等优点，并且为了尽可能的 提高蛋白药物的生物利用度以及减少其在体内的降解，通常采用静脉 给药方式，但是对于分子量小的蛋白来说，静脉给药的体内半衰期很 短。 体内的蛋白降解只是其中一方面的原因， 另一个重要的方面是， 小分子蛋白能够通过肾过滤的途径很快的被消除。 研究发现，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很快的被消除。所以，为维持小分子量蛋 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抑素同样存在半衰期短，体内清除率高的缺 点。本发明的目的就在于改善该蛋白的体内代 ¾特征，使其具有更高 的稳定性和更长的体内半衰期， 取得更好疗效。
高分子聚合物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。它具有水溶性好、 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。 PEG分子具有两亲性，既可以溶解于 7 ， 又可以溶解于大多数的有机溶剂， 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之一。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，这种方法是延长蛋白质药物体内半衰期的一种有效方 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 PEG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 α-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 N端的 PEG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 PEG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，可以在蛋白任的何部位引 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. 2005Nov
l;10(21):1451-8.)o PEG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（Graham ML Adv. Drug Deliv. Rev. 55， 1293- 1302 Avramis Vassilios I. et al. W09939732 and
US6689762)、 PEG-腺酐脱氨酶（ Levy Y et al. J.Pediatr. 113, 312- 317; Davis S et al. ClinExp. Immunol. 46: 649-652; HershfieldMS et al. N Engl J Med 316： 589-596)和 PEG-干扰素（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.WO
2004076474， Ballon Pascal Sebastian et al. US2004030101, Karasiewicz Robert et al. EP0593868)等。本领域希望得到半衰期较长以及生物活 性更高的血管抑素片段或含有其的复合物。
除 PEG外， 本领域常用的其他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， 也是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 l受体蛋白 (Kitajewsky Jan et al. WO2005- 111072), 促红细胞生成素 (EPO) (Gillies Stephen D et al. WO2005- 063808)、 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)。
另一种延长蛋白体内半衰期的方法是^^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. Pharai. 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))。 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。 发明内容
本发明中描述的 "Kl "、 "K2"、 "K3"、 "K5"、 "K1-2"、 "Kl-3"、 "Kl-5"、 "Κ2-3"、 "Κ2-5"、 "Κ3-5"、 "kringlesl-3 "， 除特殊说明外， 是指血纤溶酶原 （Plasminogen) 的片段， 其中人血 纤溶酶原的序列如 SEQ ID NO:7所示。（参见 U.S. Patent NO:7， 157,556 和 WO 02/26782 A2)—其中 "K1-3"是指 "Kringlesl-3"。 PEG除特 殊说明外， 是指聚乙二醇， 优选单甲基聚乙二醇（methylpolyethylene glycol, mPEG)。
在本发明的一方面，本发明人令人惊讶地发现了一种修饰物与血 管抑素或其片段形成的复合物，所述复合物具有比未修饰的血管抑素 或其片段更长的体内半衰期，所述修饰物选自高分子聚合物、蛋白质 分子或其片段、肽链、小分子或其他任何形式的化学物质。根据本发 明，该复合物不仅保持原有的抑制血管生成的活性，而且具有更高的 生物活性和更长的体内半衰期， 并且对细胞以及动物没有毒副作用， 长期使用， 也不会产生耐药性等副作用。 另外， Henkin等人公开了 PEG与 K5所形成的复合物保持 K5原有的生物活性， 其半衰期提高 到 5.53 h (WO 02/26782 A2)， 但本发明中所描述的 PEG与 K1-3所 形成的复合物体内半衰期延长至 81.5 h,远远高于 PEG与 K5所形成 的复合物（参见本发明实施例 4) 。 对于临床应用来说， 具有更长的 给药间隔或者是更少的给药剂量，这对于患者无论是经济上还是精神 上， 都是具有非常重要的意义。
在一个优选的实施方案中，本发明的血管抑素是人、 鼠或其他哺 乳动物来源的。 更优选地， 所述血管抑素是人血管抑素。
在另一个优选的实施方案中， 本发明的血管抑素是重组血管抑 素。 更优选地， 所述重组血管抑素是重组人血管抑素。
在一个优选的实施方案中，本发明的血管抑素的片段选自血管抑 素 Kl、 K2、 K3、 K5、 Kl-2、 Kl-3、 Kl-5、 Κ2-3、 Κ2-5和 Κ3-5。 更 优选地， 所述血管抑素片段是具有 SEQ ID NO:3所示序列的血管抑 素 K1、 具有 SEQ ID NO:4所示序列的血管抑素 K2或具有 SEQ ID NO:5所示序列的血管抑素 K3。最优选地， 本发明所述血管抑素的片 段是具有 SEQ ID ΝΟ:1所示序列的血管抑素 K1-3, 并且当所述人血 管抑素是由大肠肝菌表达的重组人血管抑素 K1-3时，其具有 SEQ ID NO:2的序列， 其中 N末端的 Met在大肠肝菌表达时任选地被删除。
在优选的实施方案中，用于本发明的复合物的血管抑素或其片段 还包括血管抑素或其片段的具有活性的突变体、衍生物、异构体或是 它们的组合。在一个优选的实施方案中，所述血管抑素或其片段的衍 生物是在血管抑素或其片段的 N端或 C端附加一段长度为 1-15个氨 基酸的肽链形成的。在一个进一步优选的实施方案中，所述血管抑素 或其片段的衍生物是在人血管抑素的 N端附加一段 7个氨基酸组成 的含有 His-tag的肽链 MHHHHHH,该衍生物具有 SEQ ID NO:6的序 列， 并且当由大肠杆菌表达时其 N末端的 Met任选地被删除。 在另 一个优选的实施方案中，所述血管抑素或其片段的衍生物是在人血管 抑素的 N端附加一段序列为 MGGSHHHHH或 MGGSHHHHHH的肽 链， 并且当由大肠杆菌表达时其 N末端的 Met任选地被删除。
在本发明的一个实施方案中，所述复合物中的修饰物与血管抑素 或其片段通过共价键连接。在本发明的另一个实施方案中，所述复合 物中的修饰物与血管抑素或其片段通过非共价键连接。优选地，修饰 物可以通过化学偶联的方法连接到血管抑素或其片段上，也可以通过 融合表达的方式和血管抑素或其片段连接到一起。本发明的高分子聚 合物可以有生物活性，也可以没有生物活性。合适的聚合物包括但不 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、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、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-Hydroxyethyl)-DL-aspartamide))、 聚幾酸
(polycarboxylates)、 氧化乙烯和甲醛的共聚物
(polyoxyethylene-oxymethylenes) ^ 聚两稀酰吗啉 (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 (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。
本发明的聚醚类化合物包括但不限于聚烷撑二醇 (HO((CH2)xO)„H)、 聚丙二醇、 聚氧化乙烯 (HO((CH2)20)nH)、 聚乙烯 醇 (XCH2CHOH)n)或它们的衍生物， 所述聚烯醇类化合物包括但不限 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、聚丁烯醇或它们的衍生物。在一个优选的实施方案中， 所 述修饰物是聚乙二醇。一个更优选的方案中，所述聚乙二醇选用单甲 基聚乙 _ >醇。
本发明使用的聚乙二醇分子是线性的或是分叉的。优选地，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。 更优 选地，所述聚乙二醇分子的平均分子量位于 5,000到 40,000道尔顿之 间。最优选地， 所述聚乙二醇是分子量为 20 kDa的单甲基聚乙二醇。
在本发明的一个实施方案中，一个血管抑素分子或其片段和一个 或多个聚乙二醇分子偶联。优选地，所述修饰是位点特异性的， 不会 影响血管抑素或其片段的生物活性。该修饰产物具有抗新生血管生成 的活性， 并且相比于无修饰物的血管抑素或其片段在代谢上更稳定， 具有更长的血液半衰期，可以作为抗新生血管生成疾病或抗肿瘤的药 物。优选地，所述血管抑素分子或其片段与聚乙二醇分子偶联的位点 选自血管抑素或其片段的 N端 α-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 链的羧基中的一或多种。更优选地，所述血管抑素分子或其片段与聚 乙二醇分子偶联的位点选自血管抑素或其片段 Ν端 a-氨基或 SEQ ID ΝΟ:1所示序列第 2、 7、 15、 17、 24、 69、 94、 97、 121、 125、 128、 129、 150、 175、 215、 228、 246位的赖氨酸残基侧链上的 ε-氨基中 的一或多个。更优选地，一个血管抑素分子或其片段与一个聚乙二醇 分子偶联，偶联位点为血管抑素或其片段 Ν端的 α-氨基。更优选地， 在本发明的复合物中， 一个具有 SEQ ID NO:2的重组人血管抑素分 子的片段 K1-3与一个聚乙二醇分子偶联， 偶联位点为血管抑素片段 Kl-3 N端的 a-氨基。最优选地，在本发明的复合物中，一个具有 SEQ ID NO:2的重组人血管抑素分子的片段 Kl-3与一个 20 kDa单甲基聚 乙二醇分子偶联， 偶联位点为血管抑素片段 K1-3 N端的 a-氨基。在 一个优选的实施方案中， 本发明使用 PEG特异修饰 K1-3 N端的 α- 氨基， 其产物具有抑制内皮细胞增殖及迁移， 抑制肿瘤细胞增殖， 抑 制小鼠肿瘤生长的生物活性。
在本发明的另一个实施方案中，一个血管抑素分子或其片段与一 个或多个聚乙二醇分子偶联，偶联方法为在血管抑素分子或其片段内 部或其 N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，合适的聚氨基酸包括，但不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。
根据本发明，适合用作修饰载体的蛋白分子优选天然存在的蛋白 或其片段，包括但不限于甲状腺结合蛋白、转甲状腺蛋白、转铁蛋白、 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却保持了载体 功能的部分。在一个优选的实施方案中，本发明的复合物是白蛋白偶 联的血管抑素或其片段，其特征为一个血管抑素分子或其片段和一个 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。在另一个优选的实施方案中，本发明的复合物是 免疫球蛋白 Fc片段偶联的血管抑素或其片段， 其特征为一个血管抑 素分子或其片段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。
本发明的修饰物还包括小分子或是小肽或是其他化合物。在一个 实施方案中，本发明的复合物是小分子或是小肽或是其他化合物修饰 的血管抑素或其片段，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， 其中所述活性是由可以和血液成分的氨基、羟基、巯基反应 形成共价键的反应活性基团产生的，优选地所述反应活性基团是马来 酰亚胺， 它可以和血液成分， 例如白蛋白中的巯基反应。在一个优选 的实施方案中，本发明的复合物是血管抑素或其片段被糖基化、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。该复合物没有很大的分子量， 但是因为修饰改变了 K1-3的性质， 从而延长了其半衰期。
本发明的血管抑素或其片段与上述修饰物直接或间接共价相连， 直接连接指的是血管抑素或其片段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。间接相连指的血管抑素 或其片段和载体蛋白通过一定的化学基团或几个基团的组合连接。当 本发明的血管抑素或其片段与修饰物通过非共价键相互作用时，该复 合物具有特定的组成式，其中的修饰物优选地是蛋白、小分子或其他 化学物质。
本发明的另一方面提供一种缓释制剂， 它是由血管抑素或其片 段， 优选片段 Kl-3， 或上述的任一复合物与生物相容性物质形成的。 该组合物中的血管抑素或其片段仍然具有生物活性，但却可以依靠该 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 剂包括但不限于微胶囊、 水凝胶、 微球、 微型渗透泵、 脂质体等。
在本发明的另一方面，提供了一种药物组合物，其由上述复合物 或缓释制剂与药学上可接受的载体形成，所述复合物由一种修饰物与 血管抑素或其片段， 优选血管抑素片段 K1-3形成。 本发明使用的药 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 ρΗ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、 和 PLURONICS®等非离子 表面活性剂。赋形剂优选无菌且一般不含不良物质。这些组合物可通 过常规的灭菌技术进行灭菌。
本发明的另一方面还提供一种试剂盒，其包括上述复合物、缓释 制剂或药物组合物和使用说明，其中所述复合物由一种修饰物与血管 抑素或其片段形成， 优选地是聚乙二醇修饰的血管抑素片段 Kl-3。
本发明还涉及到制备本发明的复合物的方法，所述复合物由一种 修饰物与血管抑素或其片段， 优选片段 K1-3形成。 特别涉及制备聚 乙二醇 (PEG)修饰的血管抑素或其片段的方法， 即将活化的 PEG 与血管抑素或其片段混合， 在适当的溶液、 温度、 pH、 摩尔比条件 下反应， 并任选地用阳离子柱或分子筛纯化偶联产物。 其中反应的 pH优选 pH 3-10之间， 更优选 pH5-7之间， 聚乙二醇与血管抑素摩 尔比为 1:1到 10:1。
本发明还提供一种上述复合物、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，其中所述复合物由一种修饰物与 血管抑素或其片段形成， 优选地是聚乙二醇修饰的血管抑素片段 Kl-3。本方法适合的癌症包括但不限于肺癌、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抑素或其片段， 优选 K1-3 的复合物、药物组合物或缓释制 ¾1在制备抗肿瘤药物中的用途，其中 所述药物适于静脉注射、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， 给药时间 由每天到每 21天给药， 优选每天至每周给药。
本发明还提供一种上述复合物、缓释制剂、药物组合物或试剂盒 在预防、诊断或治疗非肿瘤疾病的药物中的用途，其中所述复合物由 一种修饰物与血管抑素或其片段形成，优选地是聚乙二醇修饰的血管 抑素片段 Kl-3。 所述非肿瘤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，所述药物适于静脉注射、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，给药时间由每天到每 21天给药， 优选每天至每周给药。
本发明还提供一种延长血管抑素或其片段， 优选延长片段 K1-3 半衰期的方法，所述方法包含将修饰物与血管抑素或其片段形成复合 物的步骤以及任选地包含制备由上述复合物和生物相容性物质形成 的缓释制剂的步骤。 实验表明， 本发明优选的实施方案中的 PEG与 Kl-3 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 K1-3相比， 活性明显提高， 并且体内药代学研究表明 修饰后的 K1-3能有效的减缓 K1-3在体内的代谢，延长体内代谢时间。
本发明中提到的血管抑素或其片段， 除特殊说明以外，包括野生 型的血管抑素或其片段，即体内自然存在的形式或其具有活性的突变 体、 片段、异构体、衍生物等或它们的组合。血管抑素或其片段的来 源可以是但不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或酵母细胞表达的人血管抑素片 段 K1-3具有如 SEQ ID NO:l所示的氨基酸序列，或在此序列的 N端 和 /或 C端有 10个氨基酸以内的增加或缺失的序 来源于大肠肝菌 表达的 K1-3具有 SEQ ID NO:2的序列，但其 N末端的 Met在表达后 有时会被删除。
K1-3的突变体指的是通过氨基酸的取代、 缺失、 增加而得到的 Kl-3 o 其片段是指序列属于 SEQ ID Ο: 1或 SEQ ID NO:2的任何更 小的部分， 可 ¾是但不限于通过酶切得到的， 或基因工程表达的， 或 通过多肽合成的方法得到的。 优选地， 所述突变体与 SEQ ID NC 具有 60%氨基酸序列同源性， 或是具有 70%氨基酸序列同源性， 更 期望的是具有 80%以至 90%的同源性， 特别是有 95%的序列同源性。 所述片段是指包含 SEQ ID NO: 1的一部分的序列， 例如具有 SEQ ID NO:3、 SEQ ID NO:4、 SEQ ID NO:5的序列。 曾有报道具有 SEQ ID NO:3、 SEQ ID NO:4、 SEQ ID NO:5的序列的片段具有抑制内皮细胞 增殖的活性（Cattaneo MG et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY. Vol. 271, No. 46， Issue of November 15， pp. 29461 - 29467, 1996) 。 Kl -3的异构体是指具有和 SEQ ID NO:l相同的氨基 酸序列或分子式，但是却有不同的构象，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。 K1-3的衍生物是指在 SEQ ID NOrl 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 30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。 K1-3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 以上上述特征变化的产物，但不限于片段的突变体、突变体的修饰产 物等。 附图说明：
图 1： 显示人血管抑素片段 K1-3的氨基酸序列 SEQ ID ΝΟ:1。 图 2: 显示大肠杆菌表达的人血管抑素片段 K1-3的氨基酸序列 SEQ ID NO:2。 这里 N末端的 Met在表达后有时会被删除。 图 3: 显示人血管抑素 k 1的氨基酸序列 SEQ ID NO:3。
图 4: 显示人血管抑素 k 2的氨基酸序列 SEQ ID NO:4。
图 5: 显示人血管抑素 k 3的氨基酸序列 SEQ ID NO:5。
图 6: 显示一种具有活性的 N端附加氨基酸的 K1-3的氨基酸序 列 SEQ ID NO: 6。 N末端的 Met在表达后有时会被删除。
图 7: PEG与具有 SEQ ID NO:2序列的 K1-3的 N端的偶联。 反 应条件是 4°C， 10h。 反应后溶液用 SDS- PAGE检测， 泳道 1 : 蛋白 分子量标准； 泳道 2: K1-3; 泳道 3〜8分别是 PEG与 K1-3摩尔比均 为 5:1， pH分别为 5.0、 5.5、 6.0、 6.5、 7.0、 8.0条件修饰后的 K1-3; 泳道 9〜12分别是 pH5.5、 20 kDa PEG与 K1-3摩尔比分别为 1 :1、 2:1、 5:1、 10:1条件修饰后的 K1 -3。
图 8: PEG与具有 SEQ ID NO:2序列的 K1-3的 N端的偶联。 反 应条件是室温， 10 h。 反应后溶液用 SDS- PAGE检测， 泳道 1 : 蛋白 分子量标准； 泳道 2: K1-3; 泳道 3~8分别是 PEG与 K1-3摩尔比 均为 5:1， pH分别为 5.0、 5.5、 6.0、 6.5、 7.0、 8.0条件修饰后的 K1-3; 泳道 9~12分别是 pH5.5、 PEG与 K1-3摩尔比分别为 1:1、 2:1、 5:1、 10:1条件修饰后的 Kl-3。
图 9: PEG与具有 SEQ ID NO:2序列的 K1-3的 Ν端的偶联。 反 应前后溶液用 SDS-PAGE检测， 泳道 1 : 蛋白分子量标准； 泳道 2: K1-3; 泳道 3: PEG修饰的 Kl-3。
图 10: PEG修饰具有 SEQ ID NO:2序列的 K1-3的 N端偶联后， 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 : 蛋白分 子量标准； 泳道 2: 纯化前； 泳道 3: 上样穿透的溶液； 泳道 4-13: 分布收集的洗脱液。
图 11 : PEG与 Kl-3 N端偶联的产物的体内代谢试验结果。 K1-3: 具有 SEQ ID NO:2序列的 Kl-3; PEG- K1-3 : PEG修饰的具有 SEQ IDNO:2序列的 Kl-3。
图 12: PEG与 K1-3N端偶联的产物抑制人脐静脉血管内皮细胞 (HUVEC)迁移的活性。 K1-3:具有 SEQIDNO: 2序列的 Kl-3; PEG- Kl-3: PEG修饰的具有 SEQ IDNO:2序列的 Kl-3。
图 13: PEG与 K1-3N端偶联的产物抑制人脐静脉血管内皮细胞 (HUVEC)损伤的自我修复活性。 K1-3: 具有 SEQIDNO:2序列的 Kl-3; PEG-K1-3: PEG修饰的具有 SEQIDNO:2序列的 Kl-3。
图 14: PEG与 K1-3N端偶联的产物抑制人脐静脉血管内皮细胞 (HUVEC)增殖的活性。 K1-3:具有 SEQIDNO:2序列的 Kl-3; PEG- Kl-3: PEG修饰的具有 SEQ IDNO:2序列的 Kl-3。
图 15: PEG与 K1-3N端偶联的产物抑制人微血管内皮细胞 (HMEC)增殖的活性。 K1-3: 具有 SEQIDNO:2序列的 Kl-3; PEG- Kl-3: PEG修饰的具有 SEQIDNO:2序列的 Kl-3。
图 16: PEG与 K1-3N端偶联的产物抑制肿瘤细胞（Hela)增殖 的活性。 K1-3: 具有 SEQIDNO:2序列的 Kl-3; PEG-K1-3: PEG 修饰的具有 SEQ ID NO:2序列的 Kl-3。
图 17: PEG与 Kl-3 Ν端偶联的产物抑制小鼠黑色素瘤（B16) 的活性。 K1-3- 4.5-1： 具有 SEQIDNO: 2序列的 Kl-3， 4.5mg/kg体 重， 每天给药； PEG-K1-3-1.5-1: PEG修饰的具有 SEQIDNO: 2序 列的 Kl-3， 1.5mg/kg体重， 每天给药； PEG- Kl-3-4.5-1: PEG修饰 的具有 SEQ ID NO: 2序列的 Kl-3， 4.5mg/kg体重，每天给药； PEG- Kl-3-4.5-3: PEG修饰的具有 SEQIDNO: 2序列的 K1-3, 4.5 mg/kg 体重， 每 3天给药； PEG-K1-3-4.5-6: PEG修饰的具有 SEQ ID NO :2 序列的 Kl-3， 4.5 mg/kg体重， 每 6天给药。 PEG- K1 -3-4.5-9: PEG 修饰的具有 SEQ ID NO: 2序列的 Kl-3, 4.5 mg/kg体重，每 9天给药。
图 18: PEG与 Kl-3 N端偶联的产物抑制小鼠肝癌（H22)的活 性。 K1-3- 4.5-1: 具有 SEQIDNO: 2序列的 K1-3, 4.5 mg/kg体重， 每天给药； PEG- K1-3-1.5-1 : PEG修饰的具有 SEQ ID NO: 2序列的 Kl-3, 1.5 mg/kg体重， 每天给药； PEG- K1-3-4.5-1 : PEG修饰的具 有 SEQ ID NO: 2序列的 Kl-3， 4.5 mg/kg体重， 每天给药； PEG- Kl-3-4.5-3: PEG修饰的具有 8£(^ 10 ^[0: 2序列的1^1-3， 4.5 mg/kg 体重，每周给药 2次; PEG- Kl-3-4.5-7: PEG修饰的具有 SEQ ID NO:2 序列的 Kl-3， 4.5 mg/kg体重， 每周给药 1次。 具体实施方式
为了进一步说明本发明，提供下述实施例。下述实施例仅是为了 说明本发明而非对本发明进行具体限制。本领域技术人员清楚本发明 同时还包括对下述实施例所述的技术方案进行任何无需创造性劳动 的改变所产生的技术方案。
除非特别说明， 下述实施例中所用聚乙二醇 (PEG)是 082M0P01 MPEG-BUTY ALD-20K, NETKTAR™, Lot#: PT-03G -05。 实施例 1、 不同条件下 PEG与 K1-3 N端的偶联。
本实施例涉及的 Kl-3(购自普罗吉公司， 中国)是具有 SEQ ID NO:2序列的 Kl-3。 将 Kl-3透析到 ρΗ 5.5的 30 mM醋酸钠 (购自北 京化学试弃公司， 中国)溶液中。 用紫外分光光度计 (8453， Agilent) 在 280 nm波长下测量蛋白浓度， 然后将浓度调节到 1 mg/ml。 Kl-3 与特异修饰蛋白 N端的 PEG偶联。第一种情况,在 pH值 5.5时，取 10 ml上述含有 K1-3的透析后的溶液（含蛋白 10 mg) ， 按照 PEG与 K1-3的摩尔比分别为 1:1、 2:1、 5:1、 10:1， 加入 20 kDa PEG固体， 并室温搅拌，直到完全溶解， 加入还原剂 CH3BNNa (Sigma), 浓度为 20 mM, 并最后调节溶液的 pH值为 5.5。 第二种情况，取 10 ml上述 含有 K1-3的透析后的溶液 （含蛋白 10 mg) ， 按照 PEG与 K1-3的 摩尔比为 5:1， 加入 PEG固体， 室温搅拌， 直到完全溶解，加入还原 剂 CH3BNNa，浓度为 20 mM,最后调节 pH值分别为 5.0, 5.5, 6.0， 6.5, 7.0, 8.0ο 分别在室温和 4°C静置不同时间以后， 观察比较 K1-3被单 一 PEG修饰的情况， 即一个 PEG与一个 K1-3偶联， 偶联的位点是 K1-3 N端的 a-氨基， 少量 K1-3会被非特异性的多位点修饰。 这样 就可以找出 PEG与 K1-3反应的最佳条件。反应前后溶液电泳结果如 图 7-8所示。 实施例 2、 PEG与 K1-3 N端的偶联。
本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 K1-3。将 K1-3 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 1 mg/ml。 与特异修饰蛋白 N端的 PEG偶联时，取 20∞1上述含有 1-3的透析后的溶液（含蛋 白 20 mg) ， 加入 PEG固体 100 mg， 并室温搅拌， 直到完全溶解， 使得 PEG与 K1-3的摩尔比为 5:1。加入还原剂 C¾BNNa,浓度为 20 mM, 并最后调节溶液的 pH值为 5.5。 室温静置 6 h以后， 高于 80% 的 K1-3被单一 PEG修饰， 即一个 PEG与一个 K1-3分子偶联， 并且 偶联的位点是 Kl-3 N端的 α-氨基， 少量 K1-3会被非特异性的多位 点修饰。 这时反应液可直接上柱纯化。 反应前后溶液电泳结果如图 9 所示。 实施例 3、 PEG修饰 K1-3 N端后用阳离子柱 SP纯化。
本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 Kl-3。将 PEG 修饰的 Kl-3用 SP层析柱（Amersham)纯化。 将反应后的混合液调 节 pH为 5。 层析柱用含有 20 mM醋酸钠， pH值为 5.0 的平衡缓冲 液平衡后上样。 上样后再用含有 20 mM醋酸钠， 1M氯化钠， pH 5.0 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 Kl-3、 单修饰 Kl-3、 Kl-3。 根据 280nm的紫外检测可以收集不同的分离组分。 纯 化结果如图 10所示。 实施例 4、 PEG与 K1-3 N端偶联的产物在血液中的长效效应。 本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 Kl-3。测定 Κ 1-3和 PEG修饰的 K1-3在 Wistar大鼠 (维通利华实验动物中心)体内 的代谢速率来检验 PEG修饰后产物在血液中的长效效益。 选用 2只 200克左右体重的健康大鼠， 分别尾静脉注射 K1-3和 PEG修饰的 K1-3 N端偶联产物， 4.5 mg/kg体重。 按照 0、 5、 10、 30分钟、 1、 2、 4、 8、 16、 24、 36、 48、 72 、 96、 120、 144 h的时间间隔眼眶取 血。 收集血浆， 用夹心法 ELISA分别测 K1-3和 PEG修饰的 K1-3 的浓度。体内药代动力学显示， PEG修饰的 K1-3在大鼠体内的半衰 期为 81.5 h，而 K1-3在大鼠体内的半衰期仅为 12.2 h，这表明偶联高 分子量的 PEG能有效的增加 K1-3在体内代谢的半衰期，以达到长效 的目的。 结果如图 11所示。 实施例 5、 PEG与 Kl-3 N端偶联的产物抑制人脐静脉血管内皮 细胞（HUVEC)迁移的活性。
本实施例涉及的 K1 -3是具有 SEQ ID NO:2序列的 Kl-3。 将 HUVEC (普罗吉公司， 北京)在含有 20%胎牛血清 (Hyclone)的 M199 培养液 (Hyclone)中培养至对数生长期。然后饥饿 12 h。之后加入测细 胞迁移皿 (Millipore ,USA)中， 每孔细胞数 104个。 迁移条件为含 5% 胎牛血清的 M199培养液。加药组分别加入 K1-3和 PEG修饰的 K1-3, 浓度分别为 0.004 g/ml、 0.04 g/ml、 0.4 g/ml、 4 g/ml。 37。C培养 8 h， 用 1%戊二醛 (北京化工厂， 中国)固定细胞， 刮掉膜上层的未迁 移细胞， 用苏木精、 伊红 (北京化学试剂公司， 中国)染色。 在显微镜 下， 取大小相同三个视野， 计算细胞数， 最后算出抑制率。 结果显示 在 0.04-4 g/ml的范围内， 随着给药浓度的增加， 对细胞迁移的抑制 增强， 结果显示具有 SEQ ID NO:2序列的 K1-3对细胞迁移的抑制率 分别为 43%、 68%、 76%, PEG修饰后的 K1-3对细胞迁移的抑制率 达到 58%、 65%、 69%。 以上结果表明经过 PEG修饰的 K1-3保持其 原有的生物活性。 结果如图 12所示。 实施例 6、 PEG与 Kl-3 N端偶联的产物抑制人脐静脉血管内皮 细胞（HUVEC)损伤修复的活性。
本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 K1-3对 HUVEC损伤修复的影响。 将 HUVEC以 2xl05个 /ml 的密度接种在 12孔板 (Coming公司)中， 在含 20%胎牛血清的 M199完全培养液中 培养至对数生长期。然后饥饿 12 h， 在孔中轻轻划一十字交叉线， 洗 净悬浮细胞。试验设为阴性对照组（PBS)， 阳性对照组（K1-3浓度 分别为 0.04 g/ml、 0.4
Figure imgf000020_0001
， 给药组（PEG修饰的 K1-3 的浓度分别为： 0.04 g/ml、 0.4 g/ml、 4 μδ/ηι1) 。 37°C培养 16 h， 测细胞的迁移量。通过对细胞迁移的抑制来计算其药效。结果显示在 0.04-4 g/ml的范围内， 随着给药浓度的增加， 对细胞迁移的抑制增 加， 其抑制率分别为 0%、 20%、 25%； PEG修饰的 K1-3的抑制率分 别为 35%、 40%、 105%。 结果表明经 PEG修饰后的 K1-3不仅保持 而且大大加强其生物活性， 同时在 4 g/ml浓度时， 伴随一些细胞的 皱缩死亡。 结果如图 13所示。 实施例 7、 PEG与 Kl-3 N端偶联的产物抑制人脐静脉血管内皮 细胞（HUVEC)增殖的活性。
本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 Kl-3。采用饥 饿过夜的 HUVEC,以 2xl04个 /ml的密度接种在 96孔板 (Coming)中， 含 20%胎牛血清的 M199培养液中培养至对数生长期。然后饥饿 12 ho 在按照试验设计加入不同浓度的 K'l -3以及 PEG修饰的 Kl-3。 实验 设为阴性对照组（PBS)、阳性对照组（K1-3浓度分别为 0.04 g/ml、 0.4 4 g/ml)、给药组（PEG修饰的 Kl-3的浓度分别为： 0.04 μ^πιΚ 0.4 μ^ηιΚ 4 μ^ηιΐ) 。 37°C培养 48 h， 加入 MTT (Amresco 公司）， 37°C孵育 4 h， 加入 DMSO (生工生物工程公司， 中国），酶 标仪 （Model 550，Biorad公司） 570 nm测其吸收值。 结果如图 14所 示。 实施例 8、 PEG与 Kl-3 N端偶联的产物抑制人微血管内皮细胞 (HMEC)增殖的活性。
本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 Kl-3。采用饥 饿过夜 HMEC (ATCC# CRL 10636， USA), 以 2xl04个 /ml的密度 接种在 96孔板中， 在含 10%血清的 DMEM中培养至对数生长期。 然后饥饿 12 h。在预先试验设计加入不同浓度的未修饰以及修饰后的 Kl-3。 实验设阴性对照组（PBS) 、 阳性对照组 （K1-3浓度分别为 0.04 μ^πιΚ 0.4 μ^πιΚ 4 μδ/πι1)、 给药组（PEG修饰的 Kl-3的浓度 分别为： 0.04 g/ml、 0.4 g/ml、 4 g/ml)。 37°C培养 48 h,加入 MTT, 37°C孵育 4 h， 加入 DMSO，酶标仪 570 nm测其吸收值。 结果如图 15所示。 实施例 9、 PEG与 Kl-3 N端偶联的产物抑制肿瘤细胞（人宫颈 癌细胞， Hela)增殖的活性。
本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 Kl-3。以 2χ 104 的密度将 Hela细胞（ATCC# CCL-2， USA)接种在 96孔板中， 在含 10%血清的 DMEM中培养至对数生长期。 然后饥饿 12 h。 在按照试 验设计加入不同浓度的 K1-3以及 PEG修饰的 Kl-3。 实验设阴性对 照组 （PBS) 、 阳性对照组 （K1-3浓度分别为 0.4 g/ml、 4 g/ml、 40 g/ml、 150 g/ml) 、 给药组 （PEG修饰的 Kl-3的浓度分别为： 0.4 μ§/πύ, 4 μξ/πύ, 40 g/ml、 150 μ^/πύ) 。 37°C培养 48 h， 加入 MTT， 37°C孵育 4 h， 加入 DMSO，酶标仪 570 nm测其吸收值。结 果显示， PEG修饰后的 K1-3比 K1-3具有更好的抑制肿瘤细胞增殖 的活性。实验中还发现， K1-3和 PEG修饰的 K1-3在浓度为 40 μ§/πι1 时的抑制肿瘤细胞增殖的活性最好， 明显的高于 150 μ§/πι1以及 0.4 μ^πι 4 g/ml。 实验结果如图 16所示。 实施例 10、 PEG与 Kl-3 端偶联的产物在小鼠黑色素瘤 (B16) 模型晚期治疗中的活性。
本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 Kl-3。实验观 察了 PEG修饰的 K1-3对小鼠 B16肿瘤的体内抑制活性。实验材料选 用 20克体重的 C57B/L小鼠 (维通利华实验动物中心)， 背部接入 I xlO6个黑色素瘤细胞 (ATCC# CRL-6475TM， USA)。 待肿瘤长起分 组， 每组 8只， 分别设阴性对照组 （20ιηΜ醋酸钠溶液） 、 阳性对 照组（K1-3 4.5 mg/kg体重，每天给药）、给药组（PEG修饰的 K1-3 1.5 mg/kg体重， 每天给药； PEG修饰的 K1-3 4.5 mg/kg体重， 分别 每 1、 3、 6、 9天给药) 。 待肿瘤长至 l cm3开始分组， 分组后即可 给药， 给药方式为皮下注射。 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。 对于具有 SEQ ID NO:2序列的重组 人血管抑素 K1-3 , 实验结果显示： 阳性对照组的抑瘤率为 45%， 给 药组： 1.5 mg/kg体重， 每天给药组的抑瘤率为 28%; 4.5 mg/kg体 重每 1、 3、 6、 9天给药组的抑瘤率分别为 60%、 34%、 25%、 24%。 表明同剂量的 PEG修饰的 K1-3 , 比 K1-3具有体内更高的抑瘤活性； 并且较低给药剂量的 PEG修饰的 K1-3也具有一定的抑瘤效果。结果 如图 17所示。 实施例 11、 PEG与 K1-3 N端偶联的产物在小鼠肝癌 (H22)模 型早期治疗中的活性。
本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 K1-3。实验观 察了 PEG修饰的 K1-3对小鼠肝癌的体内抑制活性。 实验材料选用 20克体重的 Babl/c小鼠 (维通利华实验动物中心)， 背部接入 lxlO6 个肝癌细胞。 分别设定阴性对照组 （20mM醋酸钠溶液）、 阳性对 照组（K1-3 4.5 mg/kg体重，每天给药）、给药组（PEG修饰的 K1-3 1.5 mg/kg体重， 每天给药； 4.5 mg/kg体重， 分别每天、每周 2次、 每周 1次给药） 。接瘤的第 2天分组给药。给药方式为皮下注射。给 药周期为 3周， 第 22天处死小鼠， 称瘤重， 以抑瘤率来评价药效。 对于具有 SEQ ID NO:2序列的 Kl-3， 实验结果显示： 阳性对照组的 抑瘤率为 51%，给药组： 1.5 mg/kg体重，每天给药组的抑瘤率为 54%; 4.5 mg/kg体重每天、每周 2次、每周 1次给药组的抑瘤率分别为 60%、 84%、 50% 表明同剂量的 K1-3, 比 K1-3具有体内更高的抑制肝癌 生长的活性； 并且低剂量修饰的 K1-3组对肝癌生长的抑制率高于高 剂量 K1-3组； 同时由于 PEG修饰的 K1-3在体内代谢速度减慢， 因 此在延长给药间隔的情况下，相同剂量的 PEG修饰的 K1-3高于 K1-3 的生物活性， 其中每周给药 2次的抑瘤效果最好， 达到了 80%以上。 结果如图 18所示。 实施例 12、 PEG 与 Kl-3 N 端偶联的产物 (PEG-K1-3)在 NCI-H226非小细胞肺癌异种移植模型中的体内抗肿瘤作用
本实施例涉及的 K1-3是具有 SEQ ID NO:2序列的 Kl-3。将 2><106 NCI-H226细胞（人类肺腺癌细胞系，获自 ATCC,保藏号 CRL-5826) 皮下接种于 6-8周龄的体重为 18-20g的雌性 Balb/c nu/nu小鼠（中国 医学科学院实验动物研究所）右侧。当肿瘤大小达到大约 80-125 mm3 时将动物随机分为 3组 （每组 6只）。 如表 1所示的方案进行处理。 每周 2次监测动物的体重和肿瘤大小。 在处理的第 28天处死动物收 集肿瘤并拍照。 表 1 : 动物分组及处理方案
Figure imgf000024_0001
注： Taxol购自北京协和药厂， 批号 060408 实验结果显示： 如图 19A所示， 在处理后第 22天和第 25天， 以 4.5 mg/kg PEG-K1-3处理的动物（第 2组） 与对照组动物 （第 1 组）相比， 体重存在明显的差异 （第 2组动物的体重明显高于第 1 组，单变量 ANOVA, *p<0.05 )。在第 11天和第 25天，以 20 mg/kg Taxol 处理的动物（第 3组）与对照组动物相比， 体重存在明显的差异（第 3组动物的体重明显低于第 1组， # p<0.05)。
如图 19B所示， 以 4.5 mg/kg PEG-K1-3处理的动物（第 2组） 与对照组相比， 肿瘤的体积明显缩小（p<0.05)。 第 3组动物在第 11、 15和 18天的肿瘤体积与对照组相比有明显的差异。 此外， 在第 4、 11、 18和 25天， 第 2组（4.5mg/kg PEG-K1-3 i.v.x 21天）的肿瘤的 T/C比分别为 73%、 63%、 62%和 66%,而第 3组（20 mg/kg Taxol)分 别为 69%、 39%、 28%和 18%。
如图 19C所示， 以处理 21天后的肿瘤重量计算， 与对照组 （第 1组）相比， 以 4.5 mg/kg PEG-K1-3处理的动物（第 2组）和以 20 mg/kg Taxol处理的动物（第 3 组） 的肿瘤抑制率分别为 17.18%和 84.43%。 接受 4.5 mg/kg PEG-K1-3的动物和对照组动物的肿瘤重量 与 Taxol处理组的动物相比具有明显的差异 （分别为 P=0.008 和 Ρ=0·026)。

Claims

权 利 要 求 书
1. 一种修饰物与血管抑素片段 K1-3形成的复合物，所述复合物具有 比未修饰的血管抑素片段 K1-3更长的体内半衰期，所述修饰物选 自聚乙二醇或其他高分子聚合物、 蛋白质分子或其片段、 肽链、 小分子或其他任何形式的化学物质。
2. 权利要求 1的复合物， 其中所述血管抑素片段 K1-3是人、 鼠或其 他哺乳动物来源的血纤溶酶原片段 K1-3或其具有活性的片段、突 变体、 衍生物、 异构体或是它们的组合。
3. 权利要求 2的复合物，其中所述血管抑素片段 K1-3是具有 SEQ ID ΝΟ:1所示序列的人血管抑素片段 K1-3或其具有活性的片段、 突 变体、 衍生物、 异构体或是它们的组合。
4. 权利要求 1的复合物，其中所述血管抑素片段 K1-3是重组血管抑 素片段 Kl-3。
5. 权利要求 4的复合物， 其中所述重组血管抑素片段 K1-3是具有 SEQ ID NO:2所示序列的重组人血管抑素片段 K1-3或其具有活性 的片段、 突变体、 衍生物、 异构体或是它们的组合。
6. 权利要求 5的复合物，其中所述重组人血管抑素片段 K1-3由大肠 肝菌表达， 其中 N末端的 Met在大肠肝菌表达时任选地被删除。
7. 权利要求 1-6任一项的复合物， 所述修饰物与血管抑素片段 K1-3 通过共价键连接。
8. 权利要求 1的复合物， 所述修饰物是聚乙二醇。
9. 权利要求 8的复合物， 所述聚乙二醇是单甲基聚乙二醇。
10.权利要求 8的复合物， 其中所述聚乙二醇是线性的或是分叉的。
11.权利要求 8-10的复合物， 其中所述聚乙二醇分子量位于 1，000到 100,000道尔顿之间， 优选分子量为 20 kDa的单甲基聚乙二醇。
12.权利要求 8-10任一项的复合物，其特征为一个血管抑素片段 K1-3 与一个或多个聚乙二醇分子偶联， 偶联的位点是血管抑素的 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其组 合。
13.权利要求 12的复合物， 其特征为一个血管抑素片段 K1-3与一个 或多个聚乙二醇分子偶联， 偶联的位点是血管抑素片段 K1-3的 Ν 端 α-氨基或 SEQ ID NO:l所示序列第 2、 7、 15、 17、 24、 69、 94、 97、 121、 125、 128、 129、 150、 175、 215、 228、 246位的赖氨酸 残基侧链上的 ε-氨基或其组合。
14.权利要求 12的复合物， 其特征为一个血管抑素片段 K1-3与一个 聚乙二醇分子偶联，偶联位点为血管抑素片段 K1-3 N端的 a-氨基。
15.权利要求 14的复合物，其特征为一个具有 SEQ ID NO:2的重组人 血管抑素分子的片段 K1-3与一个聚乙二醇分子偶联，偶联位点为 血管抑素片段 K1-3 N端的 a-氨基。
16.权利要求 15的复合物，其特征为一个具有 SEQ ID NO:2的重组人 血管抑素分子的片段 K1-3与一个 20 kDa单甲基聚乙二醇分子偶 联， 偶联位点为血管抑素片段 K1-3 N端的 α-氨基。
17.权利要求 12的复合物， 其特征为一个血管抑素片段 K1-3与一个 或多个聚乙二醇分子偶联， 偶联的位点是血管抑素天冬氨酸或谷 氨酸残基侧链上的羧基。
18.权利要求 8-11任一项的复合物，其特征为一个血管抑素片段 K1-3 与一个或多个聚乙二醇分子偶联， 偶联方法为在血管抑素片段 K1-3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。
19.权利要求 1-18任一项的复合物与生物相容性物质形成的缓释制 剂。
20.权利要求 19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。
21.—种药物组合物， 其包含权利要求 1-20中任一项的复合物或缓释 制剂和药学上可接受的载体。
22.—种试剂盒， 其包含权利要求 1-21的复合物、 缓释制剂或药物组 合物和使用说明。
23.制备权利要求 8-18任一项的复合物的方法， 其特征是在足以使活 化的聚乙二醇与血管抑素片段 K1-3发生反应的溶液、 温度、 pH、 摩尔比的条件下将活化的聚乙二醇与血管抑素片段 Kl-3混合。
24.权利要求 23的方法， 其中 pH为 5-7， 聚乙二醇与血管抑素片段 K1-3摩尔比为 1:1到 10:1。
25.权利要求 24的方法， 进一步包括将偶联产物用阳离子柱纯化。
26.权利要求 1-21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。
27.权利要求 26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。
28.权利要求 1-21的复合物、 缓释制剂或药物组合物在制备治疗非肿 瘤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。
29.权利要求 26-28的用途， 其中所述药物适于静脉注射、 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。
30.—种延长血管抑素片段 K1-3半衰期的方法，所述方法包含将修饰 物与血管抑素片段 K1-3形成复合物的步骤，所述修饰物选自聚乙 二醇或其他高分子聚合物、 蛋白质分子或其片段、 肽链、 小分子 或其他任何形式的化学物质。
31.—种延长血管抑素片段 K1-3半衰期的方法，所述方法包含将血管 抑素片段 K1-3或由一种修饰物与血管抑素片段 K1-3形成的复合 物与生物相容性物质形成缓释制剂的步骤， 所述修饰物选自聚乙 二醇或其他高分子聚合物、 蛋白质分子或其片段、 肽链、 小分子 或其他任何形式的化学物质。
PCT/CN2008/000067 2007-01-10 2008-01-10 Complexes comprenant de l'angiostatine et ses fragments, leurs procédés de préparation et leurs utilisations WO2008083615A1 (fr)

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US12/522,661 US20100184661A1 (en) 2007-01-10 2008-01-10 Conjugate comprising angiostation or its fragment, the method for producing the conjugate and use thereof
AU2008204647A AU2008204647A1 (en) 2007-01-10 2008-01-10 Complexes comprising angiostatin and its fragments, preparation preparing methods and uses thereof
JP2009545050A JP2010515694A (ja) 2007-01-10 2008-01-10 アンギオスタチンあるいはその断片を含む複合体、その調製法および応用
CA2675231A CA2675231C (en) 2007-01-10 2008-01-10 A conjugate comprising angiostatin or its fragment, the method for producing the conjugate and use thereof
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