WO2016115732A1 - 一种抑制血管新生或生长的多肽及其应用 - Google Patents

一种抑制血管新生或生长的多肽及其应用 Download PDF

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WO2016115732A1
WO2016115732A1 PCT/CN2015/071438 CN2015071438W WO2016115732A1 WO 2016115732 A1 WO2016115732 A1 WO 2016115732A1 CN 2015071438 W CN2015071438 W CN 2015071438W WO 2016115732 A1 WO2016115732 A1 WO 2016115732A1
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seq
amino acid
acid sequence
nucleotide sequence
sequence
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PCT/CN2015/071438
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French (fr)
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许迅
柯潇
郑颖
罗德伦
邬智刚
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上海市第一人民医院
成都康弘药业集团股份有限公司
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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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the invention belongs to the field of biomedicine, and in particular to a polypeptide for inhibiting angiogenesis or growth.
  • the formation of new blood vessels is an extremely complex process that includes: expansion of existing blood vessels, increased vascular permeability, degradation of perivascular matrix, activation and proliferation of endothelial cells, migration, and formation of new capillary-like lumens.
  • the formation of blood vessels is a process that is closely regulated by multiple links, and plays an important role in repairing and maintaining the normal function of the body.
  • the blood vessels inside the tumor grow rapidly, it is one of the common clinical phenomena.
  • a large number of animal models and human clinical trials have shown that blocking the formation of neovascularization in tumors can effectively prevent tumor growth and cause tumor cell death, thereby achieving therapeutic effects on tumors.
  • angiogenesis is one of the important directions in the current research of new anti-tumor drugs.
  • Macromolecular anti-angiogenic drugs such as Avastin have been approved by the US FDA, and more drugs are in different preclinical and clinical research stages.
  • pathological neovascularization such as corneal neovascularization caused by herpetic keratitis, choroidal neovascularization in age-related macular degeneration, and diabetic retinopathy.
  • Retinal neovascularization in retinopathy of prematurity At present, clinically, the pathological neovascularization of the eye is conventionally treated by laser photocoagulation, photodynamic therapy (PDT), and thermal transpupillary therapy (TTT).
  • PDT photodynamic therapy
  • TTT thermal transpupillary therapy
  • angiogenesis inhibitors In the development of effective angiogenesis inhibitors, the specificity of ophthalmic medication should be fully considered.
  • systemic administration often fails to achieve sufficient drug concentration in the ocular tissue due to the blood-aqueous barrier and the blood-retinal barrier; topical administration, such as vitreous cavity Injection, macromolecules larger than 76.5kDa are theoretically difficult to penetrate the retina and act on the retina and choroidal neovascularization; for ocular surface administration, the drug must penetrate the lipophilic corneal epithelial cells tightly and hydrophilic cornea.
  • the matrix therefore, only drugs with appropriate fat-soluble, low molecular weight or binding to transporters in the ocular surface tissue (eg, amino acid transporters, oligopeptide transporters, etc.) can reach the anterior chamber.
  • the degree of dissolution of the drug in hydrophilic tears, aqueous humor, and vitreous humor is positively correlated with its effectiveness.
  • the bioavailability of ophthalmic drugs is very low; to increase it, it is necessary to increase the concentration of administration.
  • the toxic side effects of the compounds used for the treatment of tumor neovascularization are more obvious, and systemic and local administration are not possible at high doses.
  • exogenous proteins with larger molecular weights are also sensitive foreign sources that can cause immune damage to ocular tissues such as the uvea.
  • angiostatin consists of plasminogen Kringle 1-4 (plasminogen Kringle 1-4). It can significantly inhibit the growth of vascular-dependent tumors, but due to its large molecular weight and complex spatial conformation, there are deficiencies in the process of preparation and purification and the endotoxin residues.
  • the present invention provides the following technical solutions:
  • the invention provides a polypeptide that inhibits angiogenesis or growth, the polypeptide comprising the sequence set forth in SEQ ID NO:1.
  • the polypeptide is any one or more amino acids starting from the C-terminus of SEQ ID NO: 2 (AVSLLRATG) in the order preceding the sequence of SEQ ID NO: 1; or in the aforementioned SEQ ID Any one or more amino acids starting from the N-terminus of SEQ ID NO: 3 (HAVPV) are sequentially added in order of NO:1; or both of SEQ ID NO: 2 and SEQ ID NO: 3 are added in the above-described order. One or more amino acids.
  • polypeptide of the invention is:
  • SK001 its amino acid sequence is SEQ ID NO: 1; SK002: its amino acid sequence is SEQ ID NO: 5; SK003: its amino acid sequence is SEQ ID NO: 7; SK004: its amino acid sequence is SEQ ID NO: 9; SK005: Its amino acid sequence is SEQ ID NO: 11; SK006: its amino acid sequence is SEQ ID NO: 13; SK007: its amino acid sequence is SEQ ID NO: 15; SK008: its amino acid sequence is SEQ ID NO: 17; SK009: its amino acid The sequence is SEQ ID NO: 19; SK010: its amino acid sequence is SEQ ID NO: 21; SK011: its amino acid sequence is SEQ ID NO: 23; SK012: its amino acid sequence is SEQ ID NO: 25; SK013: its amino acid sequence is SEQ ID NO:27; SK014: its amino acid sequence is SEQ ID NO:29; SK015: its amino acid sequence is SEQ ID NO:31
  • the present invention provides a mutant of the above polypeptide, which also has an effect of inhibiting angiogenesis or growth, the polypeptide mutant being based on SEQ ID NO:
  • the amino acid residue in the 1 sequence is mutated, and the specific mutation is as follows: SEQ ID NO: 1:
  • the first bit A is replaced by V or L; or the second bit A is replaced by G or V; or the third bit G is replaced by A, P or D; or the fourth bit D is replaced by E; or the fifth bit E is D Substituted; or the sixth position N is replaced by Q; or the seventh position L is replaced by Q, M, F or I.
  • polypeptide mutant of the invention is:
  • SK019 its amino acid sequence is SEQ ID NO: 39; SK020: its amino acid sequence is SEQ ID NO: 41; SK021: its amino acid sequence is SEQ ID NO: 43; SK022: its amino acid sequence is SEQ ID NO: 45; SK023: Its amino acid sequence is SEQ ID NO: 47; SK024: its amino acid sequence is SEQ ID NO: 49; SK025: its amino acid sequence is SEQ ID NO: 51; SK026: its amino acid sequence is SEQ ID NO: 53; SK027: its amino acid The sequence is SEQ ID NO: 55; SK028: its amino acid sequence is SEQ ID NO: 57; SK029: its amino acid sequence is SEQ ID NO: 59; SK030: its amino acid sequence is SEQ ID NO: 61; SK031: its amino acid sequence is SEQ ID NO: 63; SK032: its amino acid sequence is SEQ ID NO: 65; SK033: its amino acid sequence is S
  • the invention provides a nucleotide sequence encoding a mutant of the above polypeptide or polypeptide.
  • the nucleotide sequence is:
  • the invention provides an expression vector or host cell expressing the polypeptide or a mutant thereof, comprising the nucleotide sequence of the polypeptide or mutant described above.
  • the expression vector may be a recombinant eukaryotic expression vector, preferably a mammal
  • the cell expression vector may also be a recombinant viral expression vector, preferably an adeno-associated virus or an adenoviral vector.
  • the expression vector is capable of replicating expression in a transformed host cell, preferably the host cell is a CHO cell and a subline thereof or a 293 cell and a subline thereof.
  • the present invention provides a method of producing the polypeptide or mutant, which is a chemical synthesis method or a recombinant expression method, which comprises introducing the above expression vector into a suitable host cell. Expression of a polypeptide or mutant.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above polypeptide or a mutant thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient; preferably, the pharmaceutical composition
  • the dosage form is an injection, eye drops, ophthalmic gel or eye ointment.
  • the invention provides the use of a polypeptide of the invention, or a mutant or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prevention of a disease caused by angiogenesis or growth.
  • the disease is preferably an eye disease, a tumor, an ischemic heart disease, a non-inflammatory cardiomyopathy, coronary arteriosclerosis, an occlusive arteriosclerosis, an arterial embolism, an arterial thrombosis, Berger's disease, chronic inflammation, inflammatory bowel disease, Ulcer, rheumatoid arthritis, scleroderma, psoriasis, infertility or sarcomatosis;
  • the eye disease is preferably age-related macular degeneration, diabetic retinopathy, choroidal neovascularization, cystoid macular edema, diabetes Macular edema, retinal vascular occlusion, corneal neovascularization, corneal transplantation, neovascular glaucoma,
  • the invention provides a method of inhibiting angiogenesis or growth in a mammal comprising: administering to a subject in need thereof a polypeptide as described above, or a mutant thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above;
  • the mode of administration is ocular surface administration or intravitreal injection.
  • the polypeptide of the present invention has a small molecular weight and is permeable to the ocular tissue barrier;
  • (d) can be prepared by solid phase synthesis, with high purity, large yield and low cost;
  • polypeptide of the present invention has good stability.
  • Fig. 1 is a diagram showing the results of purity identification of small peptide SK004 by high performance liquid chromatography and mass spectrometry.
  • Fig. 1a is a chromatogram of SK004
  • Fig. 1b is a first-order mass spectrum of SK004
  • Fig. 1c is a secondary mass spectrum of SK004.
  • Figure 2a is a graph showing the effect of small peptide SK004 on the proliferation of human umbilical vein endothelial cells HUVEC.
  • Figure 2b is a graph showing the effect of small peptide SK018 on the proliferation of human umbilical vein endothelial cells HUVEC.
  • Figure 3 is a graph showing the effect of small peptide SK004 on neovascularization in the chorioallantoic membrane of chicken embryos, wherein Figures 3a-3c show 3-5 microvessel counts in the range of 2.5 mm around the filter paper:
  • Figure 3a is the PBS group;
  • Figure 3b is the Avasin (5 ⁇ l/tablet) group;
  • Fig. 3c is SK004 (5 ⁇ l/tablet) group;
  • Fig. 3d is the effect of PBS group, Avastin group and different concentration of SK004 on neovascularization of chick embryo chorioallantoic membrane.
  • Figure 4 is a graph showing the effect of small peptide SK018 on neovascularization in the chick chorioallantoic membrane, wherein Figures 4a-4c show filter paper 3-5 grade microvessel counts in the range of 2.5 mm per week: Figure 4a is the PBS group; Figure 4b is the Avasin (5 ⁇ l/tablet) group; Figure 4c is the SK018 (5 ⁇ l/tablet) group; Figure 4d is the PBS group, the Avastin group, and The effect of the concentration of SK018 on the neovascularization of the chicken embryo chorioallantoic membrane.
  • Figure 5a is a graph showing the effect of small peptide SK004 on pathological neovascularization of rat cornea.
  • Figure 5b is a graph showing the effect of small peptide SK018 on pathological neovascularization of rat cornea.
  • the polypeptide of the present invention or a mutant thereof refers to a protein or polypeptide having an angiogenesis inhibitory activity.
  • the term includes a polypeptide comprising the sequence SEQ ID NO: 1 and variants thereof having an inhibitory angiogenesis or growth function. These variants include, but are not limited to, 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, and additions at the C-terminus and/or N-terminus or One or several amino acids are missing.
  • the term also encompasses polypeptides of the invention, linear and non-linear polypeptides (e.g., cyclic peptides), in monomeric and multimeric forms.
  • fragment refers to a polypeptide that substantially retains an angiogenic function or activity.
  • a polypeptide fragment, derivative or analog of the invention may be (i) a polypeptide having one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more a polypeptide having a substituent group in one amino acid residue, or (iii) a polypeptide formed by fusing a polypeptide of the present invention with another compound (such as a compound that extends half-life of the polypeptide, such as polyethylene glycol), or (iv) additional A polypeptide formed by fusing an amino acid sequence to the polypeptide sequence (a protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His).
  • the difference between the polypeptide analog of the present invention and the polypeptide of the present invention may be a difference in amino acid sequence, a difference in a modified form which does not affect the sequence, or a combination thereof.
  • Analogs also include analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above. Modifications (usually without altering the primary structure) include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro.
  • Modifications also include glycosylation, such as those accomplished by glycosylation enzymes, such as mammalian glycosylation enzymes or deglycosylation enzymes, in the synthesis and processing of polypeptides or in further processing steps. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the polypeptides of the invention may also be used in the form of a pharmaceutically or physiologically acceptable acid or a derivatized salt.
  • These salts include, but are not limited to, salts formed with hydrofluoric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, malay Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid or isethionic acid.
  • Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, carbamates or other conventional "prodrugs".
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the DNA can be a coding strand or a non-coding strand.
  • the full-length nucleotide sequence of the present invention or a fragment thereof can usually be obtained by PCR amplification, recombinant method or artificial synthesis method. Got it.
  • DNA sequences encoding the polypeptide of the present invention or a mutant thereof (or a fragment thereof, or a derivative thereof) have been obtained by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and to host cells genetically engineered using the vectors of the invention or the coding sequences of the polypeptides of the invention.
  • the polypeptide of the present invention or a mutant thereof may be a recombinant polypeptide or a synthetic polypeptide, and accordingly, the polypeptide of the present invention or a mutant thereof may be artificially synthesized by a conventional method or may be produced by a recombinant method.
  • a preferred method is to use liquid phase synthesis techniques or solid phase synthesis techniques such as Boc solid phase method, Fmoc solid phase method or a combination of both methods. The solid phase synthesis can quickly obtain samples, and the appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the peptide of interest.
  • a preferred solid phase support in the Fmoc system is a Wang resin to which a C-terminal amino acid is attached to the peptide, a Wang resin structure is polystyrene, an arm with an amino acid is 4-alkoxybenzyl alcohol; and 25% hexahydropyridine is used; /Dimethylformamide was treated at room temperature for 20 minutes to remove the Fmoc protecting group and extended from the C-terminus to the N-terminus according to the given amino acid sequence. After the completion of the synthesis, the synthesized proinsulin-related peptide was cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol, and the protecting group was removed.
  • the resin was removed by filtration and the diethyl ether was precipitated to obtain a crude peptide. After the solution of the obtained product was lyophilized, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography.
  • the resin be a PAM resin to which a C-terminal amino acid is attached, the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxymethyl phenylacetamide;
  • the protecting group Boc was removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA) dichloromethane in a deprotection, neutralization, and coupling cycle.
  • the peptide chain was cleaved from the resin by treatment with hydrogen fluoride (HF) containing p-cresol (5-10%) at 0 ° C for 1 hour while removing the protecting group.
  • HF hydrogen fluoride
  • the peptide was extracted with 50-80% acetic acid (containing a small amount of mercaptoethanol), and the solution was lyophilized and further purified by molecular sieve Sephadex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain a desired peptide.
  • Various amino acid residues can be coupled using various coupling agents and coupling methods known in the art of peptide chemistry, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1 can be used. Direct coupling was carried out with 1,3,3-tetraurea hexafluorophosphate (HBTU).
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • HBTU 1,3,3-tetraurea hexafluorophosphate
  • Another method is to produce a polypeptide of the invention using recombinant techniques.
  • the polynucleotide of the present invention can be utilized to express or produce a recombinant SK series polypeptide by conventional recombinant DNA techniques. Generally there are the following steps:
  • the recombinant polypeptide can be expressed in the cell or on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid
  • polypeptide of the present invention is short, it is conceivable to connect a plurality of polypeptides in series, and to obtain an expression product in a multimeric form after recombinant expression, and then to form a desired small peptide by enzymatic cleavage or the like.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a safe and effective amount of the polypeptide of the present invention or a mutant thereof or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient .
  • the amount of the polypeptide of the present invention or a mutant thereof is usually from 10 ⁇ g to 100 mg/dose, preferably from 100 to 1000 ⁇ g/dose.
  • an effective dose is from about 0.01 mg/kg to 50 mg/kg, preferably from 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the invention or a mutant thereof.
  • the polypeptide of the present invention or a mutant thereof may be used singly or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • the term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration.
  • Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers can contain liquids such as water, saline, glycerol and ethanol.
  • the therapeutic compositions can be formulated as injectables, such as liquid solutions or suspensions; solid forms such as liquid carriers, which may be formulated in solution or suspension prior to injection.
  • composition of the invention can be administered by conventional routes including, but not limited to, ocular surface, periocular, intraocular (especially intravitreal), intramuscular, intravenous, Subcutaneous, intradermal or topical administration.
  • the subject to be prevented or treated may be an animal; especially a human.
  • a pharmaceutical composition of various dosage forms may be employed depending on the use.
  • eye drops, injections (especially intravitreal injections), ophthalmic gels and eye ointments are exemplified.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers, and solubilizers, and the formulation process can be carried out according to the dosage form in a conventional manner.
  • suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers, and solubilizers
  • the preparation of eye drops can be carried out by dissolving the polypeptide of the present invention or a mutant thereof or a pharmaceutically acceptable salt thereof together with a basic substance in sterile water (a surfactant is dissolved in sterile water), and adjusting The osmotic pressure and pH are brought to a physiological state, and a suitable pharmaceutical additive such as a preservative, a stabilizer, a buffer, an isotonic agent, an antioxidant, and a tackifier may be arbitrarily added, and then completely dissolved.
  • a suitable pharmaceutical additive such as a preservative, a stabilizer, a buffer, an isotonic agent, an antioxidant, and a tackifier may be arbitrarily added, and then completely dissolved.
  • compositions of the invention may also be administered in the form of sustained release agents.
  • the polypeptide of the present invention or a mutant thereof or a salt thereof can be incorporated into a pellet or microcapsule in which a sustained-release polymer is used, and then the pellet or microcapsule is surgically implanted into a tissue to be treated.
  • the polypeptide of the present invention or a mutant thereof or a salt thereof can also be applied by inserting a drug-coated intraocular lens.
  • the sustained-release polymer include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, and lactic acid polymer.
  • a lactic acid-glycolic acid copolymer or the like is preferably exemplified by a biodegradable polymer such as a lactic acid polymer and a lactic acid-glycolic acid copolymer.
  • a polypeptide of the present invention or a mutant thereof as an active ingredient when the pharmaceutical composition of the present invention is used for actual treatment The dose of the pharmaceutically acceptable salt thereof or its pharmaceutically acceptable salt can be reasonably determined depending on the weight, age, sex, and degree of symptoms of each patient to be treated. For example, when the eye drops are partially instilled, the concentration is usually about 0.1 to 10% by weight, preferably 1 to 5% by weight, and can be administered 2 to 2 times a day, 1-2 drops each time.
  • VEGF Vascular endothelial growth factor
  • the VEGF gene can produce at least 7 VEGF variants through transcriptional cleavage, of which VEGF165 (VEGF-A, ie Prototype VEGF) is the most common and dominant type. Its main biological functions are: selectively enhancing mitosis of vascular endothelial cells, stimulating the proliferation of vascular endothelial cells and promoting angiogenesis; enhancing the permeability of blood vessels, especially small blood vessels, and making plasma proteins and other macromolecules (mainly fibrinogen) The osmotic deposition in the extravascular matrix provides nutrients for the establishment of a new capillary network.
  • Bevacizumab is a recombinant anti-human VEGF monoclonal antibody that is the first anti-angiogenic drug approved by the US FDA for the treatment of patients with metastatic colon cancer. Vitreous injection of bevacizumab can also effectively inhibit retinal and choroidal neovascularization.
  • Another VEGF inhibitor, Ranibizumab is a recombinant anti-human VEGF monoclonal antibody fragment. It is the first anti-angiogenic drug approved by the US FDA for the treatment of ocular neovascular macular degeneration. At present, people are still constantly looking for new safer and more effective drugs for the treatment of ocular neovascularization.
  • the inventors have extensively and intensively studied for the first time to prepare a class of small molecule polypeptides having a molecular weight of less than 5 kD (e.g., only about 1-2 kD).
  • the present inventors applied bioinformatics methods, based on homology analysis and biological characteristics analysis, designed several candidate sequences, synthesized by solid phase method, and isolated and purified to obtain high purity of the present invention.
  • Peptides and their mutants, VEGF-induced human umbilical vein endothelial cell proliferation model, chicken embryo chorioallantoic membrane model and rat corneal suture model were screened to obtain a new type of small, preventive and therapeutic angiogenic function.
  • Molecular peptides were screened to obtain a new type of small, preventive and therapeutic angiogenic function.
  • Angiogenesis refers to the process of forming new blood vessels by budding on the basis of primitive vascular plexuses or existing blood vessels. Under pathological conditions, the existing blood vessels in the body are stimulated by pro-angiogenic factors, dilated, vascular endothelial cell activation, proliferation, migration, and tubule-like structure formation. Among them, endothelial cell self-proliferation is the basis of angiogenesis, which provides the necessary number of cells for the formation of new blood vessels.
  • the present invention uses endothelial cell proliferation as a target of drug intervention, and uses human CCK-8 method (purchased from Dojindo) to detect human umbilical vein endothelial cells (HUVEC) to investigate the inhibition of endothelial cells by polypeptide SK004 during anti-angiogenesis.
  • human CCK-8 method purchased from Dojindo
  • HAVEC human umbilical vein endothelial cells
  • the model was first created by experimental embryologists 60 years ago and was originally used for the study of embryonic organ development. It is currently a commonly used experimental technique for studying blood vessel growth at home and abroad. It is widely used to study the induction of blood vessel growth or the inhibition of angiogenesis. The effect on microvessels.
  • the rat corneal suture model is a classic ocular neovascularization model with good reproducibility, quantitative analysis and low cost. It is commonly used to detect the activity of anti-angiogenic drugs.
  • a pharmaceutical composition containing the peptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient has significant inhibitory activity against angiogenesis or growth. It has been confirmed by animal experiments that the polypeptide of the present invention not only inhibits angiogenesis of chicken embryo chorioallantoic membrane, but also inhibits corneal neovascularization in rats, and can inhibit the proliferation of human umbilical vein endothelial cells. Therefore, the polypeptide of the present invention is expected to be developed into a medicament for the treatment of neovascular eye diseases and related neovascular diseases such as tumor neovascularization.
  • the polypeptide SK050 has the amino acid sequence of SEQ ID NO: 101 and the nucleotide sequence of SEQ ID NO: 102;
  • Peptide SK051 the amino acid sequence of which is SEQ ID NO: 103, and the nucleotide sequence is SEQ ID NO: 104;
  • Peptide SK052 the amino acid sequence of which is SEQ ID NO: 105, and the nucleotide sequence is SEQ ID NO: 106;
  • the polypeptide SK053 has the amino acid sequence of SEQ ID NO: 107 and the nucleotide sequence of SEQ ID NO: 108.
  • the SK004 polypeptide represented by SEQ ID NO: 9 was synthesized using a commercially available SYMPHONY type 12-channel polypeptide synthesizer (Protein Technologies, USA). The specific method is as follows: according to the software of the peptide synthesizer (Version.201 version), the configuration reagent is calculated, and 2-Chlorotrityl Chloride Resin resin (Tianjin Nankai Synthetic Technology Co., Ltd.) is placed in the reaction tube, and DMF (15 ml/g) is added ( Dikma), shaking for 30 min.
  • the solvent was filtered off through a sand core, and a 3-fold molar excess of Fmoc-LH-OH (Fmoc-leucine-histidine-OH) (small peptide SK004) amino acid (Suzhou Tianma Pharmaceutical Group Fine Chemical Co., Ltd.) was added. Then add a 10-fold molar excess of DIEA (N,N-diisopropylethylamine) (Shenzhen Pharmaceutical Group Shanghai Chemical Reagent Co., Ltd.), finally add DMF to dissolve, and shake for 30 min.
  • Fmoc-LH-OH Fmoc-leucine-histidine-OH
  • DIEA N,N-diisopropylethylamine
  • the polypeptide was cleaved from the resin (cutting solution (10/g): TFA (trifluoroacetic acid) (JT Baker) 94.5%, water 2.5%, EDT (1,2-ethanedithiol) (ALDRICH) 2.5%, TIS ( Benzoyl sulfide (ALDRICH) 1%; cutting time: 120 min).
  • the lysate was blown dry with nitrogen (Shanghai Bio-Industry) and washed twice with diethyl ether (Shanghai Test Chemical Reagent Co., Ltd.) and then dried at room temperature. Finally, the purified solution was lyophilized to obtain a high purity (>95%) small peptide SK004.
  • a small amount of the finished small peptide SK004 was taken for purity identification by HPLC analysis and molecular weight identification by ESI-MS.
  • the conditions are as follows: electrospray ionization, positive ion mode detection, nitrogen as sheath gas, auxiliary gas and purge gas.
  • the fog voltage is 4.7kV: the sheath gas is 15arb, the auxiliary gas is 5arb, the purge gas is 0arb, the capillary temperature is 275°C, the capillary voltage is 40V, and the lens voltage is 120V.
  • Full scan quality range 300-2000 amu.
  • the second-order mass spectrum was acquired by data-dependent mode.
  • the +1 valence molecular ion peak in the full scan spectrum was used for the second-order mass spectrometry, and the high-purity helium gas was used as the collision gas with a collision energy of 35 ev.
  • the small peptide in the form of a white powder was sealed and packaged and stored at -20 ° C for a long period of time.
  • Example 3 The polypeptides shown in Table 3 were prepared as in Example 1 and the polypeptides were identified as in Example 2. The structural information of these peptides is correct and consistent with the designed amino acid sequence. The activity was tested as follows.
  • VEGF has the activity of promoting the proliferation of HUVEC cells, and the polypeptide of the present invention has the effect of inhibiting VEGF. Therefore, the HUVEC cells are used for biological activity comparison in this test.
  • HUVEC cells are inoculated, and each sample is diluted with 2% FBS-ECM. Each sample was mixed with VEGF. After the cells were attached to the cells, the supernatant in each well was discarded, the sample was added, and the dilution control and VEGF control were added. After 4 days of culture, 20 ⁇ l of CCK-8 color solution was added to each well to develop color, and then the cells were colored. The color-developed cells were measured by a microplate reader, and the absorbance was measured.
  • the magnitude of the degree directly reflects the number of cells, and it is inferred that each sample inhibits VEGF-mediated proliferation of HUVEC cells according to the number of viable cells of HUVEC.
  • the dilution control group was used as a negative control, and the VEGF control group was used as a positive control.
  • HUVEC ScienCell
  • Each sample was diluted with ECGS-ECM medium to 2.5 mM and 1.25 mM, respectively.
  • VEGF was diluted to 40 ng/ml with 2% FBS-ECM for later use.
  • the cells were observed to confirm that the morphology was normal and non-contaminated, and 25 ⁇ l of CCK-8 working solution was added to each well, and reacted at 37 ° C for 4 hours, and the absorbance was measured at 450 nm with a microplate reader.
  • Inhibition rate (%) dilution control result / sample result * 100
  • Example 3 According to the detection method of Example 3, the activity detection of the sample of SK004 and its optical isomer was carried out, and the test results are shown in Table 4.
  • SK004-1 (SEQ ID NO: 9) GAAGDEnLH 73.44
  • SK004-2 (SEQ ID NO: 9) GAaGDENLH 61.61
  • Example 5 Effect of polypeptide on proliferation of human umbilical vein endothelial cells
  • HUVEC Human umbilical vein endothelial cells HUVEC (purchased from ScienCell) were inoculated into 96-well plates at a concentration of 2 ⁇ 10 4 /ml; cells were attached to the serum-free culture medium ECM at 37 ° C for 24 hours; The serum-free culture ECM was separately added as a negative control, VEGF (20 ng/ml) (purchased from R&D) as a positive control, VEGF (20 ng/well) + Avastin (purchased from Roche), VEGF (20 ng/well) + Different concentrations of small peptides SK004 or SK018 were used as treatment groups; after 96 hours of incubation, 20 ⁇ l of CCK-8 solution (purchased from Dojindo) was added to each well; after incubating at 37 ° C for 4 hours, using a microplate reader (Molecular Device) The company measured the absorbance of each well at 450 nm, and determined the proliferative activity of the cells according to OD
  • the small peptide SK004 significantly inhibited the proliferation of human umbilical vein endothelial cells HUVEC in a concentration-dependent manner, **P ⁇ 0.01, and the difference was statistically significant.
  • Example 6 Determination of neovascularization effect of small peptide against chicken embryo chorioallantoic membrane
  • Figures 3a-3d show 3-5 grade microvessel counts in the 2.5 mm range of filter paper.
  • Figure 3a shows the PBS group;
  • Figure 3b shows the Avastin (5 ⁇ l/tablet) group;
  • Figure 3c shows the SK004 (5 ⁇ l/tablet) group;
  • Figure 3d shows that the small peptide SK004 group at each concentration significantly inhibits the chicken embryo allantoic capsule relative to the PBS group.
  • Figures 4a-4d show 3-5 grade microvessel counts over a 2.5 mm range of filter paper.
  • Figure 4a is the PBS group;
  • Figure 4b is the Avasin (5 ⁇ l/tablet) group;
  • Figure 4c is the SK018 (5 ⁇ l/tablet) group;
  • Figure 4d is the relative concentration of each of the PBS groups.
  • the peptide SK018 group significantly inhibited the number of neovascularization in chick embryo chorioallantoic membrane, and the inhibition was concentration-dependent, **P ⁇ 0.01, and the difference was statistically significant.
  • Example 7 Determination of the effect of small peptide on the pathological neovascularization of rat cornea
  • the rat corneal suture model was used as follows: healthy SD rats, 160-180 g, male, with the right eye as the experimental eye. All experimental eyes were 0.3% of ofloxacin eye drops 3 days before surgery, 2 times a day. The experimental eyes were examined before the experiment, and the eye lesions were excluded and the body weight was measured. General anesthesia was given by intraperitoneal injection of 1% sodium pentobarbital at 3 ml/kg body weight, and ocular anesthesia was given. First, a 3mm diameter corneal lap was used to make an indentation in the center of the rat cornea, and the corneal stroma was sutured with the 10-0 nylon thread in the direction of vertical indentation and the midline as the midline.
  • the span of each needle is about 1 mm, and the outermost end of the suture is about 1 mm from the limbus.
  • the conjunctival sac is coated with ofloxacin eye ointment.
  • Each group was treated with eye drops 4 times daily (10 ⁇ l/time/eye. Eye drops were prepared as in Example 9, in which the contents of Avastin and small peptides are shown in the group).
  • the small peptide SK004 has a significant inhibitory effect on the pathological neovascularization of the rat cornea.
  • the small peptide SK004 group significantly inhibited the pathological neovascularization of the cornea on the 3rd, 5th and 7th postoperative day, *P ⁇ 0.05, **P ⁇ 0.01, the difference was statistically significant. significance.
  • the small peptide SK018 0.5 mg/ml significantly inhibited the pathological neovascularization of the cornea on the third day after surgery, *P ⁇ 0.05, **P ⁇ 0.01, the difference has Statistical significance.
  • the anti-rat corneal pathological neovascularization effects of other polypeptides were determined by the above methods.
  • the CNV area S (mean ⁇ SE) of each group was compared using one-way ANOVA, and statistical analysis was performed using SPSS 16.0.1. The test results are shown in Table 5:
  • the polypeptides shown in Table 6 were prepared according to the method of Example 1, and the polypeptides were identified as in Example 2. The results showed that the structural information of these polypeptides was correct, and the amino acid sequence was consistent with the designed sequence.
  • the CCK-8 method is used to detect the activity of the polypeptide, and the specific method is as follows:
  • HUVEC Human umbilical vein endothelial cells HUVEC (purchased from ScienCell) were inoculated into 96-well plates at a concentration of 2 ⁇ 10 4 /ml; cells were attached to the serum-free culture medium ECM at 37 ° C for 24 hours; The serum-free culture ECM was separately added as a negative control, VEGF (20 ng/ml) (purchased from R&D) as a positive control, VEGF (20 ng/well) + different small peptides SK001, SK005, SK018, SK019-049 small peptide (1 ⁇ g/ ⁇ l) as a treatment group; after continuing to culture for 96 hours, 20 ⁇ l of CCK-8 solution (purchased from Dojindo Co., Ltd.) was added to each well; after incubation at 37 ° C for 4 hours, it was measured by a microplate reader (Molecular Device).
  • the best corrected visual acuity, corneal neovascularization (including: neovascular area, total length of new blood vessels, average diameter of new blood vessels) as the main indicators.
  • the treatment situation is divided into three levels: markedly effective, effective and ineffective, and scored separately.
  • corneal neovascularization ⁇ 30% reduction in neovascular area, ⁇ 20% reduction in total length of new blood vessels, and ⁇ 10% reduction in average diameter of new blood vessels.
  • Effective 1 increase visual acuity more than 2 lines; 2 corneal neovascularization: ⁇ 15% reduction of neovascular area, ⁇ 10% reduction of total length of new blood vessels, and ⁇ 5% reduction of average diameter of new blood vessels.
  • Invalid 1 no improvement in visual acuity; 2 corneal neovascularization: ⁇ 5% reduction in neovascular area, ⁇ 3% reduction in total length of new blood vessels, and reduction in average diameter of neovascularization ⁇ 1%.
  • CNV choroidal neovascularization
  • the CNV was divided into 4 grades by color fundus photography, optical coherence section scanning (OCT), and fundus fluorescein angiography (FFA) to obtain CNV lesion regression, leakage or absorption.
  • OCT optical coherence section scanning
  • FFA fundus fluorescein angiography

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Abstract

提供了一种抑制血管新生或生长的肽及其突变体、其编码序列、包含该编码序列的载体、宿主细胞、药物组合物以及所述肽的制药和治病用途。本发明的肽分子量小，可通过固相合成的方法制备。

Description

一种抑制血管新生或生长的多肽及其应用 技术领域
本发明属于生物医药领域，具体地，涉及一种抑制血管新生或生长的多肽。
背景技术
新生血管的形成是一个极其复杂的过程，它包括：现存血管的扩张、血管通透性的增加、血管周围基质的降解、内皮细胞的激活增殖、迁移以及新的毛细血管样管腔的形成。在正常生理条件下，血管的生成是一个受多个环节严密调节的过程，对修复和维持机体的正常功能具有重要的作用。然而，如果肿瘤内部血管快速生长，则是临床常见的现象之一。大量的动物模型和人体临床试验表明，阻断肿瘤内新生血管的形成可以有效地阻止肿瘤的生长和引发肿瘤细胞的死亡，从而达到对肿瘤的治疗作用。因此，抑制血管新生是目前抗肿瘤新药研究的重要方向之一。大分子抗新生血管药物如Avastin已经通过美国FDA的上市批准，同时还有更多的药物处于不同的临床前和临床研究阶段。
在眼部，约2/3的致盲性疾病均与病理性新生血管有关，例如：单纯疱疹性角膜基质炎引起的角膜新生血管、年龄相关性黄斑变性中的脉络膜新生血管以及糖尿病视网膜病变或早产儿视网膜病变中的视网膜新生血管等。目前临床上，对于眼部病理性新生血管常规运用激光光凝、光动力学疗法(Photodynamic therapy，PDT)以及经瞳孔温热疗法(Thermal transpupillary therapy，TTT)等进行治疗。然而，这些治疗手段不仅对局部组织易造成破坏，其远期疗效也并不令人十分满意。因此，近年来人们不断尝试开发治疗眼部病理性新生血管更有效的方法；对于老年性视网膜血管病变(Age-related macular degeneration，简称为AMD)、糖尿病视网膜病变、关节炎等多种与血管新生相关的疾病，抑制血管新生的治疗新药也有广泛和重要的作用。
在开发有效的血管新生抑制剂时，应充分考虑到眼科用药的特殊性。第一，眼部存在多个解剖性和功能性的屏障：全身给药常常由于血-房水屏障和血-视网膜屏障而无法在眼组织局部达到足够的药物浓度；局部给药，如玻璃体腔注射，大于76.5kDa的大分子在理论上很难穿透视网膜作用于视网膜和脉络膜新生血管；对于眼表给药，药物必须要先后穿透亲脂性的角膜上皮细胞紧密连接和亲水性的角膜基质，因此只有具备适当脂溶性、低分子量或能与眼表组织内的转运体(如：氨基酸转运体、寡肽转运体等)结合的药物才能到达前房发挥作用。第二，药物在亲水的泪液、房水、玻璃体液中溶解的程度与其有效性呈正相关。第三，基于上述主要原因，眼科用药的生物利用度很低；要使之提高，需加大给药的浓度。但是用于治疗肿瘤新生血管的化合物毒副作用较为明显，全身和局部均无法高剂量给药。此外，分子量较大的外源性蛋白质也是敏感的异物源，可对眼部组织(如：葡萄膜)造成免疫损伤。第四，目前虽然已经有一系列相对安全的内源性血管新生抑制剂被先后证实，如血管抑素(angiostatin)，它由纤溶酶原Kringle结构域1-4(plasminogen Kringle1-4)组成，可明显抑制血管依赖性肿瘤的生长，但由于其分子量较大且空间构象复杂，故在制备过程中存在重组表达纯化工艺繁琐和内毒素残留等不足。正是由于上述种种条件 的限制，目前用于治疗眼部新生血管的药物十分有限，比如重组抗人VEGF单克隆抗体bevacizumab(Avastin)、重组抗人VEGF单克隆抗体片段ranibizumab(Lucentis)等，但它们价格高昂，并且需反复经玻璃体腔给药，甚至可引起血管栓塞等风险。
由此可见，寻找具有特异生物学活性和生物相容性的小分子抑制剂，且价格便宜，减少局部和全身的副作用，对新生血管性疾病的临床防治具有十分重要的意义。
发明内容
本发明目的在于提供一种新的抑制血管新生或生长的多肽，该多肽属于小分子抑制剂，价格便宜，能够有效地抑制血管新生或生长，并减少局部和全身的副作用。
为了达到上述目的，本发明提供了以下技术方案：
在一个方面，本发明提供一种抑制血管新生或生长的多肽，所述多肽含有SEQ ID NO：1所示序列。
在优选的实施方式中，所述多肽为在前述SEQ ID NO：1序列的前面按次序增加SEQ ID NO：2(AVSLLRATG)的从C端开始的任一个或多个氨基酸；或在前述SEQ ID NO：1的后面按次序增加SEQ ID NO：3(HAVPV)的从N端开始的任意一个或多个氨基酸；或前后都按上述次序增加SEQ ID NO：2和SEQ ID NO：3中的任一个或多个氨基酸。
在进一步优选的实施方式中，本发明的多肽为：
SK001：其氨基酸序列为SEQ ID NO：1；SK002：其氨基酸序列为SEQ ID NO：5；SK003：其氨基酸序列为SEQ ID NO：7；SK004：其氨基酸序列为SEQ ID NO：9；SK005：其氨基酸序列为SEQ ID NO：11；SK006：其氨基酸序列为SEQ ID NO：13；SK007：其氨基酸序列为SEQ ID NO：15；SK008：其氨基酸序列为SEQ ID NO：17；SK009：其氨基酸序列为SEQ ID NO：19；SK010：其氨基酸序列为SEQ ID NO：21；SK011：其氨基酸序列为SEQ ID NO：23；SK012：其氨基酸序列为SEQ ID NO：25；SK013：其氨基酸序列为SEQ ID NO：27；SK014：其氨基酸序列为SEQ ID NO：29；SK015：其氨基酸序列为SEQ ID NO：31；SK016：其氨基酸序列为SEQ ID NO：33；SK017：其氨基酸序列为SEQ ID NO：35；或SK018：其氨基酸序列为SEQ ID NO：37。
在本方面的另一种实施方式中，本发明提供上述多肽的突变体，所述多肽突变体同样具有抑制血管新生或生长的作用，所述多肽突变体是基于上述多肽中的SEQ ID NO：1序列中的氨基酸残基进行突变，具体突变如下：SEQ ID NO：1的：
第一位A被V或L取代；或第二位A被G或V取代；或第三位G被A、P或D取代；或第四位D被E取代；或第五位E被D取代；或第六位N被Q取代；或第七位L被Q、M、F或I取代。
在优选的实施方案中，本发明的多肽突变体为：
SK019：其氨基酸序列为SEQ ID NO：39；SK020：其氨基酸序列为SEQ ID NO：41；SK021：其氨基酸序列为SEQ ID NO：43；SK022：其氨基酸序列为SEQ ID NO：45；SK023：其氨基酸序列为SEQ ID NO：47；SK024：其氨基酸序列为SEQ ID NO：49；SK025：其氨基酸序列为SEQ ID NO：51；SK026：其氨基酸序列为SEQ ID NO：53；SK027：其氨基酸 序列为SEQ ID NO：55；SK028：其氨基酸序列为SEQ ID NO：57；SK029：其氨基酸序列为SEQ ID NO：59；SK030：其氨基酸序列为SEQ ID NO：61；SK031：其氨基酸序列为SEQ ID NO：63；SK032：其氨基酸序列为SEQ ID NO：65；SK033：其氨基酸序列为SEQ ID NO：67；SK034：其氨基酸序列为SEQ ID NO：69；SK035：其氨基酸序列为SEQ ID NO：71；SK036：其氨基酸序列为SEQ ID NO：73；SK037：其氨基酸序列为SEQ ID NO：75；SK038：其氨基酸序列为SEQ ID NO：77；SK039：其氨基酸序列为SEQ ID NO：79；SK040：其氨基酸序列为SEQ ID NO：81；SK041：其氨基酸序列为SEQ ID NO：83；SK042：其氨基酸序列为SEQ ID NO：85；SK043：其氨基酸序列为SEQ ID NO：87；SK044：其氨基酸序列为SEQ ID NO：89；SK045：其氨基酸序列为SEQ ID NO：91；SK046：其氨基酸序列为SEQ ID NO：93；SK047：其氨基酸序列为SEQ ID NO：95；SK048：其氨基酸序列为SEQ ID NO：97；或SK049：其氨基酸序列为SEQ ID NO：99。
在另一方面，本发明提供编码上述多肽或多肽突变体的核苷酸序列。在优选的实施方式中，所述核苷酸序列为：
sk001：核苷酸序列为SEQ ID NO：4；sk002：核苷酸序列为SEQ ID NO：6；sk003：核苷酸序列为SEQ ID NO：8；sk004：核苷酸序列为SEQ ID NO：10；sk005：核苷酸序列为SEQ ID NO：12；sk006：核苷酸序列为SEQ ID NO：14；sk007：核苷酸序列为SEQ ID NO：16；sk008：核苷酸序列为SEQ ID NO：18；sk009：核苷酸序列为SEQ ID NO：20；sk010：核苷酸序列为SEQ ID NO：22；sk011：核苷酸序列为SEQ ID NO：24；sk012：核苷酸序列为SEQ ID NO：16；sk013：核苷酸序列为SEQ ID NO：28；sk014：核苷酸序列为SEQ ID NO：30；sk015：核苷酸序列为SEQ ID NO：32；sk016：核苷酸序列为SEQ ID NO：34；sk017：核苷酸序列为SEQ ID NO：36；sk018：核苷酸序列为SEQ ID NO：38；sk019：核苷酸序列为SEQ ID NO：40；sk020：核苷酸序列为SEQ ID NO：42；sk021：核苷酸序列为SEQ ID NO：44；sk022：核苷酸序列为SEQ ID NO：45；sk023：核苷酸序列为SEQ ID NO：48；sk024：核苷酸序列为SEQ ID NO：50；sk025：核苷酸序列为SEQ ID NO：52；sk026：核苷酸序列为SEQ ID NO：54；sk027：核苷酸序列为SEQ ID NO：56；sk028：核苷酸序列为SEQ ID NO：58；sk029：核苷酸序列为SEQ ID NO：60；sk030：核苷酸序列为SEQ ID NO：62；sk031：核苷酸序列为SEQ ID NO：64；sk032：核苷酸序列为SEQ ID NO：66；sk033：核苷酸序列为SEQ ID NO：68；sk034：核苷酸序列为SEQ ID NO：70；sk035：核苷酸序列为SEQ ID NO：72；sk036：核苷酸序列为SEQ ID NO：74；sk037：核苷酸序列为SEQ ID NO：76；sk038：核苷酸序列为SEQ ID NO：78；sk039：核苷酸序列为SEQ ID NO：80；sk040：核苷酸序列为SEQ ID NO：82；sk041：核苷酸序列为SEQ ID NO：84；sk042：核苷酸序列为SEQ ID NO：86；sk043：核苷酸序列为SEQ ID NO：88；sk044：核苷酸序列为SEQ ID NO：90；sk045：核苷酸序列为SEQ ID NO：92；sk046：核苷酸序列为SEQ ID NO：94；sk047：核苷酸序列为SEQ ID NO：96；sk048：核苷酸序列为SEQ ID NO：98；或sk049：核苷酸序列为SEQ ID NO：100。其中，上述核苷酸序列与数字编号相同的多肽或突变体相对应。
在另一方面，本发明提供一种表达所述多肽或其突变体的表达载体或宿主细胞，包含上述多肽或突变体的核苷酸序列。所述表达载体可以是重组真核表达载体，优选哺乳动物 细胞表达载体；也可以是重组病毒表达载体，优选腺相关病毒或腺病毒载体。所述表达载体能在转化的宿主细胞中复制表达，优选地，所述宿主细胞为CHO细胞及其亚系或293细胞及其亚系。
在另一方面，本发明提供一种制备所述多肽或突变体的方法，所述方法为化学合成法或重组表达法，所述重组表达法包括将上述表达载体引入合适的宿主细胞中，进行多肽或突变体的表达。
在另一方面，本发明提供一种药物组合物，包含上述多肽或其突变体或其药学上可接受的盐和药学上可接受的载体或赋形剂；优选地，所述药物组合物的剂型为注射剂、眼药水、眼用凝胶或眼药膏。
在另一方面，本发明提供一种本发明所述多肽或其突变体或药学上可接受的盐在制备治疗或预防由血管新生或生长引起的疾病的药物中的应用。所述疾病优选为眼睛疾病、肿瘤、缺血性心脏病、非炎症性心肌病、冠状动脉硬化、闭塞性动脉硬化、动脉栓塞、动脉血栓、Berger′s病、慢性炎症、炎症性肠病、溃疡、风湿性关节炎、硬皮症、银屑病、不育症或肉瘤状病；所述眼睛疾病优选为年龄相关性黄斑变性、糖尿病性视网膜病变、脉络膜新生血管、黄斑囊样水肿、糖尿病性黄斑水肿、视网膜血管闭塞、角膜新生血管、角膜移植手术新生血管性青光眼、翼状胬肉或慢性结膜炎。
在另一方面，本发明提供一种抑制哺乳动物血管新生或生长的方法，包括：给需要的对象施用上述的多肽或其突变体或其药学上可接受的盐，或上述药物组合物；优选地，所述施用方式为眼表施用或玻璃体腔内注射施用。
本发明的主要优点，包括：
(a)本发明多肽分子量小，可透过眼组织屏障；
(b)水溶性好，能在中性泪液、房水和玻璃体液中保持较高的浓度；
(c)安全性高，对生物组织毒副作用小；并且眼局部用药生物利用度高，可减少剂量，从而减小全身副作用；
(d)可通过固相合成的方法制备，纯度高，产量大，成本低；
(e)本发明多肽的稳定性好。
附图说明：
图1是小肽SK004的高效液相色谱及质谱分析的纯度鉴定结果图，其中，图1a为SK004色谱图，图1b为SK004一级质谱图，图1c为SK004二级质谱图。
图2a是小肽SK004对人脐静脉内皮细胞HUVEC增殖的影响结果图。
图2b是小肽SK018对人脐静脉内皮细胞HUVEC增殖的影响结果图。
图3是小肽SK004对鸡胚尿囊膜上新生血管的影响结果图，其中图3a-3c显示滤纸片周2.5mm范围内3-5级微血管计数：图3a为PBS组；图3b为Avasin(5μl/片)组；图3c为SK004(5μl/片)组；图3d为PBS组、Avastin组以及不同浓度的SK004对鸡胚尿囊膜上新生血管的影响。
图4是小肽SK018对鸡胚尿囊膜上新生血管的影响结果图，其中图4a-4c显示滤纸片 周2.5mm范围内3-5级微血管计数：图4a为PBS组；图4b为Avasin(5μl/片)组；图4c为SK018(5μl/片)组；图4d为PBS组、Avastin组以及不同浓度的SK018对鸡胚尿囊膜上新生血管的影响。
图5a是小肽SK004对大鼠角膜病理性新生血管的影响结果图。
图5b是小肽SK018对大鼠角膜病理性新生血管的影响结果图。
具体实施方式
活性多肽
在本发明中，本发明的多肽或其突变体指具有血管新生抑制活性的蛋白或多肽。所述术语包括具有抑制血管新生或生长功能的、包含序列SEQ ID NO：1的多肽及其变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个氨基酸。此外，所述术语还包括单体和多聚体形式的本发明多肽、线性以及非线性的多肽(如环肽)。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持抑制血管新生功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)本发明的多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的然后蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明的多肽类似物与本发明的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中通过糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成的修饰。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸、磷酸丝氨酸、磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
本发明的多肽还可以以由药学或生理学可接受的酸或破衍生的盐形式使用。这些盐包括(但不限于)与如下酸形成的盐：氢氟酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、琥珀酸、草酸、富马酸、马来酸、草酰乙酸、甲磺酸、乙磺酸、苯磺酸或羟乙磺酸。其它盐包括：与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以及以酯、氨基甲酸酯或其他常规的“前体药物”的形式。
编码序列
本发明的多核苷酸可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。本发明的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获 得。目前，已经可以完全通过化学合成来得到编码本发明多肽或其突变体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明多肽的编码序列经基因工程产生的宿主细胞。
制备方法
本发明多肽或其突变体可以是重组多肽或合成多肽，相应地，本发明多肽或其突变体可用常规方法人工合成，也可用重组方法生产。一种优选的方法是使用液相合成技术或固相合成技术，如Boc固相法、Fmoc固相法或是两种方法联合使用。固相合成可快速获得样品，可根据目的肽的序列特征选用适当的树脂载体及合成系统。例如，Fmoc系统中优选的固相载体如连接有肽中C端氨基酸的Wang树脂，Wang树脂结构为聚苯乙烯，与氨基酸间的手臂是4-烷氧基苄醇；用25％六氢吡啶/二甲基甲酰胺室温处理20分钟，以除去Fmoc保护基团，并按照给定的氨基酸序列由C端逐个向N端延伸。合成完成后，用含4％对甲基苯酚的三氟乙酸将合成的胰岛素原相关肽从树脂上切割下来并除去保护基，可过滤除树脂后乙醚沉淀分离得到粗肽。将所得产物的溶液冻干后，用凝胶过滤和反相高压液相层析法纯化所需的肽。当使用Boc系统进行固相合成时，优选树脂为连接有肽中C端氨基酸的PAM树脂，PAM树脂结构为聚苯乙烯，与氨基酸间的手臂是4-羟甲基苯乙酰胺；在Boc合成系统中，在去保护、中和、偶联的循环中，用TFA/二氯甲烷(DCM)除去保护基团Boc并用二异丙基乙胺(DIEA)二氯甲烷中和。肽链缩合完成后，用含对甲苯酚(5-10％)的氟化氢(HF)在0℃下处理1小时，将肽链从树脂上切下，同时除去保护基团。以50-80％乙酸(含少量巯基乙醇)抽提肽，溶液冻干后进一步用分子筛Sephadex G10或Tsk-40f分离纯化，然后再经高压液相纯化得到所需的肽。可以使用肽化学领域内已知的各种偶联剂和偶联方法偶联各氨基酸残基，例如可使用二环己基碳二亚胺(DCC)，羟基苯骈三氮唑(HOBt)或1，1，3，3-四脲六氟磷酸酯(HBTU)进行直接偶联。对于合成得到的短肽，其纯度与结构可用反相高效液相和质谱分析进行确证。
另一种方法是用重组技术产生本发明多肽。通过常规的重组DNA技术，可利用本发明的多核苷酸来表达或生产重组的SK系列多肽。一般来说有以下步骤：
(1)将编码本发明多肽或其突变体的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；(2)在合适的培养基中培养宿主细胞；(3)从培养基或细胞中分离、纯化蛋白质。
重组多肽可在细胞内或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
由于本发明多肽较短，因此可以考虑将多个多肽串联在一起，重组表达后获得多聚体形式的表达产物，然后通过酶切等方法形成所需的小肽。
药物组合物和施用方法
本发明还提供了一种药物组合物，它含有(a)安全有效量的本发明多肽或其突变体或其药学上可接受的盐；以及(b)药学上可接受的载体或赋形剂。药物组合物中，本发明多肽或其突变体的量通常为10微克-100毫克/剂，较佳地为100-1000微克/剂。为了本发明的目的，有效的剂量为给予个体约0.01毫克/千克至50毫克/千克，较佳地0.05毫克/千克至10毫克/千克体重的本发明多肽或其突变体。此外，本发明的多肽或其突变体可以单用，也可与其它治疗剂一起使用(如配制在同一药物组合物中)。
所述药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在Remington’s Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂及其组合。药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦制成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：眼表、眼周，眼内(尤其是玻璃体腔内)、肌内、静脉内、皮下、皮内或局部给药。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有眼药水、针剂(尤其是玻璃体腔内注射剂)、眼用凝胶和眼药膏。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型利用惯常方式进行。例如，眼药水的配制可这样进行：将本发明的多肽或其突变体或其药学上可接受的盐与基本物质一起溶解于无菌水(在无菌水中溶解有表面活性剂)中，调节渗透压和酸碱度至生理状态，并可任意地加入合适的药物添加剂如防腐剂、稳定剂、缓冲剂、等渗剂、抗氧化剂和增粘剂，然后使其完全溶解。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明的多肽或其突变体或其盐可被掺入以缓释聚合物为裁体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织中。此外，本发明的多肽或其突变体或其盐还可通过插入预先涂有药物的眼内透镜而得以应用。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明的多肽或其突变体 或其药学上可接受的盐的剂量，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定。例如，当局部滴眼时，通常其浓度约为0.1-10wt％，较佳地1-5wt％，每日可2-6次给药，每次1-2滴。
血管内皮生长因子与血管新生
血管内皮生长因子(vascular endothelial growth factor，VEGF)是新生血管形成过程中的一个主要介导因子，VEGF基因经过转录水平的剪切至少可产生7种VEGF变异体，其中VEGF165(VEGF-A，即原型VEGF)是最常见、最主要的一种。其主要的生物学功能为：选择性增强血管内皮细胞有丝分裂，刺激血管内皮细胞增殖并促进血管形成；增强血管尤其是小血管的渗透性，使血浆蛋白等大分子(主要是纤维蛋白原)外渗沉积在血管外的基质中，为新生毛细血管网的建立提供营养。
因此，科研人员针对VEGF的抑制进行了很多研究。贝伐单抗(bevacizumab)是一种重组抗人VEGF单抗，它是第一个由美国FDA批准运用于治疗转移性结肠癌患者的抗新生血管药物。玻璃体注射bevacizumab亦能有效抑制视网膜及脉络膜新生血管。另一种VEGF抑制剂Ranibizumab，是一种重组抗人VEGF单抗片段。它是第一个被美国FDA批准用于治疗眼部新生血管性黄斑变性的抗新生血管类药物。目前，人们仍在不断寻找新的更加安全有效的治疗眼部新生血管的药物。
本发明人经过广泛而深入的研究，首次制备了一类分子量小于5kD(如仅约1-2kD)的小分子多肽。具体而言，本发明人应用生物信息学的方法，基于同源性分析和生物学特性等分析，设计了数个候选序列，采用固相法将其合成，分离纯化获得高纯度的本发明的多肽及其突变体，经VEGF诱导的人脐静脉内皮细胞增殖模型、鸡胚尿囊膜血管模型以及大鼠角膜缝线模型筛选，获得了一类新型的、具有预防和治疗血管新生功能的小分子多肽。
实验模型
人脐静脉内皮细胞(HUVEC)检测
血管新生指的是在原始血管丛或已存在血管的基础上以出芽的方式形成新生血管的过程。在病理情况下，机体内已存在的血管受到促血管生成因子的刺激，发生扩张，血管内皮细胞活化、增殖、迁移、小管腔样结构形成。其中，内皮细胞自我增殖是血管新生的基础，它为形成新生血管提供所必须的细胞数量。因此，本发明以内皮细胞增殖作为药物干预的靶点，应用CCK-8方法(购自Dojindo)对人脐静脉内皮细胞(HUVEC)进行检测，以探讨多肽SK004在抗血管新生过程中抑制内皮细胞增殖的作用。
鸡胚尿囊膜(CAM)模型
该模型是由60年前的实验胚胎学家首创的，最初被用于胚胎器官发育的研究，目前是国内外研究血管生长常用的实验技术，广泛用于研究诱导血管生长或者抑制血管生长类药物对微血管的作用。
大鼠角膜缝线模型
大鼠角膜缝线模型是经典的眼部新生血管干预模型，可重复性好，可定量分析并且成本较低，普遍用于检测抗新生血管药物的活性作用。
工业应用性
含有本发明肽或其药学上可接受盐作为活性成分的药物组合物，对血管新生或生长有显著的抑制活性。经动物试验证实，本发明多肽不仅可以抑制鸡胚尿囊膜的血管新生，还可抑制大鼠角膜新生血管，而且可以抑制人脐静脉血管内皮细胞的增殖作用。因此本发明多肽有望开发成药物，用于治疗新生血管性眼病及相关的新生血管性疾病，如肿瘤新生血管等。
序列说明：
以下多肽作为对照多肽，来自中国专利CN 201010235580.4。
多肽SK050，其氨基酸序列为SEQ ID NO：101，核苷酸序列为SEQ ID NO：102；
多肽SK051，其氨基酸序列为SEQ ID NO：103，核苷酸序列为SEQ ID NO：104；
多肽SK052，其氨基酸序列为SEQ ID NO：105，核苷酸序列为SEQ ID NO：106；
多肽SK053，其氨基酸序列为SEQ ID NO：107，核苷酸序列为SEQ ID NO：108。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
实施例一：小肽SK004的合成及分离纯化
采用市售的SYMPHONY型12通道多肽合成仪(美国Protein Technologies公司)，合成序列为SEQ ID NO：9所示的SK004多肽。具体方法如下：根据多肽合成仪的软件(Version.201版)计算配置试剂，将2-Chlorotrityl Chloride Resin树脂(天津市南开合成科技有限公司)放入反应管中，加DMF(15ml/g)(Dikma)，振荡30min。通过沙芯抽滤掉溶剂，分别加入3倍摩尔过量的Fmoc-L-H-OH(Fmoc-亮氨酸-组氨酸-OH)(小肽SK004)氨基酸(苏州天马医药集团精细化学品有限公司)，再加入10倍摩尔过量的DIEA(N，N-二异丙基乙胺)(国药集团上海化学试剂公司)，最后加入DMF溶解，振荡30min。去掉DMF，加20％哌啶(国药集团上海化学试剂公司)DMF溶液(15ml/g)，5min，去掉DMF，再加20％哌啶DMF溶液(15ml/g)，15min。抽掉哌啶溶液，取十几粒树脂，用乙醇洗三次，加入茚三酮，KCN，苯酚溶液各一滴，105℃-110℃加热5min，变深蓝色为阳性反应。用DMF(10ml/g)洗两次，甲醇(10ml/g)洗两次，DMF(10ml/g)洗两次。加入保护氨基酸(FOMC-Asp-OH，即Fmoc-天冬氨酸-OH)三倍过量，HBTU(O-苯并三氮唑-四甲基脲六氟磷酸酯)(苏州天马医药集团精细化学品有限公司)三倍过量，均用尽量少的DMF溶解，加入反应管，立刻加入NMM(N-甲基吗啉)十倍过量，反应30min。用DMF(10ml/g)洗一次，甲醇(10ml/g)洗两次，DMF(10ml/g)洗两次。重复上述操作步骤，从右到左依次连接小肽SK004序列中的氨基酸。最后一个氨基酸连接后，脱保护，用DMF(10ml/g)洗两 次，甲醇(10ml/g)洗两次，DMF(10ml/g)洗两次，DCM(二氯甲烷)(10ml/g)洗两次，以此洗去树脂，再抽干10min。从树脂上切割多肽(切割液(10/g)：TFA(三氟乙酸)(J.T.Baker)94.5％，水2.5％，EDT(1，2-乙二硫醇)(ALDRICH)2.5％，TIS(苯甲硫醚)(ALDRICH)1％；切割时间：120min)。将裂解液用氮气(上海比欧气体工业公司)尽量吹干，用乙醚(上海试一化学试剂有限公司)洗六次，然后常温挥干。最后将纯化后的溶液冻干，既得到高纯度(＞95％)的小肽SK004。
用HPLC(SHIMADZU高效液相色谱仪型号：制备型，分析型，软件：Class-VP.Sevial System，厂商：SHIMADZU)纯化多肽，将粗肽用纯水或者加少量乙腈(Fisher)溶解，按照下列条件进一步纯化小肽SK004。
色谱柱：Agilent Eclipse plus C18RRHD 1.8μm 2.1×150mm
流动相A：0.1％甲酸水溶液；流动相B：0.1％甲酸乙腈溶液。梯度如表1：
表1：梯度洗脱条件
时间(min) 流动相A(％) 流动相B(％) 流速(ml/min)
0 90 10 0.2
10 90 10 0.2
25 40 60 0.2
26 90 10 0.2
30 90 10 0.2
检测波长：220nm柱温：30℃
实施例二：小肽SK004的鉴定及保存
取少量的成品小肽SK004，做HPLC分析的纯度鉴定，和ESI-MS的分子量鉴定。条件如下：电喷雾电离，正离子模式检测，氮气作为鞘气、辅助气和吹扫气。啧雾电压4.7kV：鞘气为15arb，辅助气为5arb，吹扫气为0arb；毛细管温度：275℃，毛细管电压为40V，透镜电压为120V。全扫描质量范围：300-2000amu。二级质谱用数据依赖模式采集，全扫描图谱中+1价分子离子峰用于二级质谱分析，高纯氦气用作碰撞气，碰撞能量为35ev。
结果表明，小肽SK004分子量：882.89DA，其结构信息正确，符合理论设计的氨基酸序列，无位点突变及表达后修饰(结果见图1a-c)。
将白色粉末状的小肽，密封包装，置于-20℃长期保存。
实施例三：多肽的制备、鉴定和活性检测
按实施例一的方法制备如表3中所示多肽，并按实施例二进行多肽鉴定。这些多肽的结构信息均正确，与设计的氨基酸序列一致。并按如下方法进行活性检测。
1、活性检测原理
VEGF具有促进HUVEC细胞增殖的活性，本发明多肽具有抑制VEGF的作用，因此本次检测利用HUVEC细胞进行生物学活性比较，首先接种HUVEC细胞，利用2％FBS-ECM稀释各个样品，稀释完毕后，各样品与VEGF混合。待细胞贴壁后，吸弃各上样孔中上清液，加入样品，并做稀释液对照以及VEGF对照，培养4天后，每孔加入20μl CCK-8显色液对细胞进行显色，然后利用酶标仪对显色后的细胞进行测定，测定吸光 度值的大小直接反应细胞数量，根据HUVEC活细胞数，推断各个样品抑制VEGF介导的促HUVEC细胞增殖活性。其中稀释液对照组作为阴性对照，VEGF对照组作为阳性对照。
2、实验仪器及材料(见表2)
表2：试验仪器和材料
名称 厂家 型号
酶标仪 MD VESRAmax
二氧化碳孵箱 Thermo Forma Series II 3111
生物安全柜 ESCO AC2-4E1
倒置显微镜 OLYMPUS IX51-12pn/30W
ECM ScienCell  
ECGS ScienCell  
CCK-8 Dojindo  
VEGF R&D  
FBS ScienCell  
3、实验步骤
3.1复苏细胞
从液氮中取出1支HUVEC(ScienCell公司)细胞，37℃快速融化，离心重悬，用ECGS-ECM 4ml悬浮细胞，进行细胞计数，并加ECGS-ECM培养基(ScienCell公司，货号7693)配制2.0×104个/ml的细胞悬液。
3.2接种细胞
取细胞悬液4ml，加ECGS-ECM培养基16ml，混匀后以100μl/孔加入96孔板中，37℃，5％CO2孵箱中培养1天。
3.3样品稀释上样
将各样品分别用ECGS-ECM培养基稀释至2.5mM和1.25mM备用。
3.4配制VEGF工作液
用2％FBS-ECM将VEGF稀释至40ng/ml备用。
3.5孵育
取3.3制备的各样品，加入等体积VEGF工作液，混匀，37℃培养箱孵育3小时。
3.6加样
取生长良好的96孔板，吸弃96孔板中的培养液，以100μl/孔加入样品，37℃，5％CO2孵箱中培养4天
3.7读数和分析
培养结束后，观察细胞，确认形态正常无污染，每孔加入25μl CCK-8工作液，37℃反应4小时，用酶标仪于450nm处测吸收值。
4、实验结果
所有的检测结果均以抑制率(％)表示，抑制率可反应样品对VEGF的抑制情况，计 算公式如下：
抑制率(％)＝稀释液对照组结果/样品结果*100
活性检测结果见表3。
表3：多肽及活性检测结果
Figure PCTCN2015071438-appb-000001
本次测定中，当样品浓度为1.25mM时，所有样品结果均在55％～80％，说明这些多肽在该浓度下对VEGF具有一定的抑制作用，当浓度为2.5mM时，效果更优。
实施例四：SK004小肽及其光学异构体的活性检测试验
按照实施例三的检测方法，对于SK004及其光学异构体的样品进行活性检测，检测结果见表4。
表4：氨基酸序列及活性检测结果
样品编号 序列 抑制率(％)(1.25mM)
SK004(SEQ ID NO：9) GAAGDENLH 71.47
SK004-1(SEQ ID NO：9) GAAGDEnLH 73.44
SK004-2(SEQ ID NO：9) GAaGDENLH 61.61
注：小写的字母表示为D型结构
本次测定中，两个旋光异构体在1.25mM时抑制率与正常9肽样品相当。
实施例五：多肽对人脐静脉血管内皮细胞增殖活性的影响
使用CCK-8方法，具体方法如下：
将人脐静脉血管内皮细胞HUVEC(购自ScienCell公司)接种于96孔板中，接种浓度为2×104/ml；细胞贴壁后加入无血清培养剂ECM 37℃培养24小时；之后向孔中分别加入无血清培养剂ECM作为阴性对照、VEGF(20ng/ml)(购自R&D公司)作为阳性对照、VEGF(20ng/孔)+Avastin(购自罗氏公司)、VEGF(20ng/孔)+不同浓度的小肽SK004或SK018作为处理组；继续培养96小时后，在各孔中加入20μl的CCK-8溶液(购自Dojindo公司)；37℃孵育4小时后，利用酶标仪(Molecular Device公司)测定450nm处各孔的吸光度，根据OD450判断细胞的增殖活性，OD450值越低，抑制效应越强，最后运用SPSS16.0.1进行统计分析。
由图2a可见，相对于VEGF组，小肽SK004有明显抑制人脐静脉血管内皮细胞HUVEC增殖的效应，并呈浓度依赖性，**P＜0.01，差异具有统计学意义。
由图2b可见，相对于VEGF组，小肽SK018有明显抑制人脐静脉血管内皮细胞HUVEC增殖的效应，并呈浓度依赖性，**P＜0.01，差异具有统计学意义。
实施例六：小肽抗鸡胚尿囊膜新生血管效应的测定
使用鸡胚尿囊膜模型，具体方法如下：将生后1-2天的种鸡蛋(购于上海星火农场36连华青鸡场)消毒后装入恒温恒湿箱(上海博迅实业有限公司医疗设备厂，SPX-250C)(T＝37℃，湿度H＝60-70％)孵育5天(24小时计一天)，每天早晚各翻蛋一次；之后将含有醋酸可的松(5μg/μl，5μl/片)的滤纸片(Whatman quantitative filter papers，Sigma，ashless，Grade 42，Cat No 1442-042，42.5mmΦ×100circles)分别滴加PBS(5μl/片)、低浓度(0.5μg/μl)、中浓度(2μg/μl)、高浓度(10μg/μl)的小肽SK004(或SK018)(5μl/片)以及Avastin(10μg/μl，5μl/片)，滤纸片风干后置于种鸡蛋尿囊膜大血管之间并密封种鸡蛋；继续将种鸡蛋置于恒温恒湿箱(温度T＝37℃，湿度H＝60-70％)孵育2天(24小时计一天)，不翻蛋；之后完全暴露鸡胚尿囊膜，拍照(范围为滤纸片周5mm)并对3-5级微血管计数(范围为滤纸片周2.5mm)，运用SPSS16.0.1进行统计分析。
由图3a-3d可见，相较于PBS组，小肽在各浓度时均有明显抑制鸡胚尿囊膜新生血管的作用。图3a-3c示滤纸片周2.5mm范围内3-5级微血管计数。图3a为PBS组；图3b为Avastin(5μl/片)组；图3c为SK004(5μl/片)组；图3d为相对于PBS组，各个浓度的小肽SK004组均明显抑制鸡胚尿囊膜新生血管数，且抑制作用呈浓度依赖性，**P＜0.01，差异具有统计学意义。
由图4a-4d可见，相较于PBS组，小肽在各浓度时均有明显抑制鸡胚尿囊膜新生血管的作用。图4a-4c显示滤纸片周2.5mm范围内3-5级微血管计数。图4a为PBS组；图4b为Avasin(5μl/片)组；图4c为SK018(5μl/片)组；图4d为相对于PBS组，各个浓度的小 肽SK018组均明显抑制鸡胚尿囊膜新生血管数，且抑制作用呈浓度依赖性，**P＜0.01，差异具有统计学意义。
实施例七：小肽抗大鼠角膜病理性新生血管效应的测定
使用大鼠角膜缝线模型，具体方法如下：健康SD大鼠，160-180g，雄性，以右眼为实验眼。术前3天所有实验眼点0.3％氧氟沙星滴眼液，每日2次。实验前检查实验眼，排除眼部病变并磅体重。按3ml/kg体重腹腔内注射1％戊巴比妥钠，予以全身麻醉，并予眼表麻醉。首先用直径3mm的角膜环钻在大鼠角膜中央轻作压痕，用10-0尼龙线垂直压痕方向并以其为中线作角膜基质缝合，每眼于颞侧穿入角膜基质1针，每针跨度约1mm，缝线最外端距角膜缘约1mm，术毕结膜囊涂氧氟沙星眼膏。术后各组分别每日4次滴眼液(10μl/次/眼。按实施例九制备滴眼液，其中Avastin和小肽的含量见分组中所示)。
分组如下：正常对照组(不予缝线)，空白对照组(缝线+PBS)，阳性对照组(缝线+Avastin 10mg/ml)，SK004组(或SK018)(缝线+0.5mg/ml)。每组8只大鼠(n＝8)。分别于术后第3、5、7天散瞳后观察角膜新生血管(CNV)情况，并用量规测量新生血管长度L，同时记录新生血管生长钟点数C。计算CNV面积S＝0.4*3.1416*C*L。并照相。运用one-way ANOVA比较各组CNV面积S(平均值±SE)，运用SPSS16.0.1进行统计分析。
由图5a可见，小肽SK004具有明显抑制大鼠角膜病理性新生血管作用。相对于PBS组，小肽SK004组在术后第3天、第5天及第7天具有明显抑制大鼠角膜病理性新生血管作用，*P＜0.05，**P＜0.01，差异具有统计学意义。
由图5b可见，相对于PBS组，小肽SK018(0.5mg/ml)在术后第3天具有明显抑制大鼠角膜病理性新生血管作用，*P＜0.05，**P＜0.01，差异具有统计学意义。
以上述方法对其它多肽的抗大鼠角膜病理性新生血管效应进行测定。
分组如下：空白对照组(缝线+PBS)，阳性对照组(缝线+Avastin 10mg/ml)，各多肽组(缝线+0.5mg/ml)。每组8只大鼠(n＝8)。分别于术后第7天散瞳后观察角膜新生血管(CNV)情况，并用量规测量新生血管长度L，同时记录新生血管生长钟点数C。计算CNV面积S＝0.4*3.1416*C*L。并照相。运用one-wayANOVA比较各组CNV面积S(平均值±SE)，运用SPSS16.0.1进行统计分析。检测结果见表5：
表5：氨基酸序列及活性检测结果
Figure PCTCN2015071438-appb-000002
Figure PCTCN2015071438-appb-000003
结果表明，本发明多肽组在术后第7天具有明显抑制大鼠角膜病理性新生血管作用。
实施例八：其它衍生多肽的制备、鉴定和活性检测
按实施例一的方法制备如表6所示的多肽，并按实施例二进行多肽鉴定，结果表明，这些多肽的结构信息正确，氨基酸序列与设计序列一致。使用CCK-8方法检测多肽活性，具体方法如下：
将人脐静脉血管内皮细胞HUVEC(购自ScienCell公司)接种于96孔板中，接种浓度为2×104/ml；细胞贴壁后加入无血清培养剂ECM 37℃培养24小时；之后向孔中分别加入无血清培养剂ECM作为阴性对照、VEGF(20ng/ml)(购自R&D公司)作为阳性对照、VEGF(20ng/孔)+不同的小肽SK001、SK005、SK018、SK019-049小肽(1μg/μl)作为处理组；继续培养96小时后，在各孔中加入20μl的CCK-8溶液(购自Dojindo公司)；37℃孵育4小时后，利用酶标仪(Molecular Device公司)测定490nm各孔的吸光度，根据OD490判断细胞的增殖活性，OD490值越低，抑制效应越强，最后运用SPSS16.0.1进行统计分析。其中，VEGF对照组的OD490值为1.467。检测结果见表6：
表6：氨基酸序列及活性检测结果
Figure PCTCN2015071438-appb-000004
Figure PCTCN2015071438-appb-000005
结果表明，上述衍生多肽的处理组中，HUVEC细胞增殖在多肽浓度0.625mM时即开始显著受到抑制，均具有统计学意义。
实施例九：眼药水的制备
利用常规技术，混合以下组分，制得1％眼部滴眼液，其配方如下：
SK004 10mg
羟丙基甲基纤维素  0.03g
无菌水  10mL
调节渗透压  300Osm
酸碱度(pH)  6.8～7.1
经3位稳定的角膜新生血管(新生血管长入角巩缘内至少1mm)志愿者试周3周，每日4次，每次2滴/眼。疗效判断标准如下：
以最佳矫正视力、角膜新生血管(包括：新生血管面积、新生血管总长度、新生血管平均管径)为主要判定指标。治疗情况分为显效、有效和无效3个等级，分别计分。
显效：①视力提高4行以上；②角膜新生血管：新生血管面积减少≥30％、新生血管总长度减少≥20％、新生血管平均管径减少≥10％。
有效：①视力提高2行以上；②角膜新生血管：新生血管面积减少≥15％、新生血管总长度减少≥10％、新生血管平均管径减少≥5％。
无效：①视力无提高；②角膜新生血管：新生血管面积减少≤5％、新生血管总长度减少≤3％、新生血管平均管径减少≤1％。
结果：三位患者经过3周治疗后，患眼视力均提高2行或以上，角膜新生血管面积显著减少，新生血管总长度缩短，新生血管平均管径减小。这表明，所述滴眼液可有效抑制眼部新生血管相关的疾病。
实施例十：玻璃体腔注射液的制备
利用常规技术，混合以下组分，制得眼部玻璃体腔注射液，其配方如下：
SK004 10mg
BSS眼内灌注液  0.5mL
调节渗透压  300Osm
酸碱度(pH)  7.2～7.4
经3位由年龄相关性黄斑变性引起的脉络膜新生血管(CNV)志愿者每4周行玻璃体腔内注射一次，连续注射3次，每次注射0.5ml。疗效判断标准如下：以最佳矫正视力、彩色眼底照相、光学相干段层扫描(OCT)、眼底血管荧光造影(FFA)为判定指标。最佳矫正视力：提高：①视力提高≥2行；不变：①视力提高＜2行；下降：①视力下降≥2行。
以彩色眼底照相、光学相干段层扫描(OCT)、眼底血管荧光造影(FFA)获取CNV病灶消退、渗漏或吸收的情况，将CNV分为4级：
(1)CNV完全闭合：CNV病灶完全无荧光渗漏，仅表现为CNV纤维染色，OCT检查显示水肿消失；
(2)CNV部分闭合：荧光素渗漏范围小于治疗前CNV渗漏范围的50％，OCT检查显示只有少许视网膜下或层间积液；
(3)CNV小部分闭合：荧光素渗漏范围占据治疗前CNV渗漏范围的50％以上，OCT检查显示比较明显的视网膜下积液；
(4)CNV复发：出现新的CNV或CNV病变区荧光素渗漏范围超过原病灶边界，OCT检查显示明显的视网膜下积液。
结果：三位患者经过3次治疗后，患眼视力均提高2行或以上，CNV的FFA荧光渗漏范围缩小，OCT显示的网膜下积液减少。这表明，所述滴眼液可有效抑制眼部新生血管相关的疾病。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请权利要求书所限定的范围中。

Claims (11)

  1. 一种抑制血管新生或生长的多肽，所述多肽含有SEQ ID NO：1所示序列。
  2. 根据权利要求1所述的多肽，其特征在于，所述多肽为在SEQ ID NO：1序列的前面按次序增加SEQ ID NO：2的从C端开始的任一个或多个氨基酸；或在SEQ ID NO：1的后面按次序增加SEQ ID NO：3的从N端开始的任意一个或多个氨基酸；或前后都按上述次序增加SEQ ID NO：2和SEQ ID NO：3中的任一个或多个氨基酸。
  3. 根据权利要求1所述的多肽，其特征在于，所述多肽为：
    SK001：其氨基酸序列为SEQ ID NO：1；SK002：其氨基酸序列为SEQ ID NO：5；SK003：其氨基酸序列为SEQ ID NO：7；SK004：其氨基酸序列为SEQ ID NO：9；SK005：其氨基酸序列为SEQ ID NO：11；SK006：其氨基酸序列为SEQ ID NO：13；SK007：其氨基酸序列为SEQ ID NO：15；SK008：其氨基酸序列为SEQ ID NO：17；SK009：其氨基酸序列为SEQ ID NO：19；SK010：其氨基酸序列为SEQ ID NO：21；SK011：其氨基酸序列为SEQ ID NO：23；SK012：其氨基酸序列为SEQ ID NO：25；SK013：其氨基酸序列为SEQ ID NO：27；SK014：其氨基酸序列为SEQ ID NO：29；SK015：其氨基酸序列为SEQ ID NO：31；SK016：其氨基酸序列为SEQ ID NO：33；SK017：其氨基酸序列为SEQ ID NO：35；或SK018：其氨基酸序列为SEQ ID NO：37。
  4. 根据权利要求1-3任一项所述的多肽的突变体，所述突变体是基于权利要求1-3任一项所述的多肽中的SEQ ID NO：1序列中的氨基酸残基进行突变，具体突变如下：SEQ ID NO：1的：第一位A被V或L取代；或第二位A被G或V取代；或第三位G被A、P或D取代；或第四位D被E取代；或第五位E被D取代；或第六位N被Q取代；或第七位L被Q、M、F或I取代。
  5. 根据权利要求4所述的突变体，其特征在于，所述突变体为：
    SK019：其氨基酸序列为SEQ ID NO：39；SK020：其氨基酸序列为SEQ ID NO：41；SK021：其氨基酸序列为SEQ ID NO：43；SK022：其氨基酸序列为SEQ ID NO：45；SK023：其氨基酸序列为SEQ ID NO：47；SK024：其氨基酸序列为SEQ ID NO：49；SK025：其氨基酸序列为SEQ ID NO：51；SK026：其氨基酸序列为SEQ ID NO：53；SK027：其氨基酸序列为SEQ ID NO：55；SK028：其氨基酸序列为SEQ ID NO：57；SK029：其氨基酸序列为SEQ ID NO：59；SK030：其氨基酸序列为SEQ ID NO：61；SK031：其氨基酸序列为SEQ ID NO：63；SK032：其氨基酸序列为SEQ ID NO：65；SK033：其氨基酸序列为SEQ ID NO：67；SK034：其氨基酸序列为SEQ ID NO：69；SK035：其氨基酸序列为SEQ ID NO：71；SK036：其氨基酸序列为SEQ ID NO：73；SK037：其氨基酸序列为SEQ ID NO：75；SK038：其氨基酸序列为SEQ ID NO：77；SK039：其氨基酸序列为SEQ ID NO：79；SK040：其氨基酸序列为SEQ ID NO：81；SK041：其氨基酸序列为SEQ ID NO：83；SK042：其氨基酸序列为SEQ ID NO：85；SK043：其氨基酸序列为SEQ ID NO：87；SK044：其氨基酸序列为SEQ ID NO：89；SK045：其氨基酸序列为SEQ ID NO：91；SK046：其氨基酸序列为SEQ ID NO：93；SK047：其氨基酸序列为SEQ ID NO：95；SK048：其氨基酸序列为 SEQ ID NO：97；或SK049：其氨基酸序列为SEQ ID NO：99。
  6. 一种编码如权利要求1-3任一项所述的多肽或权利要求4-5任一项所述的突变体的核苷酸序列；优选地，所述核苷酸序列为sk001：核苷酸序列为SEQ ID NO：4；sk002：核苷酸序列为SEQ ID NO：6；sk003：核苷酸序列为SEQ ID NO：8；sk004：核苷酸序列为SEQ ID NO：10；sk005：核苷酸序列为SEQ ID NO：12；sk006：核苷酸序列为SEQ ID NO：14；sk007：核苷酸序列为SEQ ID NO：16；sk008：核苷酸序列为SEQ ID NO：18；sk009：核苷酸序列为SEQ ID NO：20；sk010：核苷酸序列为SEQ ID NO：22；sk011：核苷酸序列为SEQ ID NO：24；sk012：核苷酸序列为SEQ ID NO：16；sk013：核苷酸序列为SEQ ID NO：28；sk014：核苷酸序列为SEQ ID NO：30；sk015：核苷酸序列为SEQ ID NO：32；sk016：核苷酸序列为SEQ ID NO：34；sk017：核苷酸序列为SEQ ID NO：36；sk018：核苷酸序列为SEQ ID NO：38；sk019：核苷酸序列为SEQ ID NO：40；sk020：核苷酸序列为SEQ ID NO：42；sk021：核苷酸序列为SEQ ID NO：44；sk022：核苷酸序列为SEQ ID NO：45；sk023：核苷酸序列为SEQ ID NO：48；sk024：核苷酸序列为SEQ ID NO：50；sk025：核苷酸序列为SEQ ID NO：52；sk026：核苷酸序列为SEQ ID NO：54；sk027：核苷酸序列为SEQ ID NO：56；sk028：核苷酸序列为SEQ ID NO：58；sk029：核苷酸序列为SEQ ID NO：60；sk030：核苷酸序列为SEQ ID NO：62；sk031：核苷酸序列为SEQ ID NO：64；sk032：核苷酸序列为SEQ ID NO：66；sk033：核苷酸序列为SEQ ID NO：68；sk034：核苷酸序列为SEQ ID NO：70；sk035：核苷酸序列为SEQ ID NO：72；sk036：核苷酸序列为SEQ ID NO：74；sk037：核苷酸序列为SEQ ID NO：76；sk038：核苷酸序列为SEQ ID NO：78；sk039：核苷酸序列为SEQ ID NO：80；sk040：核苷酸序列为SEQ ID NO：82；sk041：核苷酸序列为SEQ ID NO：84；sk042：核苷酸序列为SEQ ID NO：86；sk043：核苷酸序列为SEQ ID NO：88；sk044：核苷酸序列为SEQ ID NO：90；sk045：核苷酸序列为SEQ ID NO：92；sk046：核苷酸序列为SEQ ID NO：94；sk047：核苷酸序列为SEQ ID NO：96；sk048：核苷酸序列为SEQ ID NO：98；或sk049：核苷酸序列为SEQ ID NO：100。
  7. 一种表达如权利要求1-3任一项所述的多肽或如权利要求4-5任一项所述的突变体的表达载体或宿主细胞，包含如权利要求6所述的核苷酸序列；优选地，所述表达载体是真核表达载体或病毒表达载体，更优选地，所述真核表达载体为哺乳动物细胞表达载体，所述病毒表达载体为腺相关病毒或腺病毒载体；优选地，所述宿主细胞为CHO细胞及其亚系或293细胞及其亚系。
  8. 一种制备如权利要求1-3任一项所述的多肽或如权利要求4-5任一项所述的突变体的方法，所述方法为化学合成法或重组表达法。
  9. 一种药物组合物，包含如权利要求1-3任一项所述的多肽或如权利要求4-5任一项所述的突变体或其药学上可接受的盐和药学上可接受的载体或赋形剂；优选地，所述药物组合物的剂型为注射剂、眼药水、眼用凝胶或眼药膏。
  10. 如权利要求1-3任一项所述的多肽或如权利要求4-5任一项所述的突变体或其药学上可接受的盐在制备治疗或预防由血管新生或生长引起的疾病的药物中的应用；优选地，所述疾病为眼睛疾病、肿瘤、缺血性心脏病、非炎症性心肌病、冠状动脉硬化、闭塞性动 脉硬化、动脉栓塞、动脉血栓、Berger′s病、慢性炎症、炎症性肠病、溃疡、风湿性关节炎、硬皮症、银屑病、不育症或肉瘤状病；更优选地，所述眼睛疾病为年龄相关性黄斑变性、糖尿病性视网膜病变、脉络膜新生血管、黄斑囊样水肿、糖尿病性黄斑水肿、视网膜血管闭塞、角膜新生血管、角膜移植手术新生血管性青光眼、翼状胬肉或慢性结膜炎。
  11. 一种抑制哺乳动物血管新生或生长的方法，包括：给需要的对象施用如权利要求1-3任一项所述的多肽或如权利要求4-5任一项所述的突变体或其药学上可接受的盐，或如权利要求9所述的药物组合物；优选地，所述施用方式为眼表施用或玻璃体腔内注射施用。
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