WO2013060303A1 - 抑制新生血管的多肽及其应用 - Google Patents

抑制新生血管的多肽及其应用 Download PDF

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WO2013060303A1
WO2013060303A1 PCT/CN2012/083671 CN2012083671W WO2013060303A1 WO 2013060303 A1 WO2013060303 A1 WO 2013060303A1 CN 2012083671 W CN2012083671 W CN 2012083671W WO 2013060303 A1 WO2013060303 A1 WO 2013060303A1
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polypeptide
group
amino acid
peptide
pharmaceutically acceptable
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PCT/CN2012/083671
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English (en)
French (fr)
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许迅
郑颖
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上海市第一人民医院
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Priority to EP12843247.3A priority Critical patent/EP2772498B1/en
Priority to US14/370,114 priority patent/US9266933B2/en
Publication of WO2013060303A1 publication Critical patent/WO2013060303A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1891—Angiogenesic factors; Angiogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a novel class of small peptides for inhibiting neovascularization, which are polypeptides derived from Placenta Growth Factor (P1GF).
  • P1GF Placenta Growth Factor
  • the invention also relates to methods of making and using the polypeptides and pharmaceutical compositions comprising the polypeptides. Background technique
  • 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.
  • pathological neovascularization such as: corneal neovascularization caused by herpes simplex keratitis, choroidal neovascularization in age-related macular degeneration, and diabetic retinopathy Or retinal neovascularization in retinopathy of prematurity.
  • pathological neovascularization such as: corneal neovascularization caused by herpes simplex keratitis, choroidal neovascularization in age-related macular degeneration, and diabetic retinopathy Or retinal neovascularization in retinopathy of prematurity.
  • ocular ocular neovascularization is routinely treated with laser photocoagulation, photodynamic therapy (PDT), and thermal transpupillary therapy (TTT).
  • PDT photodynamic therapy
  • TTT thermal transpupillary therapy
  • the bioavailability of ophthalmic drugs is very low; to increase it, the concentration of the drug can be increased.
  • Compounds used to treat neovascularization of tumors are more toxic and side effects, and are not administered at high doses both systemically and locally.
  • exogenous proteins with larger molecular weights are also sources of sensitive foreign bodies. It can cause immune damage to eye tissues such as the uvea.
  • angiostatin consists of 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 recombinant expression and purification and endotoxin residues in the preparation process. Due to the above various conditions, the drugs currently used to treat ocular neovascularization are limited, such as recombinant anti-human VEGF monoclonal antibody bevacizumab (Avastin), recombinant anti-human VEGF monoclonal antibody fragment ranibizumab (Lucentis), etc. They are expensive and require repeated administration through the vitreous cavity, which can even cause risks such as vascular embolism.
  • Another object of the invention is to provide a process and use comprising the polypeptide.
  • a polypeptide represented by the following formula I, or a pharmaceutically acceptable salt thereof is provided in a first aspect of the invention.
  • XaaO is no, or 1-3 amino acids constitute a peptide
  • Xaal is an amino acid selected from the group consisting of Thr or Ser;
  • Xaa2 is an amino acid selected from the group consisting of: Ala, Val, Leu or lie;
  • Xaa3 is an amino acid selected from the group consisting of: Asn, Gln, His, Lys or Arg;
  • Xaa4 is an amino acid selected from the group consisting of Val, Ile, Leu, Met, Phe or Ala
  • Xaa5 is an amino acid Thr or Ser selected from the group below;
  • Xaa6 is an amino acid selected from the group consisting of Met, Leu, Phe, or lie;
  • Xaa7 is an amino acid selected from the group consisting of Gin or Asn;
  • Xaa8 is an amino acid selected from the group consisting of Leu, Ile, Val, Met, Ala or Phe;
  • Xaa9 is an amino acid selected from the group consisting of Leu, lie, Val, Met, Ala or Phe;
  • Xaal O is an amino acid selected from the group consisting of Lys, Arg, Gin or Asn;
  • Xaal l is an amino acid selected from the group consisting of lie, Leu, Val, Met, Ala or Phe;
  • Xaal2 is an amino acid selected from the group consisting of Arg, Pro, Lys, Gin or Asn;
  • Xaal 3 is an amino acid Ser or Thr selected from the group consisting of
  • Xaal4 is an amino acid selected from the group consisting of Gly, Pro, or Ala;
  • Xaal 5 is an amino acid selected from the group consisting of Asp, Glu;
  • Xaal 6 is an amino acid selected from the group consisting of Arg, Lys, Gin or Asn;
  • Xaal 7 is an amino acid Pro or Ala selected from the group consisting of
  • Xaal 8 is an amino acid Ser or Thr selected from the group consisting of;
  • Xaal 9 is an amino acid selected from the group consisting of Tyr, Trp, Phe Thr or Ser;
  • Xaa20 is an amino acid selected from the group consisting of Val, Ile, Leu ⁇ Met, Phe or Ala;
  • Xaa21 is an amino acid selected from the group consisting of Glu or Arg;
  • Xaa22 is an amino acid selected from the group consisting of Leu or Ser;
  • Xaa23 is an amino acid Thr or Arg selected from the group consisting of;
  • Xaa24 is an amino acid selected from the group consisting of Phe or Ser;
  • Xaa25 is an amino acid Ser or Arg selected from the group consisting of
  • Xaa26 is an amino acid selected from the group consisting of Gin or Ser;
  • Xaa27 is an amino acid His or Arg selected from the group consisting of;
  • Xaa28 is an amino acid selected from the group consisting of no, or 1-3 amino acids, which constitutes a peptide
  • polypeptide has an activity of inhibiting angiogenesis, and the polypeptide has a length of 27 to 33 ammonia. In another preferred embodiment, the polypeptide has a length of 28 to 31.
  • Xaa28 is a peptide consisting of three amino acids.
  • XaaO is absent, or one, two or three amino acids constitute a peptide.
  • XaaO is Lys, Glu, Pro-Ile-Lys or Ile-Lys.
  • polypeptide is selected from the group consisting of:
  • polypeptide having the amino acid sequence shown in SEQ ID NO: 1 (b) The amino acid sequence represented by SEQ ID NO: 1 is formed by substitution, deletion or addition of 1-5 (preferably 1-3, more preferably 1-2) amino acid residues, and has inhibition A polypeptide derived from (a) of angiogenic function.
  • the derivative polypeptide retains > 70% of the angiogenic activity of the polypeptide of SEQ ID NO: 1.
  • the derivative polypeptide has an identity of SEQ ID NO: 1 ⁇ 80%, preferably > 90%; more preferably ⁇ 95%.
  • the invention also provides dimeric and multimeric forms of the compounds of formula I which inhibit angiogenic function.
  • an isolated nucleic acid molecule encoding the above-described polypeptide of the invention.
  • a pharmaceutical composition comprising:
  • the composition is in the form of eye drops, injections (e.g., periocular and intraocular injections), ophthalmic gels or ophthalmic ointments.
  • the composition is a sustained release dosage form.
  • a polypeptide or a pharmaceutically acceptable salt of the invention for the preparation of a medicament for inhibiting angiogenesis or for preventing diseases associated with angiogenesis.
  • the angiogenesis-related disease is selected from the group consisting of neovascular ophthalmopathy, tumor, ischemic heart disease, non-inflammatory cardiomyopathy, coronary arteriosclerosis, arteriosclerosis obliterans, arteries. Embolism, arterial thrombosis, Berger's disease, chronic inflammation, inflammatory bowel disease, ulcers, rheumatoid arthritis, scleroderma, psoriasis, infertility or sarcoma.
  • the neovascular eye disease comprises involvement of the choroid, retina, cornea or iris, including age-related macular degeneration, proliferative diabetic retinopathy, retinal vascular occlusive disease, retinopathy of prematurity, corneal infection , neovascular glaucoma and so on.
  • a method of inhibiting angiogenesis in a mammal comprising the steps of: administering to a subject in need thereof a polypeptide of the invention or a pharmaceutically acceptable salt thereof.
  • the object is a human.
  • the angiogenesis is angiogenesis associated with neovascular eye disease. It is to be understood that within the scope of the present invention, the above various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS
  • Figure 1 shows the results of purity identification of the small peptide ZY3 by high performance liquid chromatography.
  • Figure 2 shows the effect of small peptide ZY3 on the proliferation of human umbilical vein endothelial cells HUVECs.
  • the small peptide ZY3 has an obvious inhibitory effect on endothelial cell proliferation.
  • the VEGF+ small peptide ZY3 group significantly inhibited the proliferation of HUVECs, *P ⁇ 0.05, **P ⁇ 0.01, and the difference was statistically significant.
  • Figure 3 shows the effect of small peptide ZY3 on the lumen formation of human umbilical vein endothelial cells HUVECs. It can be seen that the small peptide ZY3 has an effect of inhibiting the lumen formation of endothelial cells; Fig. 3a, Fig. 3b, Fig. 3c show small peptide ZY3 to HUVECs tube Inhibition of cavity formation; Figure 3a is the VEGF group; Figure 3b is the VEGF+ZY3 (16 ( ⁇ M) group; Figure 3c shows that the VEGF and the small peptide ZY3 groups at various concentrations have significant inhibition of HUVECs lumens relative to the VEGF group. The effect of formation, *P ⁇ 0.05, the difference was statistically significant.
  • Figure 4 shows the effect of small peptide ZY3 on neovascularization in chicken chorioallantoic membrane: small peptide ZY3 has obvious effect of inhibiting neovascularization; Figure 4a-4c shows results of 3-5 microvessel counts within 2.5 mm of filter paper; 4a is the PBS group; Fig. 4b is the ⁇ 3 (10 ⁇ 1/tablet) group; Fig. 4c is the ⁇ 3 (50 ⁇ 1/tablet) group; Fig. 4d is the VEGF+ group of small peptide ZY3 groups, which inhibits the chicken embryo allantocyst significantly relative to the VEGF group.
  • Membrane neovascularization, and inhibition was concentration-dependent, **P ⁇ 0.01, the difference was statistically significant.
  • Figure 5 shows the effect of small peptide ZY3 on pathological neovascularization in mouse cornea. It can be seen that small peptide ZY3 has an effect of inhibiting neovascularization;
  • Figures 5a-5c show corneal neovascularization area, Fig. 5a is VEGF group; Fig. 5b is ⁇ 3 (0 ⁇ 5 ⁇ 1/granule) group; Figure 5c is ⁇ 3 (2 ⁇ 1/granule) group;
  • Figure 5d is the VEGF+ small peptide ZY3 different concentration group, which has obvious inhibition of corneal pathological neovascularization in mice compared with VEGF group. **P ⁇ 0.01, the difference was statistically significant.
  • the present inventors have for the first time prepared a small molecule polypeptide derived from placental growth factor and having an angiogenic function and having a molecular weight of less than 5 kD (e.g., only about 3 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 small peptide ZY3.
  • the small peptide of the invention has small molecular weight and can penetrate various eye tissue barriers; has good water solubility, can maintain high concentration in neutral tears, aqueous humor and vitreous humor; has high safety and has low toxicity to biological tissues. Eye bioavailability is high, which reduces the dose and reduces systemic side effects. On the basis of this, the present invention has been completed. Placental growth factor
  • Placenta Growth Factor is a member of the VEGF family and was isolated and purified from human placental cDNA library by Maglione et al. in 1991. In addition to the human placenta, the presence of P1GF is also detected in the heart, lungs, thyroid, and skeletal muscle. According to the selective splicing of the P1GF gene, four different isoforms can be produced: P1GF-1 (P1GF131), P1GF-2 (P1GF152), P1GF-3 (P1GF203), P1GF-4 (P1GF224), which are different in size. Secretion properties and receptor affinity are also different.
  • P1GF The two P1GF monomers exert a biological effect by forming a secretory homodimeric glycoprotein and binding to its receptor, thereby mediating subsequent signal transduction.
  • P1GF can also form a heterodimer with VEGF, which affects the signal transduction pathway of VEGF.
  • P1GF can promote the proliferation of vascular endothelial cells, especially microvascular endothelial cells, and acts as a chemokine of endothelial cell growth factor to regulate endothelial cell growth and stimulate angiogenesis.
  • P1GF also promotes the migration of monocytes and endothelial cells and increases the permeability of endothelial cells.
  • VEGF can also induce neovascularization
  • the neovascularization induced by P1GF has normal physiological characteristics without other abnormal changes. These neovascular vessels do not undergo edema, hemangioma, and opacity caused by VEGF-induced neovascularization. Sexual increase and other phenomena. Active polypeptide
  • polypeptide of the present invention refers to a peptide ZY3 amino acid sequence having an angiogenesis inhibitory activity.
  • ZY3 polypeptide ZY3 small peptide
  • short peptide ZY3 short peptide ZY3
  • peptide ZY3 refers to a peptide ZY3 amino acid sequence having an angiogenesis inhibitory activity.
  • a protein or polypeptide of (TANVTM QLLKIRSGDRPSYVELTFSQH, as set forth in SEQ ID NO: 1).
  • the term also encompasses variant forms of the sequence of SEQ ID NO: 1 having an angiogenic inhibitory function.
  • variants include (but are not limited to): 1-5 (usually 1-4, preferably 1-3, more preferably 1-2, optimally 1) amino acid deletions, insertions And / or substituted, as well as at the C end and / Or one or several (usually 5 or less, preferably 3 or less, more preferably 2 or less) amino acids are added or deleted at the N-terminus.
  • 1-5 usually 1-4, preferably 1-3, more preferably 1-2, optimally 1 amino acid deletions, insertions And / or substituted, as well as at the C end and / Or one or several (usually 5 or less, preferably 3 or less, more preferably 2 or less) amino acids are added or deleted at the N-terminus.
  • amino acids usually 5 or less, preferably 3 or less, more preferably 2 or less
  • the term also encompasses polypeptides of the invention in both monomeric and multimeric forms.
  • the term also includes both linear as well as non-linear polypeptides (such as cyclic peptides).
  • the invention also encompasses active fragments, derivatives and analogs of ZY3 polypeptides.
  • fragment refers to a polypeptide that substantially retains an angiogenic function or activity.
  • a polypeptide fragment, derivative or analog of the invention may be a polypeptide in which omega has one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more amino acids a polypeptide having a substituent group in the residue, or (iii) a polypeptide formed by fusing a ZY polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to A polypeptide formed by the polypeptide sequence (then protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His).
  • omega has one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more amino acids a polypeptide having a substituent group in the residue, or (iii) a polypeptide formed by fusing a
  • a preferred class of reactive derivatives means that up to 5, preferably up to 3, more preferably up to 2, and optimally 1 amino acid are similar or similar amino acids to the amino acid sequence of Formula I. Substituting to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the invention also provides analogs of the ZY3 polypeptide.
  • the difference between these analogs and the native ZY3 polypeptide may be a difference in amino acid sequence, or may be a difference in the modification form that does not affect the sequence, or both.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., 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 include: chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. 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 polypeptide of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include, but are not limited to, salts formed with: hydrochloric 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: salts with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, carbamates or other conventional "prodrugs". Coding sequence
  • the invention also relates to polynucleotides encoding a ⁇ 3 polypeptide.
  • a preferred coding sequence is (SEQ ID NO: 1
  • GGCCCTCCTACGTGGAGCTGACGTTCTCTCAGCAC which encodes SEQ ID NO: 1 The short peptide ZY3 shown.
  • 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 coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 2 or a degenerate variant.
  • SEQ ID NO: 2 a "degenerate variant" in the present invention refers to a polypeptide having the sequence of SEQ ID NO: 1, but with the corresponding coding region sequence of SEQ ID NO: Differential nucleic acid sequences.
  • the full-length ZY3 nucleotide sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., 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 ZY polypeptide coding sequences.
  • the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific for a ZY3 polypeptide.
  • the polypeptide of the invention may be a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptide of the present invention can be artificially synthesized by a conventional method or can 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 linked to a C-terminal amino acid in the peptide, a Wang resin structure is polystyrene, and an arm between the amino acids is 4-decyloxybenzyl alcohol; using 25% hexahydropyridine /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 is a PAM resin to which a C-terminal amino acid in the peptide 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).
  • the p-cresol (5-10%) containing hydrogen fluoride (HF) is treated at 0 ° C for 1 hour to cut the peptide chain from the resin while removing the Protection group.
  • 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.
  • Each of the 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
  • the polypeptide ZY3 of the present invention is prepared by solid phase synthesis according to the sequence thereof, and purified by high performance liquid chromatography to obtain a high-purity peptide freeze-dried powder, which is stored at -20 °C.
  • Another method is to produce a polypeptide of the invention using recombinant techniques.
  • Polynucleotides of the invention can be utilized to express or produce recombinant ZY3 polypeptides by conventional recombinant DNA techniques. Generally there are the following steps:
  • the recombinant polypeptide can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. 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 bacteria, 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 bacteria, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromat
  • polypeptide of the present invention is short, it is conceivable to connect a plurality of polypeptides in series, 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 provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a safe and effective amount of a polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient .
  • the amount of the polypeptide of the present invention 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 of body weight of the polypeptide of the invention.
  • the polypeptides of the invention may be used alone 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 means 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.
  • These vectors are well known to those of ordinary skill in the art.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the therapeutic compositions may be in the form of 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, periocular, intraocular, intramuscular, intravenous, subcutaneous, intradermal or topical administration.
  • the subject to be prevented or treated may be an animal; especially a human.
  • compositions such as eye drops, injections, ophthalmic gels and ophthalmics which can be exemplified can be formulated by mixing, diluting or dissolving according to a conventional method, and occasionally adding a suitable pharmaceutical additive such as a shape Agent, disintegrant, binder, lubricant, diluent, buffer, isotonic agent (isot 0n iciti eS ), preservative, wetting agent, emulsifier, dispersant, stabilizer and cosolvent, and The formulation process can be carried out in the usual manner depending on the dosage form.
  • a suitable pharmaceutical additive such as a shape Agent, disintegrant, binder, lubricant, diluent, buffer, isotonic agent (isot 0n iciti eS ), preservative, wetting agent, emulsifier, dispersant, stabilizer and cosolvent, and
  • the formulation process can be carried out in the usual manner depending on the dosage form.
  • the preparation of eye drops can be carried out by dissolving the short peptide ZY or a pharmaceutically acceptable salt thereof together with the basic substance in sterile water (a surfactant is dissolved in sterile water) to adjust the osmotic pressure and pH to The physiological state, and optionally a suitable pharmaceutical additive such as a preservative, a stabilizer, a buffer, an isotonicity agent, an antioxidant and a tackifier, may be optionally added and then completely dissolved.
  • a suitable pharmaceutical additive such as a preservative, a stabilizer, a buffer, an isotonicity agent, an antioxidant and a tackifier
  • compositions of the invention may also be administered in the form of sustained release agents.
  • the short peptide ZY or a salt thereof can be intruded into a pellet or microcapsule in which the sustained-release polymer is used as a carrier, and then the pellet or microcapsule is surgically implanted into the tissue to be treated.
  • the short peptide ZY3 or a salt thereof can also be applied by inserting a drug-coated intraocular lens.
  • the sustained-release polymer ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymerization may be exemplified.
  • the 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.
  • the short peptide ZY3 as an active ingredient or
  • the dose of the pharmaceutically acceptable salt can be reasonably determined depending on the weight, age, sex, and degree of symptoms of each patient to be treated.
  • the concentration is usually about 0.1 to 10% by weight, preferably 1 to 5% by weight, and may be administered 2 to 6 times a day, 1-2 drops each time.
  • a pharmaceutical composition containing the peptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient has a remarkable inhibitory activity against angiogenesis. It has been confirmed by animal experiments that the polypeptide of the present invention can inhibit not only angiogenesis of the chicken chorioallantoic membrane, but also inhibit proliferation, migration, chemotaxis and lumen formation of human umbilical vein endothelial cells, and inhibit hypoxia-inducible mice. Retinal neovascularization.
  • the main advantages of the invention include:
  • 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, high yield and low cost;
  • polypeptide of the present invention has good stability.
  • 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 invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions.
  • Example 1 Example 1
  • the ZY3 polypeptide represented by SEQ ID NO: 1 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) (Dikma), shaking for 30 min.
  • Pipette the piperidine solution take more than a dozen resins, wash three times with ethanol, add one drop of ninhydrin, KCN, phenol solution, heat at 105 ° C - 10 ° C for 5 min, dark blue is a positive reaction. Wash twice with DMF (10 ml/g), wash twice with methanol (10 ml/g), and wash twice with DMF (10 ml/g). Add three times the protective amino acid (FOMC-Asp-OH), HBTU (Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.) three times the excess, dissolve with as little DMF as possible, add the reaction tube, immediately add NMM ten times the excess. Reaction 30min .
  • the polypeptide was cleaved from the resin (cutting solution (10/g): TFA (JT Baker) 94.5%, 7X 2.5%, EDT (ALD ICH) 2.5%, TIS (ALDRICH) 1%; cutting time: 120 min).
  • the lysate was blown dry with nitrogen (Shanghai Bio-Industry), washed twice with diethyl ether (Shanghai Test Chemical Reagent Co., Ltd.), and then evaporated at room temperature.
  • the small peptide ZY3 has 27 amino acids and has a molecular weight of 3092.55.
  • HUVECs Primary human umbilical vein endothelial cells HUVECs (purchased from ScienCell) were inoculated into 96-well plates at a concentration of 2 ⁇ 10 4 /ml; cells were adhered to the serum-free culture medium ECM at 37 ° C for 24 hours; Serum-free culture medium ECM was added to each well as a negative control, VEGF (100 ng/ml) (purchased from Sigma) as a positive control, VEGF (100 ng/well) + small concentration of small peptide ZY3 as a treatment group; continued culture After 24 hours, 20 ⁇ l of MTS solution (purchased from Promega) was added to each well; after incubation at 37 ° C for 4 hours, the absorbance of each well at 490 nm was measured using a microplate reader (Bio-Rad), and the cells were determined according to OD490. Proliferative activity, and finally statistical analysis using SPSS 11.0.1.
  • Matrigel Matrigel (purchased from BD) 50 ⁇ l/well was added to a 96-well plate and incubated at 37 ° C for 30 minutes. After being solidified, primary human umbilical vein endothelial cells HUVECs were inoculated on the surface of Matrigel at a concentration of 8 ⁇ 10 6 /ml; serum-free culture ECM was added to each well as a negative control, VEGF. (100 ng/ml) (purchased from Sigma) As a positive control, VEGF (100 ng/ml) + small concentration of small peptide ZY3 as a treatment group, the culture was continued at 37 °C.
  • FIG. 3 The results are shown in Figure 3.
  • the small peptide ZY3 significantly inhibited the lumen formation of human umbilical vein endothelial cells HUVECs at 6 hours in a concentration-dependent manner.
  • Figures 3a-3c show the inhibitory effect of small peptide ZY3 on lumen formation in HUVECs.
  • Figure 3a shows the VEGF group;
  • Figure 3b shows the VEGF+ZY3 (16 ( ⁇ M) group;
  • Figure 3c shows that compared with the VEGF group, VEGF and each concentration of the small peptide ZY3 group have a significant inhibitory effect on the lumen formation of HUVECs, *P ⁇ 0.05, the difference was statistically significant.
  • Example 5 Example 5
  • the chicken embryo chorioallantoic membrane model is used as follows:
  • filter paper containing cortisone acetate (5 g/l, 5 ⁇ 1/table) (Whatman Quantitative filter papers, Sigma, ashless, Grade 42, Cat No 1442-042, 42.5mm xl00 circles)
  • Drop PBS (5 ⁇ 1/piece) or low concentration (2 ⁇ ⁇ / ⁇ 1), high concentration (10 ⁇ 8 / ⁇ 1) Small peptide ⁇ 3 (5 ⁇ 1/tablet)
  • the filter paper is air-dried and placed between the large blood vessels of the egg urinary
  • FIG. 4 The results are shown in Fig. 4. It can be seen that compared with the PBS group, the small peptide ZY3 can significantly inhibit the angiogenesis of the chick embryo chorioallantoic membrane at low concentrations (l ( ⁇ g/tablet) and high concentration (5 ( ⁇ g/table)).
  • Figures 4a-4c show 3-5 microvessel counts in the 2.5 mm range of the filter paper.
  • Figure 4a is the PBS group;
  • Figure 4b is the ⁇ 3 (10 ⁇ 1/piece) group;
  • Figure 4c is the ⁇ 3 (50 ⁇ 1/piece) group;
  • Figure 4d Compared with the VEGF group, the small peptide ZY3 group of each concentration of VEGF+ significantly inhibited the number of neovascularization in chick embryo chorioallantoic membrane, and the inhibition was concentration-dependent, **P ⁇ 0.01, the difference was statistically significant.
  • mice C57BL/6 male mice (4-5 weeks old) were anesthetized by intraperitoneal injection of 2% pentobarbital (about 0.1 ml/mouse), and topical 4% oxybuprocaine hydrochloride eye drops. Under the stereoscopic microscope, the OT needle and the 2 ml needle were used to make a blunt separation between the corneal stroma at a distance of 0.8-lmm from the angle of the cornea to make a pouch of about 0.6*0.8 mm in size.
  • Figure 5 shows the effect of small peptide ⁇ 3 on pathological neovascularization in the cornea of mice, showing that small peptide ⁇ 3 has an effect of inhibiting neovascularization.
  • Figures 5a-5c show the area of corneal neovascularization in mice.
  • Figure 5a is the VEGF group
  • Figure 5b is the ⁇ 3 (0.5 ⁇ l/granule) group
  • Figure 5c is the ⁇ 3 (2 ⁇ 1/granule) group
  • Figure 5d is the VEGF+ small peptide ZY3 concentration group with significant inhibition compared to the VEGF group.
  • Derived polypeptide 1 The sequence is the same as SEQ ID NO: 1, wherein the fourth Val is replaced by lie; the derived polypeptide 2: the sequence is the same as SEQ ID NO: 1, wherein the 12th Arg Pro is substituted;
  • Derived polypeptide 3 the sequence is identical to SEQ ID NO: 1, wherein the 15th Asp is replaced by Glu; the derived polypeptide 4: the sequence is the same as SEQ ID NO: 1, wherein the 23rd Thr is replaced by Arg; the derived polypeptide 5: the sequence is the same as SEQ ID NO: 1, wherein the deletion 8th Leu.

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Abstract

提供了具有抑制血管生成活性的多肽，所述多肽源自胎盘生长因子1。还提供了该多肽的衍生多肽、编码它们的核酸分子、含有这些多肽的药物组合物以及这些多肽在制备与血管新生相关疾病的药物中的应用。

Description

抑制新生血管的多肽及其应用 技术领域
本发明涉及生物医药领域， 具体地， 涉及一类新的抑制新生血管的小肽， 所述的小肽是源于胎盘生长因子 (Placenta Growth Factor, P1GF)的多肽。本发明 还涉及所述多肽的制法和应用以及含所述多肽的药物组合物。 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， 其远期疗效也并不十 分令人满意。 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。
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 4见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， 可 加大给药的浓度。 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 Kringle 1-4)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， 经无 创或微创的给药途径透过各种眼组织屏障， 提高眼局部的生物利用度， 降低给 药剂量， 减少局部和全身的副作用， 对新生血管性眼病的临床防治具有十分重 要的意义。 因此， 本领域迫切需要开发一种适于眼球组织的有效安全的小分子 新生血管抑制剂。 发明内容
本发明的目的是提供一类适于眼球组织的有效安全的可抑制血管新生的 小分子多肽以及其片段、 类似物和衍生物。
本发明的另一目的是提供含所述多肽的制法和应用。 在本发明的第一方面， 提供了在本发明的第一方面， 提供了一种下式 I表 示的多肽， 或其药学上可接受的盐
[Xaa0]-[Xaal]-[Xaa2]-[Xaa3]-[Xaa4]-[Xaa5]-[Xaa6]-[Xaa7]-[Xaa8]-[Xaa9 ]-[Xaal0]-[Xaall]-[Xaal2]-[Xaal3]-[Xaal4]-[Xaal5]-[Xaal6]-[Xaal7]-[X aal8]-[Xaal9]-[Xaa20]-[Xaa21]-[Xaa22]-[Xaa23]-[Xaa24]-[Xaa25]-[Xaa26 ]-[Xaa27]-[Xaa28] (I)
式中，
XaaO是无， 或 1-3个氨基酸构成肽段；
Xaal是选自下组的氨基酸： Thr或 Ser;
Xaa2是选自下组的氨基酸： Ala、 Val、 Leu或 lie;
Xaa3是选自下组的氨基酸： Asn、 Gln、 His, Lys或 Arg;
Xaa4是选自下组的氨基酸： Val、 Ile、 Leu、 Met, Phe或 Ala; Xaa5是选自下组的氨基酸 Thr或 Ser;
Xaa6是选自下组的氨基酸 Met、 Leu、 Phe、 或 lie;
Xaa7是选自下组的氨基酸 Gin或 Asn;
Xaa8是选自下组的氨基酸 Leu, Ile、 Val、 Met, Ala或 Phe ;
Xaa9是选自下组的氨基酸 Leu, lie, Val、 Met, Ala或 Phe;
Xaal O是选自下组的氨基酸 Lys、 Arg、 Gin或 Asn;
Xaal l是选自下组的氨基酸 lie, Leu、 Val、 Met, Ala或 Phe;
Xaal2是选自下组的氨基酸 Arg、 Pro、 Lys、 Gin或 Asn;
Xaal 3是选自下组的氨基酸 Ser或 Thr;
Xaal4是选自下组的氨基酸 Gly、 Pro, 或 Ala;
Xaal 5是选自下组的氨基酸 Asp、 Glu;
Xaal 6是选自下组的氨基酸 Arg、 Lys、 Gin或 Asn;
Xaal 7是选自下组的氨基酸 Pro或 Ala;
Xaal 8是选自下组的氨基酸 Ser或 Thr;
Xaal 9是选自下组的氨基酸 Tyr、 Trp、 Phe Thr或 Ser;
Xaa20是选自下组的氨基酸 Val、 Ile、 Leu^ Met、 Phe或 Ala;
Xaa21是选自下组的氨基酸 Glu或 Arg;
Xaa22是选自下组的氨基酸 Leu或 Ser;
Xaa23是选自下组的氨基酸 Thr或 Arg;
Xaa24是选自下组的氨基酸 Phe或 Ser;
Xaa25是选自下组的氨基酸 Ser或 Arg;
Xaa26是选自下组的氨基酸 Gin或 Ser;
Xaa27是选自下组的氨基酸 His或 Arg;
Xaa28是选自下组的氨基酸 无， 或 1 -3个氨基酸构成肽段;
并且所述的多肽具有抑制血管新生的活性，且所述多肽的长度为 27-33个氨 在另一优选例中， 所述多肽的长度为 28-31个〈
在另一优选例中， Xaa28是 3个氨基酸构成的肽段。
在另一优选例中， XaaO是无， 或 1个、 两个或三个氨基酸构成肽段。 在另一优选例中， XaaO为 Lys、 Glu、 Pro-Ile-Lys或 Ile-Lys。
在另一优选例中， 所述多肽选自下组：
(a)具有 SEQ ID ΝΟ: 1所示氨基酸序列的多肽； (b)将 SEQ ID NO: l所示氨基酸序列经过 1-5个 (较佳地 1-3， 更佳地 1-2个) 氨基酸残基的取代、 缺失或添加而形成的， 且具有抑制血管新生功能的由 (a)衍 生的多肽。
在另一优选例中，所述的衍生多肽保留了≥70%的 SEQ ID NO: l的所示多肽 的抑制血管新生活性。
在另一优选例中， 所述的衍生多肽与 SEQ ID NO: l的相同性≥80%， 较佳地 >90%； 更佳地≥95%。
本发明还提供了抑制血管新生功能的、式 I化合物的二聚体和多聚体形式。 在本发明的第二方面， 提供了一种分离的核酸分子， 它编码本发明上述的 多肽。 在本发明的第三方面， 提供了一种药物组合物， 它含有：
(a) 本发明上述的多肽或其药学上可接受的盐； 和
(b) 药学上可接受的载体或赋形剂。
在另一优选例中， 所述组合物的剂型为眼药水、 针剂 (如眼周和眼内注射 液)、 眼用凝胶或眼药膏。
在另一优选例中， 所述的组合物为缓释剂型。 在本发明的第四方面， 提供了一种本发明所述多肽或药学上可接受的盐的 用途， 它们被用于制备抑制血管新生或防治与血管新生相关疾病的药物。
在另一优选例中， 所述的与血管新生相关疾病的选自下组： 新生血管性眼 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、 不育症或肉瘤状病等。
在另一优选例中， 所述的新生血管性眼病包括累及脉络膜、 视网膜、 角膜 或虹膜， 包括老年性黄斑变性、 增生性糖尿病视网膜病变、 视网膜血管阻断性 疾病、 早产儿视网膜病变、 角膜感染、 新生血管性青光眼等。 在本发明的第五方面， 提供了一种抑制哺乳动物血管新生的方法， 包括步 骤： 给需要的对象施用本发明所述的多肽或其药学上可接受的盐。
在另一优选例中， 所述的对象是人。 在另一优选例中， 所述的血管新生是与新生血管性眼病相关的血管新生。 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 (如实施例) 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。 附图说明
下列附图用于说明本发明的具体实施方案， 而不用于限定由权利要求书所 界定的本发明范围。
图 1显示了小肽 ZY3的高效液相色谱分析的纯度鉴定结果。
图 2显示了小肽 ZY3对人脐静脉血管内皮细胞 HUVECs增殖的影响， 小肽 ZY3明显具有抑制内皮细胞增殖的效应。相对于 VEGF组， VEGF+小肽 ZY3各组 具有明显抑制 HUVECs增殖的作用， *P<0.05， **P<0.01， 差异具有统计学意义。
图 3显示小肽 ZY3对人脐静脉血管内皮细胞 HUVECs管腔形成的影响， 可见 小肽 ZY3明显具有抑制内皮细胞管腔形成的效应； 图 3a、 图 3b、 图 3c显示小肽 ZY3对 HUVECs管腔形成 的抑制作用 ； 图 3a为 VEGF组 ； 图 3b为 VEGF+ZY3(16(^M)组； 图 3c为相对于 VEGF组， VEGF和各个浓度的小肽 ZY3 组都具有明显抑制 HUVECs管腔形成的作用， *P<0.05， 差异具有统计学意义。
图 4显示小肽 ZY3对鸡胚尿囊膜上新生血管的影响： 小肽 ZY3明显具有抑制 新生血管的效应； 图 4a-4c显示滤纸片周 2.5mm范围内 3-5级微血管计数结果； 图 4a为 PBS组；图 4b为 ΖΥ3(10μ1/片）组；图 4c为 ΖΥ3(50μ1/片）组；图 4d为相对于 VEGF 组， VEGF+各个浓度的小肽 ZY3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。
图 5显示小肽 ZY3对小鼠角膜病理性新生血管的影响， 可见小肽 ZY3明显具 有抑制新生血管的效应； 图 5a-5c显示小鼠角膜新生血管面积， 图 5a为 VEGF组； 图 5b为 ΖΥ3(0·5μ1/粒)组； 图 5c为 ΖΥ3(2μ1/粒)组； 图 5d为相对于 VEGF组， VEGF+ 小肽 ZY3不同浓度组， 都具有明显抑制小鼠角膜病理性新生血管作用， **P<0.01， 差异具有统计学意义。 具体实施方式
本发明人经过广泛而深入的研究， 首次制备了一类源自胎盘生长因子的、 具有抑制血管新生功能的， 分子量小于 5kD (如仅约 3kD)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小肽 ZY3， 并运用 HPLC及 MS对之进行鉴定， 再经鸡胚尿囊膜血管模型、 VEGF诱导的 人脐静脉内皮细胞增殖模型和人脐静脉内皮细胞官腔模型， 以及小鼠角膜微囊 袋模型筛选，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。
本发明的小肽的分子量小， 可透过各种眼组织屏障； 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； 安全性高， 对生物组织毒副作用小； 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。 在此基础上完 成了本发明。 胎盘生长因子
胎盘生长因子 (Placenta Growth Factor, P1GF)是 VEGF家族中的一员，最早于 1991年由 Maglione等人从人体胎盘 cDNA文库中分离纯化而来。 除了人体胎盘 夕卜， 在心、 肺、 甲状腺、 骨骼肌中也可检测到 P1GF的存在。 根据 P1GF基因的选 择性拼接， 可以产生 4种不同的异构体： P1GF-1(P1GF131)， P1GF-2(P1GF152), P1GF-3(P1GF203), P1GF-4(P1GF224), 它们大小不同， 分泌特性和受体亲和力也 不一样。 两个 P1GF单体通过形成分泌性同源二聚体糖蛋白， 再和其受体结合， 从而介导之后的信号转导， 发挥生物学效应。 此外， P1GF还能和 VEGF结合形成 异源二聚体， 影响 VEGF的信号转导通路。 P1GF能促进血管内皮细胞尤其是微 血管内皮细胞增殖， 并可作为内皮细胞生长因子的趋化因子来调节内皮细胞的 生长， 剌激血管生成。 P1GF还能促进单核细胞和内皮细胞的迁移， 增加内皮细 胞的通透性。 尽管 VEGF也能诱导新生血管形成， 可由 P1GF诱导产生的新生血 管具有正常的生理特性而无其他异常改变， 这些新生血管不会发生如 VEGF诱 导产生的新生血管所致的水肿、 血管瘤、 通透性增加等现象。 活性多肽
在本发明中， 术语"本发明多肽"、 "ZY3 多肽"、 "ZY3小肽"、 "短肽 ZY3" 或"肽 ZY3"可互换使用， 都指具有血管新生抑制活性的肽 ZY3 氨基酸序列 (TANVTMQLLKIRSGDRPSYVELTFSQH，如 SEQ ID ΝΟ: 1所示）的蛋白或多肽。 此外， 所述术语还包括具有抑制血管新生功能的、 SEQ ID NO: 1序列的变异 形式。 这些变异形式包括 (但并不限于)： 1-5个 (通常为 1-4个， 较佳地 1-3个， 更佳地 1-2个， 最佳地 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 (通常为 5个以内， 较佳地为 3个以内， 更佳 地为 2个以内)氨基酸。 例如， 在本领域中， 用性能相近或相似的氨基酸进行取 代时， 通常不会改变蛋白质的功能。 又比如， 在 C末端和 /或 N末端添加或缺 失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。 此外， 所述术语还 包括单体和多聚体形式本发明多肽。 该术语还包括线性以及非线性的多肽 (如环 肽)。
本发明还包括 ZY3多肽的活性片段、 衍生物和类似物。 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 ω有一个或多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)ZY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。
一类优选的活性衍生物指与式 I的氨基酸序列相比， 有至多 5个， 较佳地 至多 3个， 更佳地至多 2个， 最佳地 1个氨基酸被性质相似或相近的氨基酸所 替换而形成多肽。 这些保守性变异多肽最好根据表 1进行氨基酸替换而产生。
表 1
Figure imgf000008_0001
Figure imgf000009_0001
发明还提供 ZY3 多肽的类似物。 这些类似物与天然 ZY3 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 步加工步骤中进行糖基化修饰而产生的多肽。 这种修饰可以通过将多肽暴露于 进行糖基化的酶 (如哺乳动物的糖基化酶或去糖基化酶)而完成。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。
本发明多肽还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使 用。 这些盐包括 (但不限于)与如下酸形成的盐： 氢氯酸、 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。 其他盐包括： 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
本发明还涉及编码 ΖΥ3多肽的多核苷酸。 一种优选的编码序列是 (SEQ ID
GGCCCTCCTACGTGGAGCTGACGTTCTCTCAGCAC), 它编码 SEQ ID NO: 1 所示的短肽 ZY3。
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。 编码成熟多肽的编码区序列可以与 SEQ ID NO:2所示的编码区序列相 同或者是简并的变异体。 如本文所用， 以 SEQ ID NO:2为例， "简并的变异体" 在本发明中是指编码具有 SEQ ID NO: l序列的多肽， 但与 SEQ ID NO:2中相应 编码区序列有差别的核酸序列。
本发明的 ZY3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 ZY 多肽编码序列经基因工程产生的宿主细胞。
另一方面， 本发明还包括对 ZY3多肽具有特异性的多克隆抗体和单克隆抗 体， 尤其是单克隆抗体。 制备方法
本发明多肽可以是重组多肽或合成多肽。本发明的多肽可以是化学合成的， 或重组的。 相应地， 本发明多肽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-垸氧基苄醇； 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/二氯甲垸 (DCM)除去保护基团 Boc 并用二异丙基乙胺 (DIEA/二氯甲垸中和。 肽链缩合完成后， 用含对甲苯酚 (5-10%)的氟化氢 (HF)，在 0°C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。
在一个优选例中， 本发明多肽 ZY3， 按其序列，采用固相合成的方法制备， 行高效液相色谱纯化， 获得高纯度目的肽冻干粉， -20°C贮存。
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可用来表达或生产重组的 ZY3多肽。一般来说有以下 步骤：
(1) .用本发明的编码 ZY3多肽的多核苷酸 (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的宿主细胞；
(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
另一方面， 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 明多肽或其药学上可接受的盐； 以及 (b)药学上可接受的载体或赋形剂。 本发明 多肽的数量通常为 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。 此外， 本发明 的多肽可以单用， 也可与其他治疗剂一起使用 (如配制在同一药物组合物中）。
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。
治疗性组合物中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。
一旦配成本发明的组合物， 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、 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、 眼用凝胶和眼药 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 (isot0nicitieS)、 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用惯常方式进行。
例如， 眼药水的配制可这样进行： 将短肽 ZY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， 短肽 ZY或其盐可被 惨入以缓释聚合物为载体的药丸或微囊中， 然后将该药丸或微囊通过手术植入 待治疗的组织。 此外， 短肽 ZY3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、 聚乙烯吡咯垸酮、 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，作为活性成分的短肽 ZY3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 l-5wt%， 每日可 2-6次给药， 每次 1-2滴。 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， 而且可以抑制人脐静脉血管内皮细胞的增殖、 迁移、 趋化及管腔 形成， 并且可抑制缺氧诱导的小鼠视网膜新生血管。 本发明的主要优点包括：
(a)本发明多肽 ZY3的分子量小， 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。
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。 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 1
小肽 ZY3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） ， 合成序列分别为 SEQ ID NO: l所示的 ZY3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 (小肽 ZY3 ) 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 (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°C— 1 10°C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)三倍过量， HBTU (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 DMF溶解，加入反应管，立刻加入 NMM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 ZY3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(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 ( J.T.Baker) 94.5%, 7X2.5%, EDT (ALD ICH) 2.5%, TIS (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。
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， 按照下列条件分别纯化小肽 ZY3。
泵 A : 0.1% 三氟乙酸 + 超纯水
泵 B： 0.1% 三氟乙酸 + 乙腈
流速： 1.0 ml/min
检测体积： 30ul
波长： 220nm
检测柱： Column:Venusi MRC-ODS C18柱 （30x250mm)
检测过程见表 2。
表 2
时间 （min) A ( %) B ( %)
0.5 90 10
30.0 20 80 30.1 停止
最后将纯化后的溶液冻干， 既得到高纯度 （>95%) 的小肽 ZY3。 实施例 2
小肽 ΖΥ3的鉴定及保存
取少量的成品小肽 ΖΥ3，做 HPLC分析的纯度鉴定，和 ESI-MS的分子量鉴定。 结果表明， 小肽 ΖΥ3洗脱峰位于 12.800min， 纯度大于 99% (结果见图 1 )。 小肽 ZY3共有 27个氨基酸， 分子量为 3092.55。
将白色粉末状的小肽， 密封包装， 置于 -20°C长期保存。 实施例 3
小肽 ZY3对人脐静脉血管内皮细胞增殖活性的影响
使用 MTS方法， 具体方法如下：
将原代人脐静脉血管内皮细胞 HUVECs (购自 ScienCell公司） 接种于 96孔 板中， 接种浓度为 2x l04/ml; 细胞贴壁后加入无血清培养剂 ECM 37°C培养 24小 时； 之后在各孔中分别加入无血清培养剂 ECM作为阴性对照、 VEGF(100ng/ml) (购自 Sigma公司） 作为阳性对照、 VEGF(100ng/孔) +不同浓度的小肽 ZY3作为 处理组；继续培养 24小时后，在各孔中加入 20μ1的 MTS溶液 (购自 Promega公司）； 37°C孵育 4小时后， 利用酶标仪 (Bio-Rad公司）测定 490nm各孔的吸光度， 根据 OD490判断细胞的增殖活性， 最后运用 SPSS 11.0.1进行统计分析。
结果见图 2， 可见小肽 ZY3有明显抑制人脐静脉血管内皮细胞 HUVECs增殖 的效应， 并呈浓度依赖性， 相对于 VEGF组， VEGF+小肽 ZY3各组具有明显抑 制 HUVECs增殖的作用， *P<0.05， **P<0.01， 差异具有统计学意义。 实施例 4
小肽 ZY3对人脐静脉血管内皮细胞管腔形成活性的影响
使用 Matrigel基质胶方法， 具体方法如下：
在 96孔板中加入 Matrigel基质胶 (购自 BD公司）50μ1/孔， 37°C孵育 30分钟。 待其结成固体状后，将原代人脐静脉血管内皮细胞 HUVECs接种于基质胶表面， 接种浓度为 8x l06/ml; 并在各孔中分别加入无血清培养剂 ECM作为阴性对照、 VEGF(100ng/ml) (购自 Sigma公司）作为阳性对照、 VEGF(100ng/ml)+不同浓度 的小肽 ZY3作为处理组， 37°C继续培养。于处理后 6小时在 200倍镜下对孔板中细 胞随机取 3个视野进行拍照， 并利用软件 Image-Pro Plus Program 5.1 ( Media Cybernetics, Inc. ) 计算其中形成的管腔最大直径的总和， 最后运用 SPSS1 1.0.1 进行统计分析。
结果见图 3， 小肽 ZY3于 6小时即有明显抑制人脐静脉血管内皮细胞 HUVECs管腔形成的效应， 并呈浓度依赖性。 图 3a-3c显示小肽 ZY3对 HUVECs 管腔形成的抑制作用。 图 3a为 VEGF组； 图 3b为 VEGF+ZY3(16(^M)组； 图 3c为 相对于 VEGF组， VEGF和各个浓度的小肽 ZY3组都具有明显抑制 HUVECs管腔 形成的作用， *P<0.05， 差异具有统计学意义。 实施例 5
小肽 ZY3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°C， 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 (5 g/ l, 5μ1/片) 的滤纸片 (Whatman quantitative filter papers, Sigma, ashless, Grade 42, Cat No 1442-042, 42.5mm xl00 circles) 分别滴加 PBS (5μ1/片） 或低 浓度 (2μ§/μ1)、 高浓度 （10μ8/μ1) 的小肽 ΖΥ3 (5μ1/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°C， 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 SPSS 11.0.1进行统计分析。
结果见图 4， 可见相较 PBS组， 小肽 ZY3在低浓度（l(^g/片)及高浓度（5(^g/ 片） 时均有明显抑制鸡胚尿囊膜新生血管的作用。 图 4a-4c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 ΖΥ3(10μ1/片）组； 图 4c为 ΖΥ3(50μ1/ 片)组； 图 4d为相对于 VEGF组， VEGF+各个浓度的小肽 ZY3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 6
小肽 ZY3抗小鼠角膜病理性新生血管效应的测定
使用小鼠角膜微囊袋模型， 具体方法如下： 于 C57BL/6雄性小鼠 （周龄 4-5周） 腹腔注射 2%戊巴比妥 （约 0.1ml/只） 进 行麻醉， 局部用 4%盐酸奧布卡因眼液。 在体式镜下， 在小鼠角膜上用 OT针头 及 2ml针头在距角巩缘约 0.8— lmm处的角膜基质层间进行钝性分离， 制成一大 小约 0.6*0.8mm的小袋， 分别将阴性对照组 （空白颗粒） 、 阳性对照组 （VEGF 组： 320ι¾/μ1， 160ng/粒） 和处理组 （ VEGF+低浓度 0.5 g/粒、 高浓度 2 g/粒的 小肽 ZY3 ) 的缓释颗粒 （12%POLYHAME: 硫糖铝 1 : 1体积混合） 植入微囊袋 内，术后第 5天观察小鼠角膜上最长新生血管长度 (VL:最长新生血管从角巩膜缘 长入角膜的长度)、 新生血管钟点数 （CN:新生血管累及角膜的圆周钟点数） ， 计算新生血管面积（Area) , Are a(mm2)=0.5*3.14* VL(mm) * CN * 0.4(mm) , 运用 SPSS 1 1.0.1进行统计分析。
结果见图 5， 可见相对于 VEGF组， 小肽 ZY3在低浓度 (0.5μ§/粒)及高浓度 ( 2μ§/粒） 时均有明显抑制小鼠角膜新生血管的作用 （具体见注释） 。 图 5显示 小肽 ΖΥ3对小鼠角膜病理性新生血管的影响， 可见小肽 ΖΥ3明显具有抑制新生 血管的效应。 图 5a-5c显示小鼠角膜新生血管面积。 图 5a为 VEGF组； 图 5b为 ΖΥ3(0·5μ1/粒)组； 图 5c为 ΖΥ3(2μ1/粒)组； 图 5d为相对于 VEGF组， VEGF+小肽 ZY3不同浓度组， 都具有明显抑制小鼠角膜病理性新生血管作用， **P<0.01， 差异具有统计学意义。 实施例 7
眼药水的制备
利用常规技术， 混合以下组分， 制得 1%眼药水， 其配方如下：
ZY3肽 (ZY3) 10 mg
羟丙基甲基纤维素 0.03g
无菌水 加至 10 ml
调节渗透压至 300Osm， 酸碱度 (pH)至 6.8-7.1。
经 5位志愿者试用一周， 每日 3次， 每次 1滴 /眼。 结果表明该眼药水可抑 制眼部的血管新生。 实施例 8
衍生多肽的制备和活性
制备了以下数种衍生多肽， 并按实施例 3所示的方法， 测定各 ZY3衍生多 肽对人脐静脉血管内皮细胞 HUVEC的增殖的抑制作用。 衍生多肽 1: 序列同 SEQIDNO: 1， 其中第 4位 Val被 lie替换； 衍生多肽 2: 序列同 SEQIDNO: 1， 其中第 12位 Arg Pro替换；
衍生多肽 3: 序列同 SEQIDNO: 1， 其中第 15位 Asp被 Glu 替换； 衍生多肽 4: 序列同 SEQIDNO: 1， 其中第 23位 Thr被 Arg替换； 衍生多肽 5: 序列同 SEQIDNO: 1， 其中缺失第 8位 Leu。
结果表明， 上述衍生多肽 1-5的处 S组（lug/ul)中， HUVEC细胞增殖显著 受到抑制。
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 请所附权利要求书所限定的范围。

Claims

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U9£80/Zl0Z l3/13d εοεο9θ/ειοζ OAV Xaa24是选自下组的氨基酸： Phe或 Ser;
Xaa25是选自下组的氨基酸： Ser或 Arg;
Xaa26是选自下组的氨基酸： Gin或 Ser;
Xaa27是选自下组的氨基酸： His或 Arg;
Xaa28是选自下组的氨基酸： 无， 或 1-3个氨基酸构成肽段；
并且所述的多肽具有抑制血管新生的活性，且所述多肽的长度为 28-24个氨 基酸。
2.如权利要求 1所述的多肽， 其特征在于， Xaa28是 3个氨基酸构成的肽 段。
3.如权利要求 1所述的多肽， 其特征在于， XaaO为 Lys。
4.如权利要求 1所述的多肽， 其特征在于， 所述多肽选自下组：
(a)具有 SEQ ID NO: 1所示氨基酸序列的多肽；
(b)将 SEQ ID NO: l所示氨基酸序列经过 1-5个氨基酸残基的取代、 缺失或 添加而形成的， 且具有抑制血管新生功能的由 (a)衍生的多肽。
5. 一种分离的核酸分子， 其特征在于， 它编码权利要求 1所述的多肽。
6. 一种药物组合物， 其特征在于， 它含有：
(a) 权利要求 1所述多肽或其药学上可接受的盐； 和
(b) 药学上可接受的载体或赋形剂。
7.如权利要求 6所述的药物组合物， 其特征在于， 所述组合物的剂型为针 剂、 眼药水、 眼用凝胶或眼药膏。
8. 如权利要求 1 所述的多肽或药学上可接受的盐的用途， 其特征在于， 用于制备抑制血管新生或防治与血管新生相关疾病的药物。
9. 如权利要求 8 所述的用途， 其特征在于， 所述的与血管新生相关疾病 的选自下组： 新生血管性眼病、 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、 不育症和肉瘤状病。
10.—种抑制哺乳动物血管新生的方法， 其特征在于， 包括步骤： 给需要的 对象施用本发明所述的多肽或其药学上可接受的盐。
PCT/CN2012/083671 2011-10-28 2012-10-29 抑制新生血管的多肽及其应用 WO2013060303A1 (zh)

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