WO2012013110A1 - Polypeptide ayant une activité inhibitrice de l'angiogenèse - Google Patents

Polypeptide ayant une activité inhibitrice de l'angiogenèse Download PDF

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WO2012013110A1
WO2012013110A1 PCT/CN2011/076755 CN2011076755W WO2012013110A1 WO 2012013110 A1 WO2012013110 A1 WO 2012013110A1 CN 2011076755 W CN2011076755 W CN 2011076755W WO 2012013110 A1 WO2012013110 A1 WO 2012013110A1
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group
amino acid
acid selected
polypeptide
ala
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PCT/CN2011/076755
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Chinese (zh)
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许迅
徐艺
赵卉
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上海市第一人民医院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • 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
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of biomedicine, and more particularly to a novel polypeptide (H-KI series polypeptide) having an action of inhibiting neovascularization.
  • the polypeptide inhibits proliferation, migration, lumen formation and inhibition of porcine embryonic allantoic membrane and mouse retinal neovascularization in vitro.
  • the invention also relates to the preparation and use of said polypeptides and to pharmaceutical compositions containing said polypeptides. Background technique
  • Angiogenesis refers to the process of forming new blood vessels by proliferation and migration of vascular endothelial cells on the basis of the original capillary network.
  • Angiogenesis plays an important role in physiological processes such as embryonic development, injury repair, and is also a major cause of many neovascular diseases such as tumor growth and metastasis, proliferative diabetic retinopathy, retinopathy of prematurity, and rheumatoid arthritis. Pathological changes. Therefore, the research and application of neovascular inhibitors are important for some refractory neovascular related diseases.
  • Ocular neovascularization is closely related to a variety of eye diseases. It often causes severe visual impairment and eventually blindness, such as diabetic retinopathy (DR), age-related macular degeneration (AMD), corneal neovascularization, neovascularization. Glaucoma and so on.
  • Current clinical treatments include surgery, lasers, and drugs. Surgery requires certain indications, patients are suffering, and risks and complications are relatively high; laser treatment also has some pain, which can cause local visual field defects, induce new blood vessels, and the mid- and long-term effects are not ideal. Therefore, a method for treating ocular neovascularization without pain, convenience, effectiveness, and high patient compliance has become a hot topic in domestic and foreign research for more than ten years.
  • VEGF Vascular endothelial growth factor
  • the drugs that have been used in clinical practice or are still in experimental research are mainly anti-angiogenic agents against VEGF or its receptor (VEGFR).
  • VEGF antisense oligonucleotides including VEGF expression inhibition; VEGF receptor antisense oligonucleotides for inhibiting VEGF receptor expression; anti-VEGF antibody, soluble VEGF receptor, VEGF Trap, anti-VEGFR antibody for neutralizing VEGF in blood circulation , tyrosine kinase inhibitors, etc., these preparations are widely used in tumor treatment.
  • the drug For ocular surface administration, the drug must penetrate the lipophilic corneal epithelial cells in close contact with the hydrophilic corneal stroma, so that only the appropriate fat-soluble, low molecular weight or transporter with the ocular surface tissue (eg: Amino acid transporters, oligopeptide transporters, etc.) bind to the drug to reach the anterior chamber.
  • the appropriate fat-soluble, low molecular weight or transporter with the ocular surface tissue eg: Amino acid transporters, oligopeptide transporters, etc.
  • 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.
  • angiostatin can significantly inhibit the growth of vascular-dependent tumors, but due to its large molecular weight and spatial conformation Complex, so there are deficiencies in the process of preparation and purification of the recombinant expression and endotoxin residues.
  • peptide angiogenesis inhibitors Compared with the widely studied protein angiogenesis inhibitors, peptide angiogenesis inhibitors have simple synthesis methods, easy chemical modification, low immunogenicity, good solubility, high bioavailability, and strong tissue penetration. The advantages of various medicines and low prices are outstanding advantages. However, there are currently no small molecule polypeptides with satisfactory effects from hepatocyte growth factor (HGF).
  • HGF hepatocyte growth factor
  • Another object of the invention is to provide a process and use comprising the polypeptide.
  • XaaO is no, or 1-3 amino acids constitute a peptide
  • Xaal is an amino acid selected from the group consisting of He , Leu, Val, Met or Ala;
  • Xaa2 is an amino acid selected from the group consisting of He , Leu, Val, Met or Ala;
  • Xaa3 is an amino acid selected from the group consisting of Gly or Ala;
  • Xaa4 is an amino acid Lys or Arg selected from the group below;
  • Xaa5 is an amino acid selected from the group consisting of Gly or Ala;
  • Xaa6 is an amino acid selected from the group consisting of Arg, Lys or Gly;
  • Xaa7 is an amino acid selected from the group below Ser or Thr
  • Xaa8 is an amino acid selected from the group consisting of Tyr or Phe
  • Xaa9 is an amino acid selected from the group consisting of Lys or Arg
  • XaalO is an amino acid selected from the group consisting of Gly or Ala;
  • Xaal l is an amino acid Thr or Ser selected from the group below ;
  • Xaal2 is an amino acid selected from the group consisting of Val, Leu, He, Met or Ala;
  • Xaal3 is an amino acid Ser or Thr selected from the group below ;
  • Xaal4 is an amino acid selected from the group consisting of He , Leu , Val, Met or Ala;
  • Xaal5 is an amino acid Thr or Ser selected from the group below ;
  • Xaal6 is an amino acid Lys or Arg selected from the group consisting of
  • Xaal7 Ser or Thr is an amino acid selected from the group;
  • Xaal8 is an amino acid selected from the group consisting of Gly or Ala;
  • Xaal9 is an amino acid selected from the group consisting of He , Leu , Val, Met or Ala;
  • Xaa20 is an amino acid Lys or Arg selected from the group consisting of;
  • Xaa21 is no, or 1-3 amino acids constitute a peptide
  • polypeptide has an activity of inhibiting angiogenesis, and the polypeptide has a length of 20-26 In another preferred embodiment, the polypeptide is 20-23 amino acids in length.
  • XaaO and/or Xaa21 are peptides consisting of 1-3 amino acids. More preferably, XaaO is C, NC, or RNC; and/or ⁇ 3&21 is (, CQ or CQP.
  • Xaal is an amino acid selected from the group consisting of: lie or Leu;
  • Xaa2 is an amino acid selected from the group consisting of: lie or Leu;
  • Xaa3 is an amino acid selected from the group consisting of Gly or Ala;
  • Xaa4 is an amino acid selected from the group consisting of Lys or Arg;
  • Xaa5 is an amino acid selected from the group consisting of Gly or Ala;
  • Xaa6 is an amino acid selected from the group consisting of Arg or Lys;
  • Xaa7 is an amino acid selected from the group consisting of Ser or Thr;
  • Xaa8 is an amino acid selected from the group consisting of: Tyr or Phe;
  • Xaa9 is an amino acid selected from the group consisting of Lys or Arg;
  • XaalO is an amino acid selected from the group consisting of Gly or Ala;
  • Xaall is an amino acid selected from the group consisting of Thr or Ser;
  • Xaal2 is an amino acid selected from the group consisting of Val or Leu;
  • Xaal3 is an amino acid selected from the group consisting of Ser or Thr;
  • Xaal4 is an amino acid selected from the group consisting of: lie or Leu;
  • Xaal5 is an amino acid selected from the group consisting of Thr or Ser;
  • Xaal6 is an amino acid selected from the group consisting of Lys or Arg;
  • Xaal7 is an amino acid selected from the group consisting of Ser or Thr;
  • Xaal8 is an amino acid selected from the group consisting of Gly or Ala;
  • Xaal9 is an amino acid selected from the group consisting of: lie or Leu; and/or
  • Xaa20 is an amino acid selected from the group consisting of Lys or Arg.
  • Xaa6 is Gly.
  • polypeptide is selected from the group consisting of:
  • 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) angiogenic function.
  • the derivative polypeptide retains 70% of the angiogenic activity of the indicated polypeptide of SEQ I. In another preferred embodiment, the derivative polypeptide is 80% identical to SEQ ID NO: 1, 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.
  • 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 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.
  • Figure 1 shows the mean values of the 0D values for each group in the MTS cell proliferation assay.
  • Figure 2 shows the number of cells in the VEGF group and VEGF+ peptides in the Transwel l cell migration experiment through the porous membrane (bar graph) and the VEGF group and the cells in different concentrations of H-KI20 migrated through the porous membrane (color photo).
  • the number of cells in the polypeptide group is significantly lower than that in the VEGF group, and it tends to decrease with increasing concentration.
  • Asterisks indicate a statistically significant difference between this group and the VEGF group.
  • the transparent small round holes in the photograph are the pores of the porous membrane, and the blue-violet cells are the cells which are stained by the hematoxylin and migrate through the pores.
  • Figure 3 shows the average total length (bar graph) of the lumen formed by the blank control group, the VEGF group, and the VEGF+ different concentrations of the polypeptide group in the in vitro lumen formation experiment of endothelial cells, as well as the blank control group, the VEGF group, and the VEGF group. + ⁇ Formation of endothelial cells in the H-KI20 group (photograph).
  • Figure 4 shows the growth of blood vessels on the chorioallantoic membrane (CAM) of the PBS group and the polypeptide group (color photograph) and the CAM blood vessel counts of each group in the evaluation experiment (bar graph).
  • CAM chorioallantoic membrane
  • the white disc in the photo is the filter paper
  • the black coil is defined by the blood vessel counting range
  • the orange blood vessel membrane is the chicken embryo allantoic membrane.
  • the filter paper with 10 ⁇ ⁇ / ⁇ 1 ⁇ - ⁇ 20 is added.
  • the number of blood vessels was significantly less than that of the PBS group.
  • the control group was in the PBS group. It can be seen that the number of blood vessels in the 5 ⁇ ⁇ / ⁇ 1 ⁇ - ⁇ 20 group and the 10 ⁇ ⁇ / ⁇ 1 ⁇ - ⁇ 20 group decreased significantly compared with the PBS group, and decreased with increasing concentration.
  • the asterisk indicates that the difference between this group and the control group is statistically significant.
  • 762, O. OO D o Figure 5 is a retinal patch of the retinal neovascular model, a photograph of the eyeball slice, and a statistical result of the number of neovascular lumens in the eyeball section. (bar chart).
  • Neovascular group (indicated by the elliptical circle), the number of neovascular clusters in the retinal plaque of young rats in the retinal neovascular model group after intravitreal injection of H-KI20 was significantly reduced. It can be seen from the eyeball slice that the neovascular lumen is not seen in the normal group before the retina, and the retinal neovascular model can see more neovascular lumens in front of the retina (shown by black arrows).
  • HGF hepatocyte growth factor
  • the present inventors After extensive and intensive research, the present inventors have for the first time prepared a small molecule polypeptide derived from hepatocyte growth factor (HGF) having an angiogenic function and having a molecular weight of less than 5 kD (e.g., only about 2 kD).
  • HGF hepatocyte growth factor
  • the inventors applied bioinformatics methods, based on homology analysis and biological characteristics analysis, designed several candidate sequences, synthesized by solid phase method, and then passed through chicken embryo chorioallantoic membrane Model, VEGF-induced cell proliferation model and hypoxia-induced retinal neovascularization in mice, a new class of small molecule peptides with prophylactic and therapeutic angiogenic functions were obtained.
  • 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.
  • the topical application of the eye is highly bioavailable, can penetrate the blood-eye barrier, and can reduce the dose, thereby reducing systemic side effects.
  • the present invention has been completed on this basis. Active polypeptide
  • polypeptide of the present invention refers to having blood vessels.
  • H-KI20 polypeptide refers to having blood vessels.
  • SEQ ID NO: 1 amino acid sequence of the peptide H-KI20 which is a novel inhibitory activity.
  • 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 substitution, and addition or deletion of one or several (usually within 5, preferably within 3, more preferably within 2) amino acids at the C-terminus and/or N-terminus.
  • 1-5 usually 1-4, preferably 1-3, more preferably 1-2, optimally 1 amino acid deletions, insertions And/or substitution, and addition or deletion of one or several (usually within 5, preferably within 3, more preferably within 2) amino acids at the C-terminus and/or N-terminus.
  • amino acids usually within 5, preferably within 3, more preferably within 2 amino acids at the C-terminus and/or N-terminus.
  • the term also encompasses both monomeric and multimeric forms of the polypeptides of the invention.
  • the term also includes both linear as well as non-linear polypeptides (e.g., cyclic peptides).
  • the invention also encompasses active fragments, derivatives and analogs of the H-KI20 polypeptide.
  • 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) one or more conserved or a polypeptide in which a non-conservative amino acid residue (preferably a conservative amino acid residue) is substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) an H-KI20 polypeptide and another a polypeptide formed by fusing a compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by the additional amino acid sequence fused to the polypeptide sequence (with a leader sequence, a secretory sequence, or a tag sequence such as 6His) The resulting protein after fusion).
  • a compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • a polypeptide formed by the additional amino acid sequence fused to the polypeptide sequence with a leader sequence, a secretory sequence
  • 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 I.
  • the invention also provides analogs of the H-KI20 polypeptide.
  • the difference between these analogs and the native H-KI20 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, ⁇ -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 derivatized 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 ⁇ - ⁇ 20 polypeptide.
  • a preferred coding sequence is atcattggta aaggacgcag ctacaaggga acagtatcta tcactaagag tggcatcaaa (SEQ ID NO: 3), which encodes the amino acid sequence set forth in SEQ ID NO: 1.
  • the polynucleotide of the present invention may be in the form of sputum or RNA. ⁇ can be a coded chain or a non-coded chain.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 3 or may be a degenerate variant.
  • a "degenerate variant" in the present invention refers to a polypeptide encoding a sequence 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 H-KI20 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.
  • a DNA sequence encoding a polypeptide of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • 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 a vector comprising a polynucleotide of the invention, and to the use of the vector of the invention or
  • H-KI20 polypeptide coding sequence is a genetically engineered host cell.
  • the invention also encompasses polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific for the polypeptide encoded by H-KI20 DNA or a fragment thereof.
  • 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) was treated at 0 ° C for 1 hour to cut the peptide chain from the resin while removing the protecting group. 50-80% The peptide is extracted with acetic acid (containing a small amount of mercaptoethanol), and the solution is further lyophilized and further purified by molecular sieve Sprint haddex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide. It can be known in the field of peptide chemistry.
  • Various coupling agents and coupling methods are coupled to each amino acid residue, and for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1, 1, 3, 3-tetra can be used.
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • 1, 1, 3, 3-tetra 1, 1, 3, 3-tetra
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • HBTU urea hexafluorophosphate
  • the polypeptide H-KI20 of the present invention is prepared by solid phase synthesis according to its sequence, and purified by high performance liquid chromatography to obtain a high-purity peptide freeze-dried powder, and -2 CTC is stored.
  • 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 H-KI20 polypeptides by conventional recombinant DNA techniques. Generally there are the following steps: (1) using a polynucleotide (or variant) encoding an H-KI20 polypeptide of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;
  • 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 sterilizing, ultra-treatment, ultra-centrifugation, 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, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography
  • 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 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 refers to a carrier for the administration of a therapeutic agent.
  • 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. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Scences (Mack Pub. Co., N. J. 1991).
  • 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 formulated as injectables, such as liquid solutions or suspensions; solid forms such as liquid carriers, which may be suitable in the preparation of solutions or suspensions before 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 (i sotonic iti es preservative, wetting agent, emulsifier, dispersant, stabilizer and cosolvent, and the formulation process) It can be carried out in a usual manner depending on the dosage form.
  • a shape Agent i sotonic iti es preservative, wetting agent, emulsifier, dispersant, stabilizer and cosolvent
  • the preparation of eye drops can be carried out by dissolving the short peptide H-KI20 or a pharmaceutically acceptable salt thereof together with the base substance in sterile water (a surfactant is dissolved in sterile water) to adjust the osmotic pressure and
  • a surfactant is dissolved in sterile water
  • the pH is in 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 optionally added, and then completely dissolved.
  • compositions of the invention may also be administered in the form of sustained release agents.
  • the short peptide H-KI20 or a salt thereof can be intruded into a pellet or microcapsule in which a sustained-release polymer is used as a carrier, and then the pellet or microcapsule is surgically implanted into a tissue to be treated.
  • the short peptide H-KI20 or a salt thereof can also be applied by inserting a drug-coated intraocular lens.
  • sustained-release polymer examples include ethylene-vinyl acetate copolymer, polyhydroxymethacrylate (polyhydrometaacrylate polyacrylamide, polyvinylpyrrolidone, methylcellulose, 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.
  • the dose of the short peptide H-KI20 as an active ingredient or a pharmaceutically acceptable salt thereof may be based on the weight, age, sex, symptom of each patient to be treated. Determined to a reasonable extent.
  • 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 polypeptide 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 the chicken embryo allantoic sac
  • the angiogenesis of the membrane can inhibit the proliferation, migration, chemotaxis and lumen formation of human umbilical vein endothelial cells, and can inhibit hypoxia-induced retinal neovascularization in mice.
  • the main advantages of the invention include:
  • 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, 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
  • H-KI20 I IGKGRSYKGTVSITKSGIK (SEQ ID NO: 1; position 129-148 in SEQ ID NO: 2)
  • H-KA SEQ ID NO: 2 in positions 150-176;
  • H-KC 190-200 in SEQ ID NO: 2;
  • the procedure is as follows: The required Fmoc protected amino acid solution, condensation reagent and cleavage reagent were calculated and prepared according to the software (Vers i on. version 201) using a SYMI3 ⁇ 40NY 12-channel polypeptide synthesizer (Protein Technologis, USA). Editing procedure, wherein the resin swelling time is 30 min; Deprotection twice, the time was 5 min and 15 min respectively; the condensation time was 30 min; the cutting time was 2 h.
  • the polypeptide was synthesized according to the above procedure, and the polypeptide was purified by high performance liquid chromatography (SHIMADZU) to obtain a white powdery polypeptide having a purity of >95% (120 mg each of each polypeptide), which was lyophilized for use.
  • SHIMADZU high performance liquid chromatography
  • H-KI20 consists of 20 amino acid residues with a molecular weight of 2093.52D, which does not contain cysteine residues and disulfide bonds.
  • Example 2 H-KI20 polypeptide inhibits VEGF-induced proliferation of vascular endothelial cells
  • HUVECs purchased from Sci enCell
  • ECGS ScienCell
  • fetal bovine serum ScienCell
  • the third to eighth generation HUVECs cells were used in all in vitro cell experiments in the present invention.
  • the MTS cell proliferation quantitative detection method is a method for producing a living cell proliferation by colorimetric determination of a water-soluble colored product by using a tetrazolium and an electron-conjugated compound under the action of a metabolically proliferating cell mitochondrial dehydrogenase.
  • HUVECs are grown close to the fusion, passaged, and seeded in a 96-well plate at a density of 3.5 ⁇ 107 ml, cultured in a well of 100 ⁇ l, 37° C., 5% CO 2 incubator for 24 hours, and then replaced with serum-free ECM. Medium, cells starved overnight.
  • the 96-well plate medium was aspirated, and each group was added with 50 ⁇ l of serum-free medium containing 1 ⁇ , 10 nM, 100 ⁇ , ⁇ , ⁇ ⁇ - ⁇ polypeptides, pretreated at 37 °C for 30 min, and then added to each well.
  • Serum-free medium containing VEGF (R&D) was used to give a final concentration of VEGF of 10 ng/ml.
  • a blank control group no VEGF-free H-KI polypeptide group
  • a VEGF control group no H-KI polypeptide group
  • 5 parallel wells were set for each experimental group. 37.
  • H-KA and H-KB peptides at high concentrations were slightly lower than those of the control group, but the 0D values at high H-KB concentrations were statistically significant compared with the control group.
  • Difference ⁇ method, 0. 05 H-KC polypeptide in the range of 1 ⁇ -10 ⁇ , the 0D value of each hole did not change significantly, the difference is not statistically significant ⁇ (Z6Y method, ⁇ >0.05) ⁇ ⁇ - ⁇ 20 group 100 ⁇ , 1 ⁇ , ⁇
  • the 0D value of each well decreased significantly, and the difference was statistically significant, LS, P ⁇ 0.01), indicating that the H-KI20 polypeptide was at a concentration of 100 ⁇ . 1 ⁇ , ⁇ can effectively inhibit the proliferation of HUVECs induced by VEGF, and the inhibition is gradually enhanced with the increase of the concentration of H-KI20 polypeptide.
  • H-KI20 polypeptide can effectively inhibit VEGF-induced vascular endothelial cell proliferation in a dose- and sequence-dependent manner.
  • H-KA, H-KB, and H-KC polypeptides did not significantly inhibit VEGF-induced vascular endothelial cell proliferation.
  • RF/6A cells monkey chorioretinal endothelial cell line, ATCC CRL-1780
  • 96-well plates 5 ⁇ 10 4 cells/well
  • the cells were serum starved overnight
  • the experimental group was added with peptide (H).
  • -KI20 set the concentration gradient (in order of ⁇ , ⁇ , 1 ⁇ , ⁇ )
  • the positive control group was added recombinant human VEGF 165 100ng/ml (rh-VEGF 165 , Sigma, St. Louis, MO), the blank control group did not Add any reagents. Make 6 replicate wells per concentration for each group.
  • MTS (3-(4, 5- dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl) - 2-(4- sul phophenyl) - 2H-tetrazolium) assay (CellTiter 96 AQ) Promega, Madison, WI, USA), continued to incubate for 4 h, and measured absorbance (0D value) at 490 nm.
  • the 0D values of the KI20 group were 0.461 ⁇ 0.026, 0.508 ⁇ 0.026, 0.477 ⁇ 0.029, 0.469 ⁇ 0.022, 0.436 ⁇ 0.010, and 0.429 ⁇ 0.029, respectively.
  • the 0D value of each concentration group decreased with the increase of concentration, indicating that VEGF promoted. Cell proliferation is inhibited by the polypeptide.
  • the inhibitory cell proliferation activity of K1 of HDF ranged from 57 nM to 116 nM, and the activity increased slowly from 116 nM to 925 nM. After that, the activity did not change significantly with increasing concentration.
  • rhVEGF Containing 100 ng / ml rhVEGF RPMI1640 165 serum-free medium was added at 24 well plate chamber, the RF / 6A cells were added to the upper chamber (the Transwell chamber, a porous membrane pore size 8 ⁇ , Corning Corporation), a cell concentration of 4X10 6 cells / ml
  • the concentration gradient was set: ⁇ , ⁇ , ⁇ , no polypeptide was added to the upper chamber of the control group, and a blank control group was set up (the upper chamber was a cell suspension, and the lower chamber was not.
  • 165 serum-free medium containing rhVEGF. Make two duplicate wells per peptide concentration.
  • the migration ability of RF/6A cells was detected by counting the number of cells migrating through the Transwell chamber porous membrane.
  • the cells migrated from the upper chamber through the porous membrane to the direction of high VEGF concentration (lower chamber).
  • cell migration to the lower chamber was inhibited by the addition of the polypeptide to the upper chamber.
  • Vascular endothelial cell lumen formation assay was performed using Matrigel (BD) in combination with VEGF to induce lumen formation.
  • a 96-well plate is coated with matrigel (growth factor-reduced Matrigel, BD Biosciences, USA).
  • the polypeptide group RF/6A cells were pretreated with different concentrations of ⁇ - ⁇ 20 (10 ⁇ , ⁇ , 1 ⁇ , 10 ⁇ ) for 30 minutes, and then inoculated into a 96-well plate coated with Matrigel at a cell concentration of 4.5 ⁇ 10 5 cells/ml. And add 100 ng / ml VEGF.
  • a blank control group without VEGF and peptide
  • a VEGF control group containing only VEGF
  • H-KI20 has the ability to inhibit the lumen formation of vascular endothelial cells in vitro and is concentration dependent.
  • the in vivo chicken chorioallantoic membrane (CAM) assay was used to further confirm the effect of H-KI20 polypeptide on inhibiting neovascularization in vivo.
  • 5 ⁇ 1 peptide (experimental group, set concentration gradient 25 ⁇ ⁇ /5 ⁇ 1, 50 ⁇ ⁇ /5 ⁇ 1) or PBS (control group) to the 5mm diameter filter paper (Whatman quantitative filter papers, Sigma) on the ultra-clean bench and dry dry.
  • the fertilized eggs are cleaned and disinfected, placed in an incubator, and turned daily until the 6th day of embryonic age.
  • the eggs are opened, and the filter paper is placed between the large blood vessels of the chicken embryo chorioallanes, which are relatively rare, and sealed.
  • the window was opened and incubated for 48 hours.
  • the number of 3-5 blood vessels in the range of 2.5 mm around the filter paper was counted under a microscope.
  • Ten eggs were set for each concentration in each group, and the differences between the groups were statistically compared.
  • the chicken embryo allantoic membrane (CAM) evaluation experiment is a stable model for evaluating the activity of small molecules affecting angiogenesis.
  • the filter paper with or without peptide is placed on the CAM. After 48 hours of incubation, the small blood vessels around the filter paper are counted. number.
  • ⁇ - ⁇ 20 polypeptide can significantly inhibit the growth of chicken chorioallantoic capillary, and with the increase of ⁇ - ⁇ 20 polypeptide dose, the inhibition of neovascularization is enhanced, with a good dose-dependent.
  • each nest is a group.
  • Group 1 Normal control group, reared in normal air with lactating mothers;
  • Group 2 oxygen-induced retinal neovascularization model group, group 3: PBS injection group;
  • group 4 and group 5 were oxygen induced by peptide intervention
  • groups 2-5 were housed in a 75 ⁇ 2% hyperoxia environment for 5 days with lactating mothers, and the oxygen partial pressure in the container was continuously monitored with an oxygen meter. It was then returned to normal air for 5 days to induce retinal neovascularization.
  • group 2 PBS group
  • group 3, 4 polypeptide group
  • PBS and H-KI20 10 mM or 50 mM
  • the eyeballs were removed, and some of the eyeballs were used as retinal patches and Alexa Fluor 568 conjugated i solect in B4 (Molecular Probes) staining to show Retinal blood vessels, photographed under a fluorescence microscope for qualitative analysis; some eyeballs were used as paraffin sections for hematoxylin and eosin staining, photographed under a light microscope and counted in each section of the neovascular lumen (defined as growing in the inner retinal membrane) The number of vascular lumens before the vitreous cavity surface. Statistical analysis and comparison of differences between groups.
  • H-KI20 can significantly inhibit the formation of oxygen-induced retinal neovascularization, and the inhibition of neovascularization with the increase of peptide dosage, indicating that H-KI20 has a good inhibitory effect on neovascularization in vivo.
  • Derived polypeptide 1 sequence identical to SEQ ID NO 1, wherein the 6th Arg is replaced by Gly
  • Derived polypeptide 2 the sequence is the same as SEQ ID NO 1, wherein the 2nd position is replaced by Leu; the derivative polypeptide 3: the sequence is the same as SEQ ID NO 1, wherein the 15th Thr is replaced by Ser; the derivative polypeptide 4: the sequence is the same as SEQ ID NO 1, wherein the fourth Lys is replaced by Arg; the derivatized polypeptide 5: the sequence is the same as SEQ ID NO 1, wherein the 18th Gly is replaced by Ala; the derived polypeptide 6: the sequence is the same as SEQ ID NO 1, wherein the first position before the N-terminus Addition of Cys; Derived polypeptide 7: Sequence identical to SEQ ID NO: 1, wherein the addition of CQP after the 20th position of the C-terminus showed that HUVEC cell proliferation was significantly inhibited in the treatment group ( ⁇ ) of the above-derived polypeptides 1-7.
  • the H-KI20 and its derived polypeptides of the present invention all show good anti-mouse corneal pathological neovascularization, anti-retinal pathological neovascularization, and inhibition of vascular endothelial cell proliferation, migration, chemotaxis in vitro. And the role of lumen formation. Therefore, it has broad application prospects. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the the the the the the the the the the. In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the present invention.

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Abstract

L'invention concerne un polypeptide ayant une activité inhibtrice de l'angiogenèse. Le polypeptide est issu des acides aminés aux positions 129-148 d'un facteur de croissance des hépatocytes. L'invention concerne également un procédé de préparation du polypeptide et une composition pharmaceutique contenant le polypeptide.
PCT/CN2011/076755 2010-07-28 2011-07-01 Polypeptide ayant une activité inhibitrice de l'angiogenèse WO2012013110A1 (fr)

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WO2001044294A2 (fr) * 1999-12-15 2001-06-21 Entremed, Inc. Compositions et procedes d'inhibition de la proliferation de cellules endotheliales
US20030148950A1 (en) * 2001-10-09 2003-08-07 Li Xin Kringle domain 1 of human hepatocyte growth factor and uses therefor

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WO2001044294A2 (fr) * 1999-12-15 2001-06-21 Entremed, Inc. Compositions et procedes d'inhibition de la proliferation de cellules endotheliales
US20030148950A1 (en) * 2001-10-09 2003-08-07 Li Xin Kringle domain 1 of human hepatocyte growth factor and uses therefor

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Title
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SHEN, Z. ET AL.: "The kringle 1 domain of hepatocyte growth factor has antiangiogenic and antitumor cell effects on hepatocellular carcinoma.", CANCER RES., vol. 68, no. 2, 15 January 2008 (2008-01-15), pages 404 - 414 *

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