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WO2007135781A1 - Peptide capable d'accroître l'activité de l'éphrine-b2, sel de celui-ci, composition à des fins médicales et kit de thérapie - Google Patents

Peptide capable d'accroître l'activité de l'éphrine-b2, sel de celui-ci, composition à des fins médicales et kit de thérapie Download PDF

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Publication number
WO2007135781A1
WO2007135781A1 PCT/JP2007/000550 JP2007000550W WO2007135781A1 WO 2007135781 A1 WO2007135781 A1 WO 2007135781A1 JP 2007000550 W JP2007000550 W JP 2007000550W WO 2007135781 A1 WO2007135781 A1 WO 2007135781A1
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WO
WIPO (PCT)
Prior art keywords
peptide
ephrin
seq
acceptable salt
pharmacologically acceptable
Prior art date
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PCT/JP2007/000550
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English (en)
Japanese (ja)
Inventor
Yasuyuki Shimohigashi
Ayami Matsushima
Tadahisa Kagimoto
Atsushi Wada
Kaoru Azuma
Shigeyuki Yoneda
Original Assignee
Aqumen Biopharmaceuticals K.K.
Kyushu University
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Publication date
Application filed by Aqumen Biopharmaceuticals K.K., Kyushu University filed Critical Aqumen Biopharmaceuticals K.K.
Publication of WO2007135781A1 publication Critical patent/WO2007135781A1/fr

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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 a peptide that increases the activity of ephrin B2, a pharmacologically acceptable salt thereof, a pharmaceutical composition containing ephrin B2 and such a peptide, a medical kit, and the like.
  • Neovascularization is a prominent feature of various ocular pathological conditions such as age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. Therefore, a drug that inhibits neovascularization and can treat or prevent these diseases is desired.
  • the formation of blood vessels is important for the normal functioning of living organisms. Therefore, a drug that suppresses pathological angiogenesis or angiogenesis that goes outside the retina, and that enhances the formation of a vascular network in the retina and the maturation of blood vessels is desired.
  • CNV choroidal neovascularization
  • PED F pigment epithelium-derived factor
  • Patent Document 1 discloses that ephrin-2 has an inhibitory effect on the synthesis of DN ⁇ of arterial and venous endothelial cells in the eye; It has been shown to have an inhibitory effect on p44Zp42MAPK activation in arterial and venous endothelial cells; ephrin B2 has been shown to have an inhibitory effect on tube formation in arterial and venous endothelial cells in the eye.
  • Example 3 shows that ephrin B 2 is effective in treating age-related macular degeneration (AMD) using the CNV (choroidal neovascularization) model.
  • AMD age-related macular degeneration
  • CNV choroidal neovascularization
  • Patent Document 2 discloses ephrin B2.
  • ephrin is used as a gene therapy for angiogenesis of the eye (page 1, lines 4 to 12).
  • Patent Document 2 has no suggestion as to whether ephrin B 2 suppresses the growth of new blood vessels or promotes the formation of normal vascular networks.
  • Patent Document 2 describes ephrin B 2 As examples, only tumor cell inhibition experiments have been carried out, so there has been no evidence of the relationship between ephrin B 2 and new blood vessels or the relationship between ephrin B 2 and normal blood vessels.
  • the peptides represented by SEQ ID NO: 1 to SEQ ID NO: 3 of the present application are not disclosed.
  • LERK-5 in Japanese Patent Publication No. 10-502810 is considered to correspond to ephrin B2.
  • Example 1 of Patent Document 3 cDNA of human ephrin B 2 was isolated, and in Example 7, a fusion protein of human ephrin B 2 (extracellular domain) and F c stimulates phosphorylation of the receptor 1 This is demonstrated in an in vitro experiment.
  • Patent Document 3 only intends to use ephrin B2 for neurological diseases (page 21, line 23 to page 22, line 2), and does not describe its use for neovascular diseases.
  • the peptides represented by SEQ ID NO: 1 to SEQ ID NO: 3 of the present application are not disclosed.
  • Patent Document 4 The “H tk ligand” in JP-T-10-501701 (Patent Document 4) is presumed to correspond to ephrin B2. Patent Document 4 mentions the treatment of neurodegeneration as an application of H tk ligand (page 60, line 7 to page 63, line 9). However, H tk ligand is used for the suppression of neovascularization and the like. Is not described or suggested. In addition, the peptides represented by SEQ ID NO: 1 to SEQ ID NO: 3 of the present application are not disclosed.
  • Patent Document 5 Japanese National Patent Publication No. 2002-51 1 41 7 (Patent Document 5) describes that ephrin B2 enhances angiogenesis caused by tumors, etc. (paragraph [00 62] of Patent Document 5). Thus, there is nothing motivating to use ephrin B2 to inhibit new blood vessels. In addition, the peptides represented by SEQ ID NO: 1 to SEQ ID NO: 3 of the present application are not disclosed.
  • Patent Document 1 WO 2006/006079 Publication
  • Patent Document 2 W02002Z26827 Publication
  • Patent Literature 3 Japanese Patent Publication No. 10-50281 0
  • Patent Document 4 Japanese Patent Publication No. 10-501 701
  • Patent Document 5 Special Table 2002-51 1 41 7
  • Non-patent document 1 Kyoko Ohno "Molecular mechanism of choroidal neovascularization in age-related macular degeneration, especially about the role of pigment epithelium-derived factor" 1 0 7, No. 1 1, pp. 657 -673, 2003
  • Non-Patent Document 2 T. Kondoeta, "PK CZM A ⁇ S igna I ing S uppression by Retinal P ericy te ndy te p a n te p ri m ent P revents R etinal En dothelial Center Proliferation", Journalofeel I u I arphysio I ogy, 203, p. 378-386, 2005
  • An object of the present invention is to provide a novel peptide or a pharmacologically acceptable salt thereof that is effective in the treatment of a disease associated with neovascularization.
  • An object of the present invention is to provide a novel peptide or the like that enhances the activity of ephrin B2 and the like and is effective in the treatment of a disease involving ephrin B2, such as a disease associated with new blood vessels.
  • An object of the present invention is to provide a new blood vessel inhibitor containing such a novel peptide and the like, an auxiliary agent for new blood vessel inhibition that enhances the new blood vessel inhibitory ability of F and Jin B2, and the like.
  • An object of the present invention is to provide a pharmaceutical composition and the like effective for inhibiting new blood vessels including ephrin B2 and the above-mentioned peptides and the like.
  • the present invention is a medical kit effective for inhibiting new blood vessels, comprising a first composition containing ephrin B2 and the like and a second composition containing the above-mentioned peptides and the like.
  • the purpose is to provide firewood.
  • the present invention is based on the finding that, by using a low molecular weight polypeptide such as SEQ ID NO: 1 together with ephrin B2, the neovascular inhibitory activity of ephrin B2 can be enhanced. Is based. Furthermore, the present invention provides a sequence number This is based on the finding that administration of low molecular weight polypeptide such as No. 1 and ephrin B 2 separately can significantly enhance the neovascular inhibitory activity of ephrin B 2 compared to administration of these simultaneously. .
  • the first aspect of the present invention is basically a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 3 (preferably SEQ ID NO: 1 or 2), or a pharmacologically acceptable peptide thereof. Related to the salt.
  • the above peptides themselves have the ability to inhibit M A P K phosphorylation.
  • the above peptides have the effect of dramatically increasing the ability of ephrin B 2 to inhibit M A P K phosphorylation when used in combination with ephrin B 2. This means that ephrin B 2 has an increased ability to inhibit new blood vessels in vascular endothelial cells.
  • the present invention relates to a peptide consisting of an amino acid sequence in which one or two amino acids are deleted, substituted, inserted or added, a peptide that enhances the inhibitory activity of ephrin B 2 on new blood vessels, or a pharmaceutically acceptable salt thereof.
  • a peptide substantially similar to the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 demonstrated in the examples also has an effect of increasing the activity of ephrin B2. . More specifically, it can be said that a peptide containing SEQ ID NO: 3 having a certain number of residues significantly enhances the neovascular inhibitory activity of ephrin B2.
  • a preferred embodiment of the first aspect of the present invention is an auxiliary agent for inhibiting neovascularization, particularly in the eye, containing any of the peptides described above, or a pharmacologically acceptable salt thereof.
  • a preferred embodiment of this adjuvant is a neovascular inhibitor whose main agent contains ephrin B2.
  • a preferred embodiment of the first aspect of the present invention is an auxiliary agent for inhibiting DNA synthesis in vascular endothelial cells, comprising any of the peptides described above, or a pharmacologically acceptable salt thereof. is there.
  • a preferred embodiment of this adjuvant is to use an agent containing ephrin B 2 as the main agent.
  • a preferred embodiment of the first aspect of the present invention contains p4 4 Zp 4 2 in vascular endothelial cells, containing any of the peptides described above, or a pharmacologically acceptable salt thereof. It is an adjuvant for inhibiting MAPK phosphorylation activity.
  • a preferred embodiment of this adjuvant is to use an agent containing ephrin B2 as the main agent.
  • a preferred embodiment of the first aspect of the present invention is a disease or disorder related to angiogenesis or angiogenesis, comprising any of the above-mentioned peptides, or a pharmaceutically acceptable salt thereof. It is a therapeutic agent.
  • a preferred embodiment of the first aspect of the present invention is a neovascular inhibitor containing any of the peptides described above, or a pharmacologically acceptable salt thereof. This neovascular inhibitor contains an effective amount of any of the above peptides as an active ingredient.
  • the neovascular inhibitor is preferably an intraocular neovascular inhibitor, and is preferably a therapeutic or prophylactic agent for diseases associated with intraocular neovascularization.
  • the peptides of the present invention inhibit angiogenesis or inhibit angiogenesis. Therefore, it can be said that the above peptides and the like are effective as therapeutic agents for diseases or disorders related to these.
  • Diseases or disorders related to intraocular angiogenesis or neovascularization include: “Age-related macular degeneration, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, One or more selected from the group consisting of “diabetic retinal ischemia, diabetic retinal edema, diabetic retinopathy”.
  • angiogenesis or angiogenesis other than intraocular diseases or disorders related to angiogenesis or angiogenesis other than intraocular include tumors, rheumatoid arthritis, psoriasis, atherosclerosis, diseases such as force-positive sarcoma, and solid tumor metastasis. It is preferable to use an agent containing ephrin B 2 as the main agent, which preferably contains any of the above peptides, or a pharmacologically acceptable salt thereof as an active ingredient. Also good.
  • the present invention includes a step of administering an agent containing ephrin B 2 as a main agent to a human or non-human mammal, and before or after (preferably after) the above-mentioned peptide, Also provided is a method for treating diseases associated with intraocular neovascularization, wherein a pharmacologically acceptable salt is administered to a rabbit or a non-human mammal.
  • the present invention 1 or 2 in SEQ ID NO: 1 or SEQ ID NO: 2 before or after (preferably after) the step of administering an agent containing ephrin B 2 as the main agent to a rabbit or a non-human mammal.
  • a peptide having an amino acid sequence deleted, substituted, inserted or added, which enhances the inhibitory activity of ephrin B 2 on neovascularization, or a pharmacologically acceptable salt thereof, is selected from the group consisting of amino acids and non-amino acids. Also provided are methods for treating diseases associated with neovascularization in the eye administered to mammals.
  • the present invention further provides any of the above-mentioned peptides for producing an angiogenesis inhibitor (preferably an adjuvant for ocular neovascular inhibition) in a human or non-human mammal, Also provided is the use of pharmacologically acceptable salts.
  • the present invention relates to an amino acid sequence in which one or two amino acids are deleted, substituted, inserted or added in SEQ ID NO: 1 or SEQ ID NO: 2 for producing an auxiliary agent for inhibiting neovascularization in the eye. It also provides the use of a peptide that enhances the neovascular inhibitory activity of ephrin B2, or a pharmacologically acceptable salt thereof.
  • the second aspect of the present invention basically includes ephrin B2 or a pharmacologically acceptable salt thereof; and (i) a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, Or a pharmacologically acceptable salt thereof, or (ii) consisting of an amino acid sequence in which one or two amino acids are deleted, substituted, inserted or added in SEQ ID NO: 1, and the formation of the ephrin B 2 And a pharmacologically acceptable salt thereof that enhances the inhibitory activity of blood vessels.
  • the second aspect of the present invention basically includes ephrin B 2 or a pharmacologically acceptable salt thereof; any of the above-mentioned peptides or a pharmacologically acceptable salt thereof.
  • the peptide of the present invention significantly enhances the activity of ephrin B 2 when used in combination with ephrin B 2. Therefore, the pharmaceutical composition according to the second aspect of the present invention effectively inhibits angiogenesis, or effectively inhibits angiogenesis. Therefore, a disease or disorder involving angiogenesis or neovascularization (particularly in the eye) It can be effectively used for the treatment or prevention of diseases or disorders involving neovascular vessels.
  • the second aspect of the present invention relates to ephrin B 2 or a pharmaceutically acceptable salt thereof.
  • a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, or (ii) one or two amino acids deleted or substituted in SEQ ID NO: 2.
  • a peptide comprising an inserted or added amino acid sequence and enhancing the inhibitory activity of ephrin B 2 on a new blood vessel, or a pharmacologically acceptable salt thereof, and a pharmaceutical composition comprising:
  • the second aspect of the present invention further includes ephrin B2 or a pharmacologically acceptable salt thereof; a peptide comprising the amino acid sequence represented by SEQ ID NO: 3, wherein the peptide comprises
  • the present invention relates to a pharmaceutical composition comprising a peptide having 9 or more and 100 or less amino acid residues, or a pharmacologically acceptable salt thereof.
  • the above peptide has the effect of dramatically increasing the ability of ephrin B2 to inhibit MAPK phosphorylation when used in combination with ephrin B2.
  • ephrin B 2 has an increased ability to inhibit new blood vessels in vascular endothelial cells. Therefore, it is considered that a pharmaceutical composition containing ephrin B 2 and the above-mentioned peptides can be effectively used for suppressing neovascularization in the eye.
  • a preferred embodiment of the second aspect of the present invention relates to the pharmaceutical composition as described above, wherein the ephrin B 2 contains an extracellular domain of natural ephrin B 2 and does not contain a cytoplasmic domain. Since the region that contains the extracellular domain of natural ephrin B 2 but not the cytoplasmic domain is considered to be the site that acts on the pharmacophore of E ph B 4, ephrin B 2 containing such a site If so, it is considered that it has the ability to suppress neovascularization even if it is not full length.
  • a preferred embodiment of the second aspect of the present invention relates to the pharmaceutical composition according to any one of the above, wherein the ephrin B 2 is a fusion protein of human Fc and ephrin B2.
  • ephrin B 2 In order for ephrin B 2 to exert its ability to inhibit neovascularization, it is considered that ephrin B 2 should be dimerized. Therefore, a fusion protein of 2Fc and ephrin B 2 should be used.
  • Ephrin B 2 is dimerized and can exert a high ability to inhibit new blood vessels.
  • a preferred embodiment of the second aspect of the present invention relates to the pharmaceutical composition according to any one of the above, which is a neovascular inhibitor, particularly an intraocular neovascular inhibitor.
  • a preferred embodiment of the second aspect of the present invention relates to the pharmaceutical composition according to any one of the above, which is a neovascular inhibitor in the choroid in the eye.
  • a preferred embodiment of the second aspect of the present invention relates to the pharmaceutical composition according to any one of the above, which is a therapeutic / prophylactic agent for a disease associated with neovascularization in the eye. More specifically, a preferred embodiment of the second aspect of the present invention relates to the pharmaceutical composition according to any one of the above, which is a therapeutic agent for a disease or disorder associated with angiogenesis or angiogenesis.
  • a preferred embodiment of the second aspect of the present invention is “age-related macular degeneration, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retinal ischemia.
  • composition according to any one of the above, which is a therapeutic or preventive agent for one or more diseases selected from the group consisting of:
  • a particularly preferred embodiment is a treatment / prevention agent for diseases of either or both of age-related macular degeneration and diabetic retinopathy.
  • a preferable embodiment of the second aspect of the present invention is a therapeutic agent for metastasis of tumor, rheumatoid arthritis, psoriasis, atherosclerosis, force positive sarcoma, or solid cancer.
  • the present invention relates to a pharmaceutical composition.
  • the present invention relates to ephrin B 2 or a pharmacologically acceptable salt thereof; any one of the above peptides or a pharmacologically acceptable salt thereof; It also provides a method of treating diseases associated with neovascularization in the eye.
  • the present invention relates to ephrin B2 or a pharmacologically acceptable salt thereof for producing a therapeutic agent for a disease associated with neovascularization in the eye; any one of the above peptides or Also provided is the use of a pharmacologically acceptable salt thereof.
  • the third aspect of the present invention basically includes the first composition containing ephrin B2 or a pharmaceutically acceptable salt thereof, and a pharmacologically acceptable carrier.
  • a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, or a pharmacologically acceptable salt thereof, or (ii) 1 or 2 amino acids in SEQ ID NO: 1. Consists of an amino acid sequence deleted, substituted, inserted or added, and the peptide that enhances the neovascular inhibitory activity of ephrin B2, or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable And a second composition containing a carrier.
  • the third aspect of the present invention basically includes the first composition containing ephrin B 2 or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable carrier; And a second composition containing a pharmacologically acceptable salt thereof and a pharmacologically acceptable carrier.
  • the above-described medical kit basically includes the first composition for suppressing neovascularization in the eye as the main agent and the second composition as an auxiliary agent.
  • the above peptides have the effect of dramatically increasing the ability of ephrin B 2 to inhibit MAPK phosphorylation when used in combination with ephrin B 2. This means that ephrin B 2 has an increased ability to inhibit neovascularization in vascular endothelial cells.
  • administration of the first composition followed by the second composition markedly inhibits the activity of MA P K. Therefore, the kit of the present invention can be effectively used particularly for such administration method.
  • a preferred embodiment of the third aspect of the present invention relates to the kit as described above, wherein the ephrin B 2 contains the extracellular domain of natural ephrin B 2 and does not contain a cytoplasmic domain.
  • a preferred embodiment of the third aspect of the present invention relates to the kit according to any one of the above, wherein the ephrin B 2 is a fusion protein of chick F c and ephrin B 2.
  • a preferred embodiment of the third aspect of the present invention relates to the kit according to any one of the above, which is used for neovascular inhibition, particularly for intraocular neovascular inhibition.
  • a preferred embodiment of the third aspect of the present invention relates to the kit according to any one of the above, which is used for inhibiting neovascularization in the choroid in the eye.
  • a preferred embodiment of the third aspect of the present invention is a kit according to any one of the above, which is used for treatment or prevention of a disease related to neovascularization, particularly neovascularization in the eye.
  • a preferred embodiment of the third aspect of the present invention relates to any of the above kits used for the treatment of a disease or disorder associated with angiogenesis or angiogenesis.
  • Preferred embodiments of the third aspect of the present invention include age-related macular degeneration, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retinal ischemia , Diabetic retinal edema, diabetic retinopathy ”, which is used for the treatment or prevention of one or more diseases selected from the group consisting of“ a diabetic retinopathy ”.
  • a preferred embodiment of this aspect relates to the kit according to any one of the above, which is used for the treatment or prevention of one or both of age-related macular degeneration and diabetic retinopathy.
  • Preferred embodiments of the third aspect of the present invention are associated with tumors, rheumatoid arthritis, psoriasis, atherosclerosis, positive sarcoma, metastasis of solid cancer, other angiogenesis or angiogenesis It relates to a kit for the treatment of a disease or disorder.
  • a preferred embodiment of the third aspect of the present invention is that, after the first composition is administered to a rabbit or a non-human mammal, the second composition is used for a rabbit or non-human mammal.
  • a preferred embodiment of the third aspect of the present invention is that after administration of the first composition, 30 seconds to 24 hours, preferably 1 minute to 12 hours, more preferably 1 minute.
  • the present invention includes a first composition comprising ephrin B2 or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable carrier;
  • a first composition comprising ephrin B2 or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable carrier;
  • a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a pharmacologically acceptable salt thereof, or
  • SEQ ID NO: 1 or SEQ ID NO: 2 A peptide or a pharmacologically acceptable salt thereof, which consists of an amino acid sequence in which two amino acids are deleted, substituted, inserted or added, and which enhances the neovascular inhibitory activity of ephrin B 2)
  • a second composition containing a pharmacologically acceptable carrier, and a method of treating a neovascular-related disease, wherein the first composition is administered after the second composition is administered. Also provide.
  • the pharmaceutical composition comprising ephrin B2 and the peptides represented by SEQ ID NO: 1 to SEQ ID NO: 3 can be combined with each other, compared to the case where each is administered alone. It was shown that high neovascular inhibition activity can be obtained. Therefore, according to the present invention, it is possible to provide a pharmaceutical composition that is effective for inhibiting new blood vessels including ephrin B 2 and a predetermined peptide.
  • a medical kit that is effective for inhibiting new blood vessels, including a first composition containing ephrin B 2 and a second composition containing a predetermined peptide. Can be provided.
  • FIG. 1 is a western plotting photograph replacing the drawing showing the results of ephrin B2, peptidyl of SEQ ID NO: 1, and their concomitant agents for p44Z42MAPK phosphorylation in Example 3. is there.
  • FIG. 2 is a photograph replacing a drawing obtained by Western blotting analysis showing the result of administration of ephrin B 2 to p44Z42MAPK phosphorylation in Example 4.
  • FIG. 3 is a photograph replacing a drawing obtained by Western blotting analysis showing the results of ephrin B2, peptide of SEQ ID NO: 1, and their concomitant drugs for p44Z42MAPK phosphorylation in Example 5.
  • FIG. 4 is a drawing obtained by Western blotting analysis showing the results of ephrin B2, peptide of SEQ ID NO: 1, peptide of SEQ ID NO: 2, and their concomitant drugs for p44Z42MAPK phosphorylation in Example 6. It is a photo that changes The
  • FIG. 5 shows the results of ephrin B 2, peptide of SEQ ID NO: 1, peptide of SEQ ID NO: 2, and their concomitant agents for p 4 4 Z 4 2 MAPK phosphorylation in Example 7. It is a photograph that replaces a drawing analyzed by blotting.
  • FIG. 6 shows the amino acid sequence of the peptide according to SEQ ID NO: 4 and the nucleotide sequence of the gene encoding this peptide.
  • FIG. 6A shows the amino acid sequence shown in SEQ ID NO: 4. The amino acids from the 1st to 1 6 7th are shown.
  • the boxed part is the peptide part shown in SEQ ID NO: 1.
  • Figure 6B shows the continuation of Figure 6A.
  • the first aspect of the present invention is basically “a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1”, “in SEQ ID NO: 1, one or two amino acids missing, substituted, inserted or A peptide consisting of an added amino acid sequence, a peptide that enhances the inhibitory activity of ephrin B 2 on a new blood vessel, or a pharmacologically acceptable salt thereof, "a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, Or a pharmacologically acceptable salt thereof ”,“ in SEQ ID NO: 2, consisting of an amino acid sequence in which one or two amino acids have been deleted, substituted, inserted or added, and inhibits the new blood vessel of ephrin B 2 Or a pharmacologically acceptable salt thereof, or a peptide having the amino acid sequence represented by SEQ ID NO: 3, wherein the peptide contains 9 or more amino acid residues , 1 0 0 or less Rubepuchi de, or to a pharmaceutically acceptable
  • the peptide of the present invention itself has an inhibitory effect on new blood vessels, and also functions as an auxiliary agent having a function of enhancing the activity of ephrin B2. Since ephrin B 2 inhibits the development of new blood vessels, the peptide of the present invention is useful for the treatment or prevention of diseases involving new blood vessels.
  • a peptide consisting of an amino acid sequence in which 1 or 2 amino acids are deleted, substituted, inserted or added in SEQ ID NO: 1 the amino acid sequence represented by SEQ ID NO: 1 is in the 2nd to 10th positions.
  • a peptide consisting of an amino acid sequence in which one or two amino acids are deleted, substituted, inserted or added at a site other than the portion is preferred.
  • the effectiveness of the “peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a pharmacologically acceptable salt thereof” is as demonstrated in the examples described later.
  • a peptide comprising an amino acid sequence in which one or two amino acids are deleted, substituted, inserted or added in SEQ ID NO: 2 and enhancing the inhibitory activity of ephrin B 2 on new blood vessels, or a drug thereof.
  • the amino acid residue represented by SEQ ID NO: 3 is conserved, and 1 or 2 amino acids are deleted, substituted, inserted or added at other sites. I like it.
  • the amino acid sequence represented by SEQ ID NO: 1 includes the 2nd to 10th positions (SEQ ID NO: 3), a known amino acid sequence may be added as appropriate. It may consist of amino acid residues or may consist of 10 to 20 amino acid residues.
  • Such a peptide is, for example, a partial peptide (for example, 5 to 100 amino acid residues, preferably 1 from the amino acid sequence of ephrin B 2 represented by SEQ ID NO: 4). (0 to 30 amino acid residues, more preferably 10 to 20 amino acid residues).
  • one or several amino acids (specifically 2, 3, 4 or 5) were deleted, substituted, inserted or added from the partial peptide of ephrin B 2 thus extracted.
  • a peptide consisting of an amino acid sequence having the same activity as the peptide of the present invention may be used.
  • the “activity similar to the peptide of the present invention” means, for example, those having the ability to suppress neovascularization in the eye, the ability to serve as an adjunct to ephrin B2, and the like.
  • the nucleotide sequence shown in SEQ ID NO: 5 is the nucleotide sequence of DNA encoding ephrin B 2 shown in SEQ ID NO: 4.
  • the peptide (polypeptide) of the present invention is represented as the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end.
  • the C-terminus may be any of a carboxyl group (_COOH), a carboxylate (_COO_), an amide (_CON H 2 ), or an ester (one COOR).
  • R in the ester include d-6 alkyl groups such as methyl, ether, n-propyl, isopropyl, and n_butyl.
  • the carboxyl group may be amidated or esterified.
  • the peptides of the present invention, and the like which are Amino groups of the amino acid residues at the N-terminus is protected with a protecting group (e.g., formyl group, etc.
  • C _ 6 Ashiru groups such Arukanoi Le such Asechiru group
  • a salt with an inorganic acid for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • an organic acid for example, acetic acid, formic acid
  • Salts with propionic acid fumaric acid, maleic acid, succinic acid, tartaric acid, citrate, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).
  • the peptide of the present invention or a salt thereof can be produced from a cell or tissue of a human mammal by a known protein purification method. It can also be produced by culturing a transformant containing DNA encoding the peptide. It can also be produced according to the peptide synthesis method.
  • the tissue or cells are homogenized and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography or ion exchange chromatography. Chromas such as graphics By combining the graphics, the peptide of the present invention or a salt thereof can be isolated and purified.
  • Peptide synthesis methods include, for example, solid phase synthesis, liquid phase synthesis (Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi, Michinori Waki, "Basics and Experiments of Peptide Synthesis” 1 9 8 5, Maruzen ( Stock))).
  • the peptide of the present invention can be isolated and purified by combining ordinary purification methods such as solvent extraction 'distillation' and muchromography ⁇ liquid chromatography or recrystallization.
  • the peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method.
  • the free form or other substances can be converted by a known method. Can be converted to salt.
  • the polypeptides of the present invention when the peptide represented by SEQ ID NO: 1 is produced by the solid phase synthesis method, for example, the carboxyl group of the protected amino acid residue of the 20th residue of the amino acid sequence Is bound directly (or optionally via a spacer) to an insoluble resin having a chloromethyl or oxymethyl group. Then, each protected amino acid from position 19 to position 1 of the amino acid sequence is sequentially bound according to the solid phase synthesis method. Then, the insoluble resin and amino acid protecting groups are removed. In this way, the polypeptide of the present invention can be obtained.
  • the insoluble resin, spacer, and N-protected amino acid resin in which an N-protected amino acid is bound to an insoluble resin can be prepared by known methods, and commercially available products may be used as appropriate.
  • the insoluble resin is not particularly limited as long as it can be directly bonded to the carboxyl group of the N-protected amino acid at the C-terminal, or can be bonded via a spacer and can be detached from the carboxyl group.
  • Well-known ones can be used as appropriate.
  • an insoluble resin for example, when a peptide is synthesized by Boc (t_butyloxycarbonyl) strategy, chloromethyl resin (chloromethylated styrene-divinylbenzene copolymer), oxymethyl resin, or A 4-oxymethyl_Pam (phenylacetamidomethyl) resin into which a spacer is introduced is preferred.
  • F moc (9-fluorenylmethyloxyca When peptides are synthesized using the (Luponyl) strategy, oxymethylphenoxymethyl (Wang) resin is preferred.
  • the protected amino acid means an amino acid whose functional group is protected with a protecting group by a known method.
  • As the protected amino acid a commercially available known protected amino acid can be appropriately used.
  • the protecting group for the imidazolyl group of histidine is preferably Tos, Z, Pac (phenacyl), Bom (benzyloxymethyl), Dnp (dinitrophenyl) or Trt (trityl).
  • B z I as a protecting group for the mercapto group of cysteine (Cy s)
  • t_butyl t_Bu S (t_butylthio), and MB z I, 4-Me B z I, T rt, A cm, or N pys are preferable.
  • protecting groups for the hydroxyl group of thiocin (T yr) include B zl, CI 2 ⁇ ⁇ ⁇ I (2, 6-dichloromouth benzyl), and t_Bu.
  • the hydroxyl group of tyrosine (T yr) does not need to introduce a protective group.
  • CHO is a protecting group for the indole group of tribtophan (T rp).
  • the indole group of tribtophan does not introduce a protective group. You may leave.
  • a protecting group for the thiomethyl group of methionine (Me t) is a methyl sulfoxide group, but it is not necessary to introduce a protecting group.
  • Examples of the protecting group for the hydroxyl group of (S e r) and ⁇ leonine (T h r) include the B z I or t _Bu group.
  • protecting group for the carboxyl group of (GI u), OB z I (benzyl ester), O t Bu (t_butyl ester), Oc Hex (cyclohexyl ester), or OPac (phenacyl ester) is preferred.
  • a protecting group for the carbamide group of asparagine (A sn) and glutamine (G in) is preferable.
  • Protected amino acids can be bound by conventional condensation methods such as the DCC (dicyclohexyl carpositimide) method or the D I PCD I (diisopropyl carpositimide) method.
  • DCC dicyclohexyl carpositimide
  • D I PCD I diisopropyl carpositimide
  • Of_Amino group protecting group elimination reagent is trifluoroacetic acid Z dichloromethane, HCI Z dioxane, piperidine ZDMF or piperidine ZNMP, etc., and is selected as appropriate depending on the type of protecting group. .
  • the degree of progress of the condensation reaction at each stage of the synthesis is determined by the method of E. Kaiser et al.
  • the protected peptide resin is hydrogen fluoride, T FMSA (trifluoromethanesulfonic acid) [A ademic Press, edited by E. Goss, H Ya ji ma et al; "T he Peptides” 5, 65 (1 98 3)], TMSOTf (trimethylsilyl triflate), TMSBr (trimethylsilyl bromide) [Fujii. N et al .; Ch em. Pharm. Bu II., 35, 3880 (1 987 )], Or by treatment with trifluoroacetic acid or the like, the resin and the protecting group can be eliminated simultaneously.
  • the above elimination reagent is appropriately selected according to the strategy (B OC or F m OC ), the resin, and the type of protecting group.
  • the peptides thus obtained can be obtained by known methods such as extraction, recrystallization, various chromatography (gel filtration, ion exchange, distribution, adsorption, reverse phase), electrophoresis, countercurrent distribution, etc. It can be isolated and purified. Of these, the purification method using reversed-phase high-performance liquid chromatography is preferred. If the peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method. When the peptide is obtained in the form of a salt, it can be converted into a free form or other salt by a known method.
  • Ephrin B 2 is induced by endothelial cell (EC) DNA synthesis; extracellular signal-regulated kinase (ERK) phosphorylation in EC; both VEG F and b FG F, as shown in the Examples below.
  • EC endothelial cell
  • ERK extracellular signal-regulated kinase
  • the present method can be appropriately employed by adopting a known method for formulation. It can be used for an angiogenesis inhibitor containing the peptide of the invention.
  • This neovascular inhibitor is, for example, an agent containing the peptide of the present invention and a pharmacologically acceptable carrier, and is preferably an intraocular neovascular inhibitor, which is related to intraocular neovascularization.
  • a therapeutic or prophylactic agent for the disease is preferred. Since the peptide of the present invention is shorter than ephrin B2, it can be easily produced. Therefore, the neovascular inhibitor containing the peptide of the present invention is stable and can be produced at a relatively low cost.
  • the peptide of the present invention enhances the activity of ephrin B2, it is an ocular neovascular inhibition adjuvant, an intraocular venous endothelial cell DNA synthesis inhibition adjuvant, and an intraocular venous endothelium.
  • An adjuvant for inhibiting p44Zp42MAPK phosphorylation activity in cells can be provided.
  • an agent containing ephrin B 2 is used as the main agent.
  • MAPK When vascular endothelial cells are stimulated with VEG F, MAPK is activated by the signal transduction system downstream of the receptor, and phosphorylated MAP K is elevated (Ab edi, H. and Zachary, I., J. B iol. C he m., 272, 1 5442-1 5451 (1 997)). MAPK activation is known to play an important role in the proliferation of vascular endothelial cells in angiogenesis (Mere nmies, J. eta I., Cell Growth & Differr., 83 — 1 0 (1 997); Ferrara, N. and Da vis—Smy th, T. Endocr. Rev., 1 8, 4—25 (1 997)).
  • the peptide of the present invention has an auxiliary activity for inhibiting angiogenesis or for inhibiting angiogenesis by ephrin B2.
  • Angiogenesis at the pathological site is mainly caused by diseases such as tumors, rheumatoid arthritis, psoriasis, atherosclerosis, and positive-positive sarcoma. (F or kman, J. Nature Me d. 1: 27-31 (1 995), Bicknell, R., Harris, A. L. C. urr. Op i n. On col.
  • the peptides of the present invention may be used in diseases such as tumors, rheumatoid arthritis, psoriasis, atherosclerosis, force positive sarcoma, and solid cancer metastasis, other angiogenesis or angiogenesis-related diseases or It can be used to treat disorders, and the present invention can also provide such therapeutic agents.
  • agents and adjuvants include those containing an effective amount of the peptide of the present invention.
  • Effective amount means an amount sufficient to achieve the desired purpose.
  • Effective amount in an adjuvant for inhibiting neovascularization in the eye means an amount sufficient to enhance the neovascular inhibitory action of the neovascular inhibitor in the eye such as ephrin B2.
  • the dose of the peptide of the present invention may be appropriately adjusted depending on the target disease, administration subject, administration route and the like.
  • As a dosage of the peptide of the present invention for example, when it is administered to the eye by vitreous injection, generally 1 X 10 2 ng to 1 mg per eye is preferable for adults (weight 60 kg).
  • the 1 X 1 0 2 ng ⁇ 1 g, more preferably Ru are exemplified I xl 0 2 ng ⁇ 7 xl 0 2 ng.
  • the number of doses can be adjusted as appropriate. For example, one that is administered once a day with an agent containing ephrin B2, the main agent.
  • the adjuvant is administered orally, in the general adult (weighing 6 O kg), once diary 2 g ⁇ 1 X 1 0 2 mg , preferably 2 g to 1 mg, more preferably 1 X From 10 2 ng to 2 g, more preferably from 2 g to 1 X 10 2 g.
  • the amount converted per 6 O kg body weight may be administered as appropriate.
  • the peptide of the present invention can be administered per se or as a pharmaceutical composition (adjuvant).
  • adjuvant examples include those containing a pharmacologically acceptable carrier, diluent or excipient.
  • Adjuvants should be adjusted as dosage forms suitable for the dosage form.
  • the peptide or adjuvant of the present invention when used as an eye drop or injection, an effective amount of the peptide of the present invention and a known diluent (the diluent is a pharmacologically acceptable carrier). It may be an eye drop or injection containing As diluent, water such as sterilized water, pure water, distilled water; physiological saline; glucose solution; alcohol such as ethanol; polyalcohol such as glycerol, propylene glycol, polyethylene glycol; sterilized organic solvent; ; Any one or a mixture of two or more of PBS.
  • diluent water such as sterilized water, pure water, distilled water; physiological saline; glucose solution; alcohol such as ethanol; polyalcohol such as glycerol, propylene glycol, polyethylene glycol; sterilized organic solvent; ; Any one or a mixture of two or more of PBS.
  • Eye drops or injections containing the peptide of the present invention include decongestants such as epinephrine, epinephrine hydrochloride, ephedrine, and the like; eye regulators such as neostigmine methyl sulfate and tropicamide; zinc sulfate, zinc lactate , Allantoin, ypsilon monoaminocaproic acid, indomethacin, anti-inflammatory components such as lysozyme chloride; Local anesthetic components such as force-in, cocaine hydrochloride, cornecaine hydrochloride, dibu force-in hydrochloride, etc. may be included as appropriate.
  • decongestants such as epinephrine, epinephrine hydrochloride, ephedrine, and the like
  • eye regulators such as neostigmine methyl sulfate and tropicamide
  • zinc sulfate zinc lactate
  • Thickeners such as sodium chondroitin sulfate and sodium hyaluronate; surfactants such as benzalkonium chloride; preservatives such as sodium benzoate, ethanol, benzalkonium chloride, bactericides or antibacterial agents; hydrochloric acid, boric acid, hydroxylation PH regulators such as sodium and hydrogen carbonate sodium; tonicity agents such as hydrogen sulfite sodium sulfite, sodium sulfite sodium chloride, and chlorinated rhodium; menthol, camphor, heart power oil, peppermint oil, etc. Perfume or refreshing agent; or a buffer such as citrate buffer.
  • surfactants such as benzalkonium chloride
  • preservatives such as sodium benzoate, ethanol, benzalkonium chloride, bactericides or antibacterial agents
  • hydrochloric acid, boric acid, hydroxylation PH regulators such as sodium and hydrogen carbonate sodium
  • tonicity agents such as hydrogen s
  • the adjuvant containing the peptide of the present invention may be an orally administered agent such as a tablet, capsule, granule, powder, or syrup.
  • pharmacologically acceptable carriers include those appropriately selected from excipients, diluents, lubricants, binders, disintegrants, stabilizers, and flavoring agents.
  • the adjuvant containing the peptide of the present invention can be produced according to a known method. Eye drops Alternatively, the injection can be produced, for example, by adding the peptide of the present invention to a diluent or the like and placing it in a container such as an ampoule. Tablets can be produced, for example, by tableting a pharmaceutical composition obtained by mixing the peptide of the present invention with a known carrier using a tableting machine. The capsule can be produced, for example, by encapsulating the peptide of the present invention in a carrier in the form of a capsule or the like.
  • the peptide of the present invention enhances the activity of ephrin B2, it can be appropriately used in a medical composition or kit containing ephrin B2, as described later.
  • the second aspect of the present invention basically includes ephrin B 2 or a pharmacologically acceptable salt thereof; the peptide of the present invention, or a pharmacologically acceptable salt thereof.
  • the present invention relates to a pharmaceutical composition.
  • ephrin B2 or a pharmacologically acceptable salt thereof a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, are preferably pharmaceutical compositions containing their pharmacologically acceptable salts.
  • ephrin B 2 inhibits DNA synthesis of arterial and venous endothelial cells in the eye; p 44Zp in arterial and venous endothelial cells in the eye 42MAPK activation inhibitory effect; has an inhibitory effect on tube formation in arterial and venous endothelial cells in the eye.
  • ephrin B 2 suppresses neovascularization in the choroid in the eye and is associated with intraocular neovascularization such as age-related macular degeneration (AMD). It is effective in the treatment of diseases.
  • AMD age-related macular degeneration
  • the peptide of the present invention has the effect of dramatically increasing the ability of ephrin B2 to inhibit MAPK phosphorylation when used in combination with ephrinB2.
  • Ephrin B 2 has an increased ability to suppress neovascularization in intraocular vein (arterial) endothelial cells. Therefore, a pharmaceutical composition containing ephrin B 2 and the peptide of the present invention has a special feature. In particular, it is thought to be particularly effective in the treatment of diseases related to neovascularization in the eye, such as suppression of neovascularization in the eye.
  • the pharmaceutical composition of the present invention may contain an effective amount of ephrin B 2.
  • the ephrin B 2 used in the pharmaceutical composition of the present invention may be any ephrin B 2 including an analog and a variant.
  • Ephrin B 2 may be soluble ephrin B 2 (usually containing the extracellular domain but not the cytoplasmic domain).
  • Ephrin B 2 may be one obtained by isolating and purifying a naturally occurring protein, or one produced from a microorganism by genetic recombination.
  • Ephrin B 2 may be full length.
  • a preferred embodiment of ephrin B 2 includes the natural extracellular domain of ephrin B 2 and no cytoplasmic domain. Since the region containing the extracellular domain of natural ephrin B 2 but not the cytoplasmic domain is considered to be the site that acts on the pharmacophore of Eph B 4, ephrin B 2 containing such a site If present, it is thought that it has the ability to inhibit neovascularization even if it is not full length.
  • ephrin B 2 those consisting of amino acid residues from 1 to 25 to 30 8 in SEQ ID NO: 4, those consisting of amino acid residues from 1 to 30 8 in SEQ ID NO: 4,
  • SEQ ID NO: 4 consisting of an amino acid residue at positions 1 to 199, and having at least 90% homology with them (preferably Are those having a homology of 95% or more, or those having a homology of 90% or more including the site shown in SEQ ID NO: 3, and having the same activity as ephrin B 2 It is done. Note that homology can be You can find it using version 6 of the GAP computer program with optimized parameters.
  • the peptide represented by SEQ ID NO: 4 consists of an ephrin B 2 N-terminal signal peptide (amino acids 1 to 25 to 1 of SEQ ID NO: 4), an extracellular domain (amino acids 1 to 19 9), a transmembrane region ( A peptide consisting of amino acids 20 0 to 2 2 5) and a cytoplasmic domain (amino acids 2 2 6 to 3 0 8) (The amino acid numbers shown in SEQ ID NO: 4 are The amino acid sequence and the base sequence of the gene coding for this peptide are shown in FIG.
  • ephrin B2 may be a fusion protein of human Fc and ephrin B2.
  • ephrin B 2 In order for ephrin B 2 to exert its ability to inhibit neovascularization, it is considered that ephrin B 2 should be dimerized. Therefore, if it is a fusion protein of ⁇ Fc and ephrin B2, , Ephrin B 2 is dimerized and can exhibit high ability to inhibit new blood vessels.
  • the ephrin B 2 used in this fusion protein preferably contains the extracellular domain of ephrin B 2 or the extracellular domain but does not contain the cytoplasmic domain.
  • N-terminal signal peptide and the transmembrane region may or may not be included in all of them, and only some of them may be included.
  • a fusion protein of ephrin B 2 and rabbit F c can be produced, for example, by the method described in Japanese Patent Publication No. Hei 10—5 0 2 8 10 (Patent Document 3).
  • a preferred embodiment of the second aspect of the present invention relates to the pharmaceutical composition according to any one of the above, which is a neovascular inhibitor, particularly an intraocular neovascular inhibitor.
  • a preferred embodiment of the second aspect of the present invention relates to the pharmaceutical composition according to any one of the above, which is a neovascular inhibitor in the choroid in the eye.
  • These pharmaceutical compositions may appropriately contain known pharmacologically acceptable carriers and the like. As such a carrier, those described above can be used as appropriate.
  • a neovascular inhibitor can be produced in the same manner as the adjuvant described above.
  • a preferred embodiment of the second aspect of the present invention relates to the pharmaceutical composition according to any one of the above, which is a therapeutic / prophylactic agent for a disease associated with neovascularization in the eye. More specific Specifically, the preferred embodiment of the second aspect of the present invention is “age-related macular degeneration, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema. , Diabetic retinal ischemia, diabetic retinal edema, diabetic retinopathy ”, the pharmaceutical composition according to any one of the above, which is a therapeutic or preventive agent for one or more diseases About.
  • a particularly preferred embodiment is a therapeutic / prophylactic agent for the treatment of either or both of age-related macular degeneration and diabetic retinopathy.
  • the peptide of the present invention has an angiogenesis-inhibiting action or an angiogenesis-inhibiting action by ephrin B2.
  • Angiogenesis at the pathologic site is largely associated with tumors, rheumatoid arthritis, psoriasis, atherosclerosis, force positive sarcoma, and metastasis of solid tumors, except in the eye (F or kman, J. Nature Me d. 1: 27 -3 1 (1 995); Bicknell, R., Harris, A. I_. C urr. O pin.
  • a preferred embodiment of the second aspect of the present invention relates to a pharmaceutical composition which is a therapeutic agent for tumor, rheumatoid arthritis, psoriasis, atherosclerosis, force positive sarcoma, or solid cancer metastasis.
  • the pharmaceutical composition of the present invention can be used as a therapeutic agent for diseases related to neovascular vessels in the eye that are already affected, or to prevent these diseases, or these diseases Can also be used as a preventive agent to delay the occurrence of
  • the ratio (molar ratio) of ephrin B2 and the peptide of the present invention contained in the pharmaceutical composition or therapeutic / preventive agent according to the second aspect of the present invention may be adjusted as appropriate.
  • the molar ratio of ephrin B 2 to the peptide of the present invention can be from 1: 1 X 10_ 3 to 1: 1 X 1 0 3 , and 1: 1 X 1 0- 2 ⁇ 1: 1 X 1 0 2 is preferred instrument 1: 1 X 1 0 - 1 ⁇ 1: 1 X 1 0 is preferably tool 1: 1 ⁇ 1: 1 X 1 0 3 is preferably tool 1: 1 to 1: 1 X 1 0 2 is preferred 1: 1 to 1: 1 X 1 0 is preferred 1: 1 to 1: 5 X 10 is preferred ⁇ , 1: 1 X 1 0 ⁇ 1: 5 X 10 is more preferable.
  • the third aspect of the present invention basically includes the first composition containing ephrin B 2 or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable carrier; Or a pharmacologically acceptable salt thereof, and a medical kit containing a second composition containing a pharmacologically acceptable carrier (hereinafter referred to as “a pharmacologically acceptable salt”).
  • the present invention relates to a medical kit comprising a first composition containing ephrin B 2 and the like and a second composition containing the peptide of the present invention.
  • a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, or a pharmacologically acceptable salt thereof is preferred. .
  • the above-mentioned medical kit is basically composed of the first composition for inhibiting new blood vessels, in particular for inhibiting new blood vessels in the eye, and the second composition as an auxiliary agent. Is included.
  • the peptide of the present invention has the effect of dramatically increasing MAPK phosphorylation inhibitory activity of ephrin B 2 when used in combination with ephrin B 2. This means that ephrin B 2 has an increased ability to inhibit new blood vessels in venous (arterial) endothelial cells in the eye.
  • administration of the second composition after administration of the first composition significantly inhibits the activity of MAPK. Therefore, the kit of the present invention can be effectively used for such administration method.
  • a preferred embodiment of the medical kit of the present invention is used for neovascular inhibition, particularly for neovascular inhibition in the eye, and a more preferred embodiment is for neovascular inhibition in the intraocular choroid. It is used for.
  • a preferred embodiment of the third aspect of the present invention relates to the kit according to any one of the above, which is used for treatment or prevention of a disease associated with neovascularization in the eye.
  • aspects include age-related macular degeneration, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retinal ischemia, diabetic retinal edema, diabetic retinopathy
  • the invention relates to a kit according to any one of the above, which is used for the treatment or prevention of one or more diseases selected from the group consisting of:
  • a preferred embodiment of the third aspect of the present invention is The present invention relates to a kite according to any one of the above, which is used for the treatment or prevention of age-related macular degeneration and diabetic retinopathy or both diseases.
  • the peptide of the present invention has an angiogenesis-inhibiting action or an angiogenesis-inhibiting action by ephrin B2.
  • Angiogenesis at the pathological site is deeply associated with tumors, rheumatoid arthritis, psoriasis, atherosclerosis, force positive sarcoma, and metastasis of solid tumors, mainly outside the eye (F or kma n, J. Nature Me d. 1: 27 -3 1 (1 995); Bicknell, R., Harris, AL Curr. O pin n. Onco 8: 60—65 (1 996 )).
  • a preferred embodiment of the third aspect of the present invention is a kit according to any one of the above, which is used for the treatment of tumor, rheumatoid arthritis, psoriasis, atherosclerosis, force positive sarcoma, or solid cancer metastasis.
  • a preferred embodiment of the third aspect of the present invention relates to a kit for treating tumor, rheumatoid arthritis, psoriasis, atherosclerosis, force positive sarcoma, or solid cancer metastasis.
  • the first composition may be a therapeutic or prophylactic agent for a disease related to neovascularization such as neovascularization in the eye
  • the second composition may be an adjuvant of the first composition.
  • therapeutic agents, preventive agents and adjuvants those described above can be used as appropriate.
  • the contents of ephrin B 2 and the peptide of the present invention contained therein can be appropriately used.
  • a preferred embodiment of the third aspect of the present invention relates to the kit according to any one of the above, wherein the second composition is administered after the first composition is administered. 30 seconds to 24 hours after administration of the first composition, preferably 1 minute to 12 hours later, more preferably 1 minute to 10 minutes later, the second composition is The kit according to any one of the above, which is administered.
  • a preferred embodiment of the third aspect of the present invention relates to the kit according to any one of the above, wherein the first composition is administered after the second composition is administered. 30 seconds to 24 hours after administration of the second composition, preferably 1 minute to 12 hours later, more preferably 1 minute to 10 minutes later, The kit according to any one of the above, which is administered.
  • peptides of the present invention and ephrin B 2 are effective as therapeutic agents for diseases or disorders associated with intraocular angiogenesis or neovascularization, such as AMD, has been shown, for example, by laser-induced choroidal neovascularization (CNV). ) Can be demonstrated using a model.
  • the laser-induced CNV model is considered to be an AMD ecological model.
  • the following is an example of the CNV model protocol.
  • a CNV model experiment is carried out using 12 eyes derived from 6 force diquan monkeys (OS 3-6 kg). All of these animals are housed in a pathogen free state so that they are not painful. Therefore, the experiment is performed under general anesthesia with ketamine hydrochloride.
  • CNV experimental choroidal neovascularization
  • a lens for an ophthalmic laser (3-MI nfant Laser OG 3M IA, Ocular I nstru me nts, Inc.) is used, and the posterior pole of the fundus is krypton laser.
  • a lens for an ophthalmic laser 3-MI nfant Laser OG 3M IA, Ocular I nstru me nts, Inc.
  • the posterior pole of the fundus is krypton laser.
  • laser For example, the light intensity is about 700 mW.
  • the coagulation size should be 100 m, the coagulation time should be 0.1 sec, and 8 coagulations per eye should be performed.
  • mice Examples of administration methods and concentrations are shown in Table 1 below.
  • the arrow means that the next peptide is administered after the administration of one peptide.
  • the following intravitreal concentrations of PBS, ephrin B2, and the peptide of the present invention are administered into the vitreous immediately after laser irradiation and 5 mg after irradiation.
  • 0.1 mL per administration is injected into the eye through the ciliary flat area using a 30G needle.
  • 1.66 nM ephrin B2 is used.
  • Animal numbers 3 to 5 should be administered at 3 minute intervals.
  • animal number 3 (right eye) receives the peptide of SEQ ID NO: 1 (1 00 L) 3 minutes after administration of ephrin B 2 (1 00 L).
  • a total of 200 L will be administered.
  • the CNV model experiment is evaluated as follows. Fluorescent fundus angiography is performed 10 days after laser irradiation to evaluate choroidal neovascularization. The examination should be carried out with both eyes at different times. In both eyes, fluorescein (fluorescein fundus fluorescein (registered trademark) injection solution No. 1, Nippon Alcon Co., Ltd., 0.1 mL mLk g) 2 minutes in succession in the order of 5 to 10 minutes after administration. Take pictures one by one. An ophthalmologist will make an evaluation based on the pictures taken.
  • the test animal is anesthetized by intravenous administration of (68.8 mgZmL, 0.4 ml LZ kg) on the cephalic vein, the body weight is measured, the blood is euthanized, and the outer surface, internal organs and tissues are observed visually.
  • a polypeptide represented by SEQ ID NO: 1 was synthesized as follows. In other words, solid-phase synthesis using Fmo c (9_fluorenylmethyloxycarbonyl) strategy using the automated peptide synthesizer “Syro II” from Mu I ti Syntech. Adjusted by law.
  • the synthesis involves HBTU (2- (1 H) —benzotriazole 1 _yl) _ 1, 1, 3, 3 —tetramethyl uronium hexafluorphosphine 1) HOB t (1-hydroxybenzo (Riazole) method (Knorr, R. et al., T etrahedron Lett., 1 927 (1 989)) was used.
  • the synthesis scale was 25 micromolar.
  • the resins and amino acids used were as shown in Table 3 and Table 4, respectively.
  • DIEA means ⁇ , ⁇ '-disopropylethylamine.
  • the synthetic peptide obtained as described above was reacted at room temperature for 2 hours using TF ⁇ (trifluoroacetic acid) Z water / triethylsilane (Triethysylalan) [90: 5: 5].
  • the resin was filtered from the reaction mixture, washed twice with 1 ml of trifluoroacetic acid, 100 ml of ether was added to the combined filtrate and washings under ice cooling, and the resulting precipitate was centrifuged. The residue was separated from the supernatant by decantation.
  • the resulting residue was washed with ether under ice-cooling, centrifuged, and the supernatant was purified by reversed-phase high-performance liquid chromatography.
  • the reversed-phase high-performance liquid chromatograph uses HP LC “LC 1 0AD” manufactured by Shimadzu Corporation.
  • the column is a chromaxil (Kroma si I) KR “1 00_ 1 0C 1 8” 250 x 3 Omm column.
  • the conditions for reversed-phase high-performance liquid chromatography were a two-component gradient of 0.1% TFA aqueous solution (A solution) and 80% acetonitrile in 0.1% TFA aqueous solution (B solution). 5%, 30 minutes later, B solution was 80%, and the detection wavelength was 210 nm.
  • the fraction containing the purified peptide was collected and lyophilized.
  • the final purified peptide was obtained from Shimadzu HP LC “LC 10 AD” and Kromasil (KRoma si I) KR “1 00_ 1 0C 1 8” 250 x This was confirmed by high performance liquid chromatography using a 4 (or 6) mm column. The final purified peptide was also confirmed by the MA LDI-TO F mass spectrometer “Re flex II” manufactured by Bruker.
  • HUVEC human umbilical vein endothelial cells
  • the cell line was thawed in a 37 ° C thermostat and dissolved in 10 ml of vascular endothelial cell medium (H ume dia, EG_2 kit, Kurabo Industries), then type I collagen-coated 10 cm culture dish (I WAK I made) )
  • I WAK I made type I collagen-coated 10 cm culture dish
  • the medium was replaced with 1 Om I of EG-2 medium.
  • Subculture was performed when 80-90% confluent.
  • the cell concentration at this time was about 5 X 10 6 ZD ish.
  • a medium obtained by removing cell growth factors (h EG F, h FG F- B) from vascular endothelial cell medium (H ume dia, EG-2 kit, Kurabo Industries) (6 we II
  • the medium was changed to 2 ml) and cultured for 24 hours (star Vation).
  • the medium was removed, and the peptide-degrading enzyme inhibitor bacitracin (final concentration 100 microgZm I) was added to the vascular endothelial cell medium (H ume dia, EG-2 kit, Kurabo Industries) to which the cell growth factor was removed. Things were added as controls.
  • the peptide and ephrin B 2 were diluted with this Toncolol medium and added to the cells. After 10 minutes (or an appropriate time), the cells were washed twice with PBS, then lysed buffer (Lysis buffer). Buffer) (0.2 ml Zwell I for 6 we II) was used to collect the whole cell lysate.
  • Example 3 lysed buffer (Lysis buffer). Buffer) (0.2 ml Zwell I for 6 we II) was used to collect the whole cell lysate.
  • Vascular endothelial cells were prepared by adding the peptide-degrading enzyme inhibitor bacitracin (final concentration 100 gZm I) to a medium obtained by removing cell growth factor from vascular endothelial cell medium (H ume dia EG_2 kit, manufactured by Kurabo). And the peptide obtained in Example 1 (the peptide represented by SEQ ID NO. 1. Hereinafter, also referred to as “peptide of SEQ ID NO. 1”) and ephrin B 2 (extracellular of ephrin B 2) It was treated for 10 minutes using a mixture of a domain and Fc fusion protein).
  • bacitracin final concentration 100 gZm I
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide electrophoretic electrophoresis
  • SDS-PAGE uses 10% acrylamide, separated at 200 ports for 40 minutes, and wet for 1 hour under 100 pol water conditions. Transferred to P VDF membrane. After blocking with 3% skim milk for 1 hour, an antibody against phosphorylated P44 / 42MAPK (Cell Signa II ngtechnology, Danvers, A, U t> A) was used as the primary antibody with 4 ° Incubate in C (1: 2, 2.00).
  • the membrane was washed with horse radish peroxidase-labeled secondary antibody (Bio_Rad, Rickmond, CA, USA) (1: 20,000) for 40 minutes at room temperature. Incubated. Visualization was performed using an Amersham enhanced chemiluminescence (ECL) detection system according to the manufacturer's instructions.
  • ECL Amersham enhanced chemiluminescence
  • FIG. 1 is a photograph of Western blotting showing the results of ephrin B2, peptidyl of SEQ ID NO: 1, and their concomitant agents for p44Z42MAPK phosphorylation in Example 3.
  • the first lane shows the control; the second lane shows that treated with 100 nM peptide of SEQ ID NO: 1; the third lane shows that treated with 3 nM ephrin B 2
  • Lane 4 shows 200 ⁇ ⁇ peptide with SEQ ID NO: 1 and 6 ⁇ ⁇ ephrin ⁇ 2 processed simultaneously;
  • Lane 5 shows 200 nM with SEQ ID NO: 1 peptide.
  • lane 7 shows control; lane 8 shows that treated with the peptide of SEQ ID NO: 1 of 10 ⁇ ; lane 9 shows 3 nM ephrin B
  • the first lane shows the 20 ⁇ ⁇ peptide of SEQ ID NO: 1 and 6 nM ephrin ⁇ 2 treated simultaneously; the first lane shows the 20 nM sequence.
  • the final concentrations in the 10th, 1st and 1st and 2nd lanes at the time of recovery of whole cell lysate 10 minutes after the start of treatment are 10 nM for SEQ ID NO: 1 and ephrin B 2 respectively. 3 nM.
  • Figure 1 shows Western blotting analysis using all antibodies that recognize p44Z42MAPK, or antibodies that recognize Q? -Tubulin, regardless of whether they are phosphorylated or dephosphorylated as the primary antibody. Show photos.
  • a comparison of the first and third lanes shows that ephrin B2 inhibits p44Z42MA PK phosphorylation activity. The same can be said when comparing the 7th and 9th lanes.
  • the peptide of SEQ ID NO: 1 inhibits p44Z42MAPK phosphorylation activity by serum. The same can be said by comparing the 7th and 8th lanes.
  • FIG. 2 is a photograph of Western blotting analysis showing the result of administration of ephrin B 2 to p 44 Z42MAPK phosphorylation in Example 4.
  • the first and sixth lanes show the control
  • the second lane shows the 10 minutes after treatment with 80 nM ephrin B 2 of the present invention
  • the third lane shows the present invention.
  • the 10th minute after treatment with 30 nM ephrin B 2 is shown, the 4th lane shows 10 minutes after treatment with 9 nM ephrin B 2 of the present invention, the 5th lane 10 minutes after the treatment with 3 ⁇
  • ephrin 82 of the present invention is shown.
  • Lane 7 shows 30 minutes after treatment with 80 nM ephrin B 2 of the present invention, and Lane 8 shows 30 minutes after treatment with 30 nM ephrin B 2 of the present invention.
  • Lane 9 shows 30 minutes after treatment with 9 nM ephrin B 2 of the present invention, and Lane 10 shows 30 minutes after treatment with 3 nM ephrin B 2 of the present invention. Indicates what has passed.
  • Figure 2 shows that the same whole cell lysate as above was Western blotted using an antibody that recognizes P44Z42MAPK, regardless of whether it is phosphorylated or dephosphorylated, or an antibody that recognizes Q? -Tubulin.
  • the analyzed photograph is shown. Comparing the 1st lane with the 2nd, 3rd, 4th, and 5th lanes, it can be seen that ephrin B 2 from 3 ⁇ 1 ⁇ 1 to 80 ⁇ 1 ⁇ 1 inhibits MAPK phosphorylation activity. The same can be said by comparing the 7th lane with the 8th, 9th, 10th, 10th, 1st, 12th lanes.
  • Example 5 Example 5
  • Example 3 and Example 3 except that the amounts of ephrin B 2 and the peptide of SEQ ID NO: 1 were changed. Similarly, Western plotting was performed. Figure 3 shows p44 in Example 5.
  • FIG. 3 This is a photograph of Western blotting analysis showing the results of Ephrin B2, the peptide of SEQ ID NO: 1, and their concomitant drugs for Z42MA P K phosphorylation.
  • the first and seventh lanes show controls
  • the second lane shows 10 minutes after treatment with 100 M peptide of SEQ ID NO: 1
  • the third lane shows 3 nM.
  • 10 minutes after treatment with ephrin B 2 of the present invention is shown
  • the fourth lane shows 10 minutes after treatment with 200 M SEQ ID NO: 1 peptide and 6 nM ephrin B 2 simultaneously.
  • Lane 5 shows the treatment with 200 M of SEQ ID NO: 1 peptide, 3 minutes later, 6 n M of ephrin B 2 of the present invention, and 10 minutes after treatment.
  • Lane 6 shows treatment with 6 nM ephrin B 2 of the present invention, and 10 minutes after treatment with 200 M of SEQ ID NO: 1 peptide after 3 minutes.
  • Lane 8 shows 10 minutes after treatment with 1 M SEQ ID NO: 1 peptide
  • Lane 9 shows 10 minutes after treatment with 3 nM ephrin B 2
  • the 10th lane shows 2 M of the peptide of SEQ ID NO: 1 and 6 nM of the ephrin B 2 of the present invention at the same time after 10 minutes.
  • the 11th lane shows 2 M In the second lane, 10 minutes after the treatment with 6 nM ephrin B 2 of the present invention, and the second lane of 6 nM of the present invention was treated with the peptide of SEQ ID NO: 1. Shown is 10 minutes after treatment with ephrin B 2 and 3 minutes later with 2 M SEQ ID NO: 1 peptide.
  • the final concentration in the 4th, 5th and 6th lanes at the time of recovery of whole cell lysate 10 minutes after the start of treatment is 1 ⁇ ⁇ ⁇ ⁇ for SEQ ID NO: 1 and 3 ⁇ for ephrin ⁇ 2 respectively. It was a spear.
  • the final concentrations in the 10th, 1st and 1st and 2nd lanes at the time of recovery of whole cell lysate 10 minutes after the start of treatment were 1 ⁇ for the peptide of SEQ ID NO: 1 and 3 ⁇ for ephrin ⁇ 2, respectively. It was rare.
  • Figure 3 also shows Western blotting analysis using antibodies that recognize all ⁇ 44 ⁇ 42 ⁇ ⁇ ⁇ regardless of phosphorylated and dephosphorylated types as primary antibodies, or antibodies that recognize Q? -Tubulin. Show photos. Lane 1 And lane 3 show that ephrin B2 inhibits MAPK phosphorylation activity. The same can be said by comparing the 7th and 9th lanes. In addition, comparing the first and second lanes, it can be seen that the peptide of SEQ ID NO: 1 inhibits MAP K phosphorylation activity. The same can be said when comparing the 7th and 8th lanes.
  • FIG. 4 is a photograph obtained by Western blotting analysis showing the results of ephrin B 2 for p44Z42MAPK phosphorylation in Example 6, the peptide of SEQ ID NO: 2, and their concomitant agents.
  • the first lane shows the control
  • the second lane shows the one after 13 minutes after treatment with 100 nM peptide of SEQ ID NO: 1 of the present invention
  • the third lane shows the present invention.
  • SEQ ID NO: 2 shows the 4th lane, 1 3 minutes after treatment with 3 nM ephrin B 2 of the present invention, 5th lane Lanes show 3 minutes after treatment with 6 nM ephrin B2 of the present invention and 10 minutes after treatment with 200 nM peptide of SEQ ID NO: 1 of the present invention. Lane 6 shows the result of treatment with 6 nM ephrin B 2 of the present invention for 3 minutes after treatment with 200 nM of SEQ ID NO: 2 peptide of the present invention.
  • the final concentration in the 5th and 6th lanes at the time of recovery of the whole cell lysate 1 to 3 minutes after the start of treatment was 100 nM for SEQ ID NO: 1 and that for SEQ ID NO: 2 respectively.
  • 1 00 nM and ephrin B 2 were 3 nM.
  • FIG. 5 is a photograph obtained by Western blotting analysis showing the results of ephrin B 2 for P 44Z42MAPK phosphorylation in Example 7, the peptide of SEQ ID NO: 2, and their concomitant drugs.
  • the first lane shows the control
  • the second lane shows that 10 minutes have passed after the treatment with the peptide of SEQ ID NO: 2 of 100 ⁇ of the present invention
  • the third lane shows the present invention.
  • Lane 5 shows the lane after treatment with 200 nM of the peptide of SEQ ID NO: 1 of the present invention for 3 minutes and further treatment with 3 nM of ephrin B2 of the present invention.
  • Lane 6 shows the result of 3 minutes after treatment with 6 nM ephrin B2 of the present invention and further treatment with 200 nM of SEQ ID NO: 1 peptide of the present invention.
  • the final concentrations in the 4th, 5th, and 6th lanes at the time of recovery of whole cell lysate 10 minutes after the start of treatment were 100 nM for the peptide of SEQ ID NO: 1 and 3 nM for ephrin B 2 Met. Comparing the first and second lanes, it can be seen that the peptide of SEQ ID NO: 2 inhibits MAPK phosphorylation activity.
  • peptides, agents, adjuvants, pharmaceutical compositions and medical kits of the present invention are particularly effective for the treatment or prevention of diseases related to neovascularization in the eye, they can be used in the pharmaceutical industry and the like.

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Abstract

L'invention concerne un nouveau peptide efficace pour le traitement d'une maladie associée à une néovascularisation ou un sel acceptable du point de vue pharmacologique de celui-ci ; un agent servant à faciliter l'inhibition de la néovascularisation ; un inhibiteur de néovascularisation ; une composition à des fins médicales ; un kit à des fins médicales ; entre autres. Plus précisément, l'invention concerne un peptide comprenant la séquence d'acides aminés décrite dans la SEQ ID n° 1 ou un sel acceptable du point de vue pharmacologique de celui-ci ; une composition ou un kit à des fins médicales comprenant de l'éphrine-B2 ou un sel acceptable du point de vue pharmacologique de celle-ci et (i) un peptide comprenant la séquence d'acides aminés décrite dans la SEQ ID n° 1 ou un sel acceptable du point de vue pharmacologique de celui-ci ou (ii) un peptide comprenant une séquence d'acides aminés ayant une délétion, une substitution, une insertion ou une addition d'un ou deux résidus d'acides aminés dans la séquence d'acides aminés décrite dans la SEQ ID n° 1 et capable d'accroître l'activité d'inhibition de la néovascularisation de l'éphrine-B2 ou d'un sel acceptable du point de vue pharmacologique de celui-ci ; entre autres.
PCT/JP2007/000550 2006-05-24 2007-05-23 Peptide capable d'accroître l'activité de l'éphrine-b2, sel de celui-ci, composition à des fins médicales et kit de thérapie WO2007135781A1 (fr)

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JP2006144728A JP2009221107A (ja) 2006-05-24 2006-05-24 エフリンｂ２の活性を高めるペプチド，その塩，医薬用組成物，治療用キット

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002026827A1 (fr) * 2000-09-29 2002-04-04 Novartis Ag Polypeptides extracellulaires de recpteurs eph b et ligands ephrine b, et molecules d'acide nucleiques correspondantes
GB2376016A (en) * 2001-05-30 2002-12-04 Nadir R Farid Thyrotropin receptor (TSHR) domain cleavage catalysed by the metalloprotease ADAM 10
WO2006006079A2 (fr) * 2004-04-05 2006-01-19 Aqumen Biopharmaceuticals K.K. Procedes permettant d'eliminer la neovascularisation au moyen de l'ephrine b2
JP2006507256A (ja) * 2002-09-24 2006-03-02 ザ バーナム インスティチュート Ｅｐｈ受容体活性を調節する新規薬剤
WO2006052409A2 (fr) * 2004-10-23 2006-05-18 Case Western Reserve University Agonistes d'epha sous forme de peptides et de petites molecules et utilisations de ceux-ci
WO2006081418A2 (fr) * 2005-01-27 2006-08-03 The Burnham Institute Peptides de liaison aux recepteurs ephb

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002026827A1 (fr) * 2000-09-29 2002-04-04 Novartis Ag Polypeptides extracellulaires de recpteurs eph b et ligands ephrine b, et molecules d'acide nucleiques correspondantes
GB2376016A (en) * 2001-05-30 2002-12-04 Nadir R Farid Thyrotropin receptor (TSHR) domain cleavage catalysed by the metalloprotease ADAM 10
JP2006507256A (ja) * 2002-09-24 2006-03-02 ザ バーナム インスティチュート Ｅｐｈ受容体活性を調節する新規薬剤
WO2006006079A2 (fr) * 2004-04-05 2006-01-19 Aqumen Biopharmaceuticals K.K. Procedes permettant d'eliminer la neovascularisation au moyen de l'ephrine b2
WO2006052409A2 (fr) * 2004-10-23 2006-05-18 Case Western Reserve University Agonistes d'epha sous forme de peptides et de petites molecules et utilisations de ceux-ci
WO2006081418A2 (fr) * 2005-01-27 2006-08-03 The Burnham Institute Peptides de liaison aux recepteurs ephb

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