WO2007135781A1 - Peptide capable of enhancing the activity of ephrin-b2, salt thereof, composition for medical purposes, kit for therapy - Google Patents

Peptide capable of enhancing the activity of ephrin-b2, salt thereof, composition for medical purposes, kit for therapy 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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WIPO (PCT)
Prior art keywords
peptide
ephrin
seq
acceptable salt
pharmacologically acceptable
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PCT/JP2007/000550
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French (fr)
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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Application filed by Aqumen Biopharmaceuticals K.K., Kyushu University filed Critical Aqumen Biopharmaceuticals K.K.
Publication of WO2007135781A1 publication Critical patent/WO2007135781A1/en

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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.

Abstract

Disclosed are: a novel peptide effective for the treatment of a disease associated with neovascularization or a pharmacologically acceptable salt thereof; an agent for assisting the inhibition of neovascularization; a neovascularization inhibitor; a composition for medical purposes; a kit for medical purposes; and others. More specifically, disclosed are: a peptide comprising the amino acid sequence depicted in SEQ ID NO:1 or a pharmacologically acceptable salt thereof; a composition or kit for medical purposes comprising: ephrin-B2 or a pharmacologically acceptable salt thereof; and (i) a peptide comprising the amino acid sequence depicted in SEQ ID NO:1 or a pharmacologically acceptable salt thereof or (ii) a peptide comprising an amino acid sequence having the deletion, substitution, insertion or addition of one or two amino acid residues in the amino acid sequence depicted in SEQ ID NO:1 and capable of enhancing the neovascularization-inhibiting activity of ephrin-B2 or a pharmacologically acceptable salt thereof; and others.

Description

明 細 書  Specification
エフリン B 2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  Peptides that enhance the activity of ephrin B 2, salts thereof, pharmaceutical compositions, treatment kits
技術分野  Technical field
[0001] 本発明は， エフリン B 2の活性を高めるペプチド， その薬理学的に許容さ れる塩；エフリン B 2とそのようなぺプチドを含む医薬組成物及び医療用キ ッ卜などに関する。  [0001] 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.
背景技術  Background art
[0002] 新生血管は， 加齢黄斑変性， 糖尿病性網膜症， 及び未熟児網膜症のような 種々の眼病理症状の顕著な特徴である。 したがって， 新生血管を阻害し， こ れらの疾患を治療又は予防できる医薬が望まれる。 一方で血管が新生される ことは， 生体が正常に機能する上でも重要である。 そこで， 網膜外に向かう 病的な血管形成または血管新生を抑制し， かつ網膜中において血管網が形成 されることや， 血管が成熟することを増強するような医薬が望まれる。  [0002] 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. On the other hand, 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.
[0003] 大野京子 "加齢黄斑変性における脈絡膜新生血管の分子機構一特に色素上 皮由来因子の役割について—" 日本眼科学界雑誌 （J o u r n a l o f Op h t h a I mo I o g ι c a I S o c i e t y) Vo l . 1 0 /, N o. 1 1， p p. 657-673, 2003 (下記非特許文献 1 ) には， 以下の内容が開示されている。 "加齢黄斑変性 （AMD) は， 先進諸 国における 50代以上の失明原因の常に上位を占める重要な疾患であり， 人 口の高齢化に伴いますますその頻度は増加しつつある。 しかしながら， AM Dにおける脈絡膜新生血管 （CNV) の分子機構は未だ完全には解明されて いない。 従来から網膜色素上皮 （RPE) が産生する血管内皮増殖因子 （V EG F) の発現上昇が CNV発生に重要であるとされてきたが， トランスジ エニックマウスの結果などから V E G F単独では C N V発生には不十分であ ると考えられる。 CNVは無血管である網膜外層に新生血管が侵入していく 現象であり， この場合には正常で血管侵入を抑制している抑制因子の発現低 下の方が重要ではないかと我々は考えた。 そして， 近年明らかになった最強 の血管新生抑制因子， 色素上皮由来因子 （PED F) に注目し， 加齢や酸化 ストレスによって生じる RPEの分化状態の崩れが PED Fの発現低下を惹 起し， その結果生じる VEG Fと P ED Fのバランスの崩れが CN V発現の 引き金を引くのではないかといぅメ力二ズムを明らかにした。 " [0003] Kyoko Ohno "Molecular mechanism of choroidal neovascularization in age-related macular degeneration, especially about the role of pigment epidermis--" The Journal of Ophthalmology in Japan (Journalof Ophtha I mo Iog oca IS ociety) Vo l. 1 0 /, No. 1 1, pp. 657-673, 2003 (the following Non-Patent Document 1) discloses the following contents. “Age-related macular degeneration (AMD) is an important disease that has always been one of the leading causes of blindness in advanced countries in the 50s and up, and its frequency is increasing with the aging of the population. The molecular mechanism of choroidal neovascularization (CNV) in AM D has not yet been fully elucidated Traditionally, increased expression of vascular endothelial growth factor (VEG F) produced by retinal pigment epithelium (RPE) is important for CNV development However, VEGF alone is considered to be insufficient for CNV development from the results of transgenic mice, etc. CNV is a phenomenon in which new blood vessels invade the outer retinal layer, which is avascular. Yes, in this case, the expression of inhibitory factors that are normal and suppress vascular invasion is low. We thought the bottom was more important. Focusing on the strongest angiogenesis-inhibiting factor, pigment epithelium-derived factor (PED F), revealed in recent years, the disruption of the differentiation state of RPE caused by aging and oxidative stress causes a decrease in the expression of PED F. It was clarified that the resulting imbalance between VEG F and PEDF might trigger CN V expression. "
[0004] T. Ko n d o e t a に ， "PK C/M A Ρ Κ S i n a l i n g S u p p r e s s i o n b y Re t i n a l P e r i c y t e Co n d i t i o n e d Me d i um P r e v e n t s Re t i n a I En d o t h e l i a l Ce l l P r o l i f e r a t i o n" , J o u r n a l o f e e l I u I a r p h y s i o l o g y, 203 ， p p. 378-386, 2005 (下記非特許文献 2 ) の F i g 6では， P 44Z42MAP Kの抑制が， 細胞増殖及び脈絡膜の血管新生の抑制と強 い相関性があることが示されている。  [0004] T. Kondoeta, “PK C / MA Κ S inaling S uppression by Re tinal P ericyte Coordinated Me di um Prevents Re tina I En dothelial Cell Proliferation”, J ournalofeel I u I arphysiology, 203 , P p. 378-386, 2005 (Non-patent Document 2 below) shows that inhibition of P44Z42MAP K is strongly correlated with inhibition of cell proliferation and choroidal neovascularization. Yes.
[0005] WO 2006/006079号公報 （下記特許文献 1 ) には， エフリン Β 2が， 眼内における動脈及び静脈内皮細胞の DN Α合成の阻害作用を有する こと；エフリン B 2が， 眼内における動脈及び静脈内皮細胞における p 44 Zp 42MAPK活性化の阻害作用を有すること；ェフリン B 2が， 眼内に おける動脈及び静脈内皮細胞における管形成の阻害作用を有することが示さ れている。 同文献では， その実施例 3において， CNV (脈絡膜新生血管） モデルを用いてエフリン B 2が加齢黄斑変性 （AMD) の治療に有効である ことが示されている。 しかしながら， 本出願の配列番号 1〜配列番号 3で表 されるぺプチドは開示されていない。  [0005] WO 2006/006079 (Patent Document 1) discloses that ephrin-2 has an inhibitory effect on the synthesis of DN の 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. In this document, Example 3 shows that ephrin B 2 is effective in treating age-related macular degeneration (AMD) using the CNV (choroidal neovascularization) model. However, the peptides represented by SEQ ID NO: 1 to SEQ ID NO: 3 of the present application are not disclosed.
[0006] エフリン B2に関して， さらに以下の文献が知られている。 W02002 Z26827号公報 （特許文献 2) には， エフリン B 2が開示されている。 そして， エフリンを目の血管新生の遺伝子治療剤として用いるとの記載があ る （1頁 4行目〜 1 2行目） 。 しかし， 特許文献 2は， エフリン B 2が新生 血管の成長を抑圧するものであるのか， 正常血管網の形成を促進するもので あるかについては何らの示唆もない。 また， 特許文献 2は， エフリン B 2の 実施例として， 腫瘍細胞の阻害実験しかしていないので， エフリン B 2と新 生血管との関係や， エフリン B 2と正常血管との関係などについては何ら実 証されていない。 まして， 本出願の配列番号 1〜配列番号 3で表されるぺプ チドは開示されていない。 [0006] The following documents are also known regarding ephrin B2. W02002 Z26827 (Patent Document 2) discloses ephrin B2. There is a description that ephrin is used as a gene therapy for angiogenesis of the eye (page 1, lines 4 to 12). However, 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. In addition, 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. In addition, the peptides represented by SEQ ID NO: 1 to SEQ ID NO: 3 of the present application are not disclosed.
[0007] 特表平 1 0— 50281 0号公報 （特許文献 3) の LERK—5は， エフ リン B2に相当するものと考えられる。 そして， 特許文献 3の実施例 1では ， ヒトエフリン B 2の c DN Aが単離され， 実施例 7ではヒトエフリン B 2 (細胞外ドメイン） と F cの融合タンパク質がレセプタ一のリン酸化を刺激 することを i n V i t r o (試験管内）実験で実証している。 しかしながら ， 特許文献 3では， エフリン B2を神経性疾病へ用いることのみが意図され ており （21頁 23行目〜 22頁 2行目） ， 新生血管関連疾患への用途は記 載されていない。 まして， 本出願の配列番号 1〜配列番号 3で表されるぺプ チドは開示されていない。  [0007] LERK-5 in Japanese Patent Publication No. 10-502810 (Patent Document 3) is considered to correspond to ephrin B2. In 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. However, 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. In addition, the peptides represented by SEQ ID NO: 1 to SEQ ID NO: 3 of the present application are not disclosed.
[0008] 特表平 1 0— 501 701号公報 （特許文献 4) の " H t kリガンド" は ， エフリン B2に相当するものと推定される。 特許文献 4では， H t kリガ ンドの用途として， 神経変性の治療があげられているが （60頁 7行目〜 6 3頁 9行目） ， H t kリガンドを新生血管など抑制などに用いる点は記載も 示唆もされていない。 まして， 本出願の配列番号 1〜配列番号 3で表される ぺプチドは開示されていない。  [0008] 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.
[0009] 特表 2002— 51 1 41 7号公報 （特許文献 5) には， エフリン B2が ， 腫瘍による血管新生を増強等するとの記載がある（特許文献 5の段落 [00 62] )。 よって， エフリン B2を， 新生血管を阻害するために用いることに ついて動機付けとなるものはない。 まして， 本出願の配列番号 1〜配列番号 3で表されるぺプチドは開示されていない。  [0009] 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.
特許文献 1 ： WO 2006/006079号公報  Patent Document 1: WO 2006/006079 Publication
特許文献 2： W02002Z26827号公報  Patent Document 2: W02002Z26827 Publication
特許文献 3：特表平 1 0— 50281 0号公報  Patent Literature 3: Japanese Patent Publication No. 10-50281 0
特許文献 4：特表平 1 0— 501 701号公報 特許文献 5：特表 2002-51 1 41 7号公報 Patent Document 4: Japanese Patent Publication No. 10-501 701 Patent Document 5: Special Table 2002-51 1 41 7
非特許文献 1 ：大野京子 "加齢黄斑変性における脈絡膜新生血管の分子機構一 特に色素上皮由来因子の役割について一" 日本眼科学界雑誌 Vo に 1 0 7, N o. 1 1， p p. 657-673, 2003  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
非特許文献 2： T. Ko n d o e t a に ， "PK CZM A Ρ Κ S i g n a I i n g S u p p r e s s i o n b y Re t i n a l P e r i c y t e Co n d i t i o n e d Me d i um P r e v e n t s R e t i n a l En d o t h e l i a l Ce l l P r o l i f e r a t i o n" , J o u r n a l o f e e l I u I a r p h y s i o I o g y， 203, p p. 378-386, 2005  Non-Patent Document 2: T. Kondoeta, "PK CZM A Ρ 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
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0010] 本発明は， 新生血管が関連する疾患の治療に有効な新規べプチド又はその 薬理学的に許容される塩などを提供することを目的とする。 [0010] 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.
[0011] 本発明は， エフリン B 2などの活性を高め， 新生血管が関連する疾患など エフリン B 2が関与する疾患の治療に有効な新規べプチドなどを提供するこ とを目的とする。 本発明は， そのような新規ペプチドなどを含有する新生血 管阻害剤や， エフ、 Jン B 2の新生血管阻害能を高める新生血管阻害用補助剤 などを提供することを目的とする。 [0011] 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.
[0012] 本発明は， エフリン B 2などと上記したペプチドなどを含む新生血管の阻 害などに有効な， 医薬組成物などを提供することを目的とする。 [0012] 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.
[0013] 本発明は， エフリン B 2などを含有する第 1の組成物と， 上記したぺプチ ドなどを含む第 2の組成物とを含む， 新生血管の阻害などに有効な， 医療用 キッ卜を提供することを目的とする。 [0013] 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.
課題を解決するための手段  Means for solving the problem
[0014] 本発明は， 基本的には， 配列番号 1などの低分子ポリペプチドをエフリン B 2とあわせて用いることで， エフリン B 2の新生血管阻害活性などを高め ることができるという知見に基づくものである。 さらに， 本発明は， 配列番 号 1などの低分子ポリべプチドとエフリン B 2を別々に投与すると， これら を同時に投与した場合に比べて， エフリン B 2の新生血管阻害活性を著しく 高めることができるという知見に基づくものである。 [0014] 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. .
[0015] 本発明の第 1の側面は， 基本的には， 配列番号 1〜配列番号 3 (好ましく は配列番号 1又は 2 ) で表されるアミノ酸配列からなるペプチド， 又はその 薬理学的に許容される塩に関する。  [0015] 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.
[0016] 後述する実施例によって実証されたとおり， 上記のペプチドは， それ自体 M A P Kリン酸化阻害能を有する。 また， 上記のペプチドは， エフリン B 2 と併用することでエフリン B 2の M A P Kリン酸化阻害能を飛躍的に高める 作用を有する。 このことは， エフリン B 2が有する， 血管内皮細胞における 新生血管の抑制能を高めることを意味する。  [0016] As demonstrated by the examples described below, the above peptides themselves have the ability to inhibit M A P K phosphorylation. In addition, 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.
[0017] 本発明の第 1の側面のある態様は， 配列番号 1又は配列番号 2において，  [0017] In an embodiment of the first aspect of the present invention, in SEQ ID NO: 1 or SEQ ID NO: 2,
1又は 2個のアミノ酸が欠失， 置換， 揷入若しくは付加されたアミノ酸配列 からなリ，エフリン B 2の新生血管の阻害活性を高めるぺプチド， 又はその薬 理学的に許容される塩に関する。  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.
[0018] このように， 実施例によって実証された配列番号 1又は配列番号 2で表さ れるアミノ酸配列からなるぺプチドと実質的に同様なぺプチドも， エフリン B 2の活性を高める作用を有する。 より具体的には， 配列番号 3を含むぺプ チドであって， ある程度の残基数のものは， エフリン B 2の新生血管阻害活 性を著しく高めるといえる。  [0018] Thus, 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.
[0019] 本発明の第 1の側面の好ましい態様は， 上記いずれかに記載のぺプチド， 又はそれらの薬理学的に許容される塩を含有する特に眼内の新生血管阻害用 補助剤である。 この補助剤の好ましい態様は， 主剤がエフリン B 2を含有す る新生血管阻害剤である。  [0019] 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.
[0020] 本発明の第 1の側面の好ましい態様は， 上記いずれかに記載のぺプチド， 又はそれらの薬理学的に許容される塩を含有する， 血管内皮細胞における D N A合成阻害用補助剤である。 この補助剤の好ましい態様は， 主剤としてェ フリン B 2を含有する剤を用いるものである。 [0021 ] 本発明の第 1の側面の好ましい態様は， 上記いずれかに記載のぺプチド， 又はそれらの薬理学的に許容される塩を含有する， 血管内皮細胞における p 4 4 Z p 4 2 M A P Kリン酸化活性阻害用補助剤である。 この補助剤の好ま しい態様は， 主剤としてエフリン B 2を含有する剤を用いるものである。 [0020] 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. [0021] 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.
[0022] 本発明の第 1の側面の好ましい態様は， 上記いずれかに記載のぺプチド， 又はそれらの薬理学的に許容される塩を含有する， 血管形成または血管新生 に関連する疾患または障害の治療剤である。 本発明の第 1の側面の好ましい 態様は， 上記いずれかに記載のペプチド， 又はそれらの薬理学的に許容され る塩を含有する新生血管阻害剤である。 この新生血管阻害剤は， 上記いずれ かに記載のぺプチドを有効成分として有効量含むものである。 この新生血管 阻害剤として， 眼内の新生血管阻害剤が好ましく， 眼内の新生血管に関連す る疾患の治療剤又は予防剤が好ましい。 また， 後述する実施例によって実証 されたとおり， 本発明のペプチドは， 血管形成を阻害し， 又は血管新生を阻 害する。 よって， 上記ペプチド等は， これらに関連する疾患又は障害の治療 剤として有効であるといえる。 眼内の血管形成または血管新生に関連する疾 患または障害として， "加齢黄斑変性， 虚血性網膜症， 眼内血管新生， 角膜 血管新生， 網膜血管新生， 脈絡膜血管新生， 糖尿病性黄斑浮腫， 糖尿病性網 膜虚血， 糖尿病性網膜浮腫， 糖尿病性網膜症" からなる群から選択される， 1種又は 2種以上があげられる。 眼内以外の血管形成または血管新生に関連 する疾患または障害として， 腫瘍， 慢性関節リウマチ， 乾癬， ァテローム性 動脈硬化症， 力ポジ肉腫のような疾患， 及び固形癌の転移があげられる。 こ れの剤は， 上記いずれかに記載のペプチド， 又はそれらの薬理学的に許容さ れる塩を有効成分として含むものが好ましぐ 主剤としてエフリン B 2を含 有する剤を用いるものであってもよい。  [0022] 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. In addition, as demonstrated by the examples described later, 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”. 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.
[0023] 本発明は， 主剤としてエフリン B 2を含有する剤をヒト又は非ヒト哺乳類 に投与する工程と， 前記工程の前又は後 （好ましくは後） に， 上記いずれか のべプチド， 又はその薬理学的に許容される塩をヒ卜又は非ヒ卜哺乳類に投 与する眼内の新生血管に関連する疾患の治療方法をも提供する。 本発明は， 主剤としてエフリン B 2を含有する剤をヒ卜又は非ヒ卜哺乳類に投与するェ 程と， 前記工程の前又は後 （好ましくは後） に， 配列番号 1又は配列番号 2 において， 1又は 2個のアミノ酸が欠失， 置換， 挿入若しくは付加されたァ ミノ酸配列からなり，エフリン B 2の新生血管の阻害活性を高めるぺプチド， 又はその薬理学的に許容される塩をヒ卜又は非ヒ卜哺乳類に投与する眼内の 新生血管に関連する疾患の治療方法をも提供する。 [0023] 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.
[0024] 本発明は， さらに， ヒト又は非ヒト哺乳類の新生血管阻害用補助剤 （好ま しくは， 眼内の新生血管阻害用補助剤） を製造するための上記いずれかのぺ プチド， 又はその薬理学的に許容される塩の使用をも提供する。 本発明は， 眼内の新生血管阻害用補助剤を製造するための配列番号 1又は配列番号 2に おいて， 1又は 2個のアミノ酸が欠失， 置換， 挿入若しくは付加されたアミ ノ酸配列からなり，エフリン B 2の新生血管の阻害活性を高めるぺプチド， 又 はその薬理学的に許容される塩の使用をも提供する。  [0024] 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.
[0025] 本発明の第 2の側面は， 基本的には， エフリン B 2又はその薬理学的に許 容される塩と； （ i )配列番号 1で表されるアミノ酸配列からなるぺプチド， もしくはその薬理学的に許容される塩， 又は（ i i )配列番号 1において， 1 又は 2個のアミノ酸が欠失， 置換， 揷入若しくは付加されたアミノ酸配列か らなり，前記エフリン B 2の新生血管の阻害活性を高めるぺプチド， もしくは それらの薬理学的に許容される塩と； を含有する医薬用組成物に関する。 す なわち， 本発明の第 2の側面は， 基本的には， エフリン B 2又はその薬理学 的に許容される塩と；上記したいずれかのぺプチド又はその薬理学的に許容 される塩とを含有する医薬組成物に関する。 後述する実施例によって実証さ れたとおり， 本発明のペプチドは， エフリン B 2と併用することで， エフリ ン B 2の活性を著しく高める。 よって， 本発明の第 2の側面に係る医薬組成 物は， 血管形成を効果的に阻害し， 又は血管新生を効果的に阻害するので， 血管形成や新生血管が関与する疾患や障害 （特に眼内の新生血管が関与する 疾患や障害） の治療又は予防に効果的に用いることができる。  [0025] 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. That is, 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 pharmaceutical composition containing these. As demonstrated by the examples described later, 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.
[0026] 本発明の第 2の側面は， エフリン B 2又はその薬理学的に許容される塩と ； ( i )配列番号 2で表されるアミノ酸配列からなるペプチド， もしくはその 薬理学的に許容される塩， 又は（ i i )配列番号 2において， 1又は 2個のァ ミノ酸が欠失， 置換， 挿入若しくは付加されたアミノ酸配列からなり，前記ェ フリン B 2の新生血管の阻害活性を高めるぺプチド， もしくはそれらの薬理 学的に許容される塩と； を含有する医薬用組成物に関する。 [0026] The second aspect of the present invention relates to ephrin B 2 or a pharmaceutically acceptable salt thereof. (I) 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:
[0027] 本発明の第 2の側面は， 更に， エフリン B 2又はその薬理学的に許容され る塩と；配列番号 3で表されるアミノ酸配列を具備するべプチドであって， 当該ペプチドに含まれるアミノ酸残基の数が 9以上， 1 0 0以下であるぺプ チド， 又はその薬理学的に許容される塩とを含有する医薬用組成物に関する [0027] 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.
[0028] 後述する実施例によって実証されたとおり， 上記のペプチドは， エフリン B 2と併用することでエフリン B 2の M A P Kリン酸化阻害能を飛躍的に高 める作用を有する。 このことは， エフリン B 2が有する， 血管内皮細胞にお ける新生血管の抑制能を高めることを意味する。 よって， エフリン B 2と上 記のぺプチドなどを含有する医薬用組成物は， 特に眼内の新生血管の抑制な どに効果的に用いることができるものと考えられる。 [0028] As demonstrated by the examples described later, 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. This means that 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.
[0029] 本発明の第 2の側面の好ましい態様は， 前記エフリン B 2が， 天然のエフ リン B 2の細胞外ドメインを含み， 細胞質ドメインを含まない， 上記に記載 の医薬用組成物に関する。 天然のエフリン B 2の細胞外ドメインを含み， 細 胞質ドメインを含まない領域が， E p h B 4のファーマコフォアに作用する 部位であると考えられるので， そのような部位を含むエフリン B 2であれば ， 全長でなくても新生血管の抑制能を有すると考えられる。  [0029] 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.
[0030] 本発明の第 2の側面の好ましい態様は， 前記エフリン B 2が， ヒト F cと エフリン B 2の融合タンパク質である上記いずれかに記載の医薬用組成物に 関する。 エフリン B 2が新生血管の抑制能を発揮するためには， エフリン B 2が二量体化していることが望ましいと考えられ， そのためヒ卜 F cとエフ リン B 2との融合タンパク質であれば， エフリン B 2が二量化し， 高い新生 血管の抑制能を発揮しうる。 [0031 ] 本発明の第 2の側面の好ましい態様は， 新生血管阻害剤， 特に眼内の新生 血管阻害剤である上記いずれかに記載の医薬用組成物に関する。 本発明の第 2の側面の好ましい態様は， 眼内の脈絡膜における新生血管阻害剤である上 記いずれかに記載の医薬用組成物に関する。 [0030] 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. 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. [0031] 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.
[0032] 本発明の第 2の側面の好ましい態様は， 眼内の新生血管に関連する疾患の 治療■予防剤である上記いずれかに記載の医薬用組成物に関する。 より具体 的に説明すると， 本発明の第 2の側面の好ましい態様は， 血管形成または血 管新生に関連する疾患または障害の治療剤である上記いずれかに記載の医薬 用組成物に関する。 本発明の第 2の側面の好ましい態様は， "加齢黄斑変性 ， 虚血性網膜症， 眼内血管新生， 角膜血管新生， 網膜血管新生， 脈絡膜血管 新生， 糖尿病性黄斑浮腫， 糖尿病性網膜虚血， 糖尿病性網膜浮腫， 糖尿病性 網膜症" からなる群から選択される， 1又は 2種以上の疾患の治療，予防剤 である上記いずれかに記載の医薬用組成物に関する。 特に好ましい態様は， 加齢黄斑変性及び糖尿病性網膜症のいずれか又は両方の疾患の治療■予防剤 である。  [0032] 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. Or a diabetic retinal edema, or a diabetic retinopathy. The pharmaceutical 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.
[0033] 本発明の第 2の側面の好ましい態様は， 腫瘍， 慢性関節リウマチ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫， 又は固形癌の転移の治療剤である上 記いずれかに記載の医薬用組成物に関する。  [0033] 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.
[0034] 本発明は， エフリン B 2又はその薬理学的に許容される塩と；上記したい ずれかのぺプチド又はその薬理学的に許容される塩； をヒ卜又は非ヒ卜哺乳 動物に投与する， 眼内の新生血管に関連する疾患の治療方法をも提供する。  [0034] 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.
[0035] 本発明は， 眼内の新生血管に関連する疾患の治療剤を製造するための， ェ フリン B 2又はその薬理学的に許容される塩と；上記したいずれかのぺプチ ド又はその薬理学的に許容される塩の使用をも提供する。  [0035] 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.
[0036] 本発明の第 3の側面は， 基本的には， エフリン B 2又はその薬理学的に許 容される塩， 及び薬理学的に許容される担体を含有する第 1の組成物と； （ i )配列番号 1で表されるアミノ酸配列からなるぺプチド， もしくはその薬理学 的に許容される塩， 又は（ i i )配列番号 1において， 1又は 2個のアミノ酸 が欠失， 置換， 挿入若しくは付加されたアミノ酸配列からなり，前記エフリン B 2の新生血管の阻害活性を高めるぺプチド， もしくはそれらの薬理学的に 許容される塩， 及び薬理学的に許容される担体を含有する第 2の組成物とを 含有する医療用キットに関する。 すなわち， 本発明の第 3の側面は， 基本的 には， エフリン B 2又はその薬理学的に許容される塩， 及び薬理学的に許容 される担体を含有する第 1の組成物と；上記したいずれかのぺプチド又はそ の薬理学的に許容される塩及び薬理学的に許容される担体を含有する第 2の 組成物とを含有する医療用キッ卜に関する。 [0036] 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. (I) 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. That is, 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.
[0037] 上記の医療用キットは， 基本的には， 眼内の新生血管の抑制のための第 1 の組成物を主剤とし， 第 2の組成物を補助剤として含むものである。 後述す る実施例によって実証されたとおり， 上記のペプチドは， エフリン B 2と併 用することでエフリン B 2の M A P Kリン酸化阻害能を飛躍的に高める作用 を有する。 このことは， エフリン B 2が有する， 血管内皮細胞における新生 血管の抑制能を高めることを意味する。 さらに， 後述する実施例によって実 証されたとおり， 第 1の組成物を投与した後に第 2の組成物を投与すると M A P Kの活性を著しく阻害することが示された。 よって， 本発明のキットは ， 特にそのような投与方法に有効に利用されうる。  [0037] 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. As demonstrated by the examples described below, 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. Furthermore, as demonstrated by the examples described below, it was shown that 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.
[0038] 本発明の第 3の側面の好ましい態様は， 前記エフリン B 2が， 天然のエフ リン B 2の細胞外ドメインを含み， 細胞質ドメインを含まない， 上記に記載 のキットに関する。 本発明の第 3の側面の好ましい態様は， 前記エフリン B 2が， ヒ卜 F cとエフリン B 2の融合タンパク質である上記いずれかに記載 のキッ卜に関する。  [0038] 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.
[0039] 本発明の第 3の側面の好ましい態様は， 新生血管阻害用， 特に眼内の新生 血管阻害用に用いられる上記いずれかに記載のキッ卜に関する。 本発明の第 3の側面の好ましい態様は， 眼内の脈絡膜における新生血管阻害用に用いら れる上記いずれかに記載のキッ卜に関する。  [0039] 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.
[0040] 本発明の第 3の側面の好ましい態様は， 新生血管， 特に眼内の新生血管に 関連する疾患の治療又は予防用に用いられる上記いずれかに記載のキッ卜に 関する。 本発明の第 3の側面の好ましい態様は， 血管形成または血管新生に 関連する疾患または障害の治療用に用いられる上記いずれかのキッ卜に関す る。 本発明の第 3の側面の好ましい態様は， 加齢黄斑変性， 虚血性網膜症， 眼内血管新生， 角膜血管新生， 網膜血管新生， 脈絡膜血管新生， 糖尿病性黄 斑浮腫， 糖尿病性網膜虚血， 糖尿病性網膜浮腫， 糖尿病性網膜症" からなる 群から選択される， 1又は 2種以上の疾患の治療用又は予防用に用いられる 上記いずれかに記載のキッ卜に関する。 本発明の第 3の側面の好ましい態様 は， 加齢黄斑変性及び糖尿病性網膜症のいずれか又は両方の疾患の治療用又 は予防用に用いられる上記いずれかに記載のキッ卜に関する。 [0040] 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. Related. 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.
[0041 ] 本発明の第 3の側面の好ましい態様は， 腫瘍， 慢性関節リウマチ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫， 固形癌の転移， その他の血管形成ま たは血管新生に関連する疾患または障害の治療用キッ卜に関する。  [0041] 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.
[0042] 本発明の第 3の側面の好ましい態様は， 前記第 1の組成物がヒ卜又は非ヒ 卜哺乳動物に投与された後に， 前記第 2の組成物がヒ卜又は非ヒ卜哺乳動物 に投与される上記いずれかに記載のキッ卜に関する。 本発明の第 3の側面の 好ましい態様は， 前記第 1の組成物が投与された後， 3 0秒後〜 2 4時間後 ， 好ましくは 1分後〜 1 2時間後， より好ましくは 1分後〜 1 0分後に前記 第 2の組成物が投与される上記いずれかに記載のキッ卜に関する。  [0042] 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. The kit according to any one of the above, which is administered to an animal. 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 kit according to any one of the above, wherein the second composition is administered after ˜10 minutes.
[0043] 本発明は， エフリン B 2又はその薬理学的に許容される塩， 及び薬理学的 に許容される担体を含有する第 1の組成物と；上記いずれかのぺプチド又は その塩 （例えば， （ i )配列番号 1又は配列番号 2で表されるアミノ酸配列か らなるペプチド， もしくはその薬理学的に許容される塩， 又は（ i i )配列番 号 1又は配列番号 2において， 1又は 2個のアミノ酸が欠失， 置換， 挿入若 しくは付加されたアミノ酸配列からなり，前記ェフリン B 2の新生血管の阻害 活性を高めるペプチド， もしくはそれらの薬理学的に許容される塩） ， 及び 薬理学的に許容される担体を含有する第 2の組成物とを用い， 前記第 2の組 成物を投与した後に前記第 1の組成物を投与する新生血管に関連する疾患の 治療方法をも提供する。 発明の効果 [0043] The present invention includes a first composition comprising ephrin B2 or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable carrier; For example, (i) 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 (ii) 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), and And 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 invention's effect
[0044] 後述する実施例により実証されたとおり， 本発明によれば， 新生血管が関 連する疾患の治療に有効な新規べプチドなどを提供できる。  [0044] As demonstrated by the examples described later, according to the present invention, it is possible to provide a novel peptide and the like effective in the treatment of diseases associated with neovascularization.
[0045] 上記のとおりであるから， 本発明によれば， エフリン B 2の活性を高め， 新生血管が関連する疾患の治療に有効な新規べプチドなどを提供できる。  [0045] As described above, according to the present invention, it is possible to increase the activity of ephrin B 2 and provide a novel peptide and the like that are effective in the treatment of diseases associated with new blood vessels.
[0046] 後述する実施例により， エフリン B2と配列番号 1から配列番号 3で表さ れるペプチドなどを含む医薬組成物は， それらを組み合わせることで， それ ぞれを単独に投与した場合に比べて， 高い新生血管の阻害活性を得ることが できることが示された。 よって， 本発明によれば， エフリン B 2と所定のぺ プチドなどを含む新生血管の阻害などに有効な， 医薬組成物を提供できる。  [0046] According to the examples described below, 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.
[0047] 後述する実施例により， エフリン B 2を投与した後に， 配列番号 1又は配 列番号 2で表されるぺプチドを投与することで， 効果的に新生血管を阻害で きることが実証された。 よって， 本発明によれば， エフリン B 2を含有する 第 1の組成物と， 所定のペプチドなどを含む第 2の組成物とを含む， 新生血 管の阻害などに有効な， 医療用キットを提供できる。  [0047] The examples described later demonstrate that neovascularization can be effectively inhibited by administering the peptide represented by SEQ ID NO: 1 or SEQ ID NO: 2 after administering ephrin B2. It was. Therefore, according to the present invention, there is provided 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.
図面の簡単な説明  Brief Description of Drawings
[0048] [図 1]図 1は， 実施例 3における p 44Z42MAPKリン酸化に対するエフ リン B 2， 配列番号 1のべプチド， 及びそれらの併用剤の結果を示す図面に 替わるウェスタンプロッティングの写真である。  [0048] [FIG. 1] 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.
[図 2]図 2は， 実施例 4における p 44Z42MAPKリン酸化に対するエフ リン B 2を投与した結果を示すウェスタンブロッテイング解析した図面に替 わる写真である。  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.
[図 3]図 3は， 実施例 5における p 44Z42MAPKリン酸化に対するエフ リン B2， 配列番号 1のペプチド， 及びそれらの併用剤の結果を示すウェス タンブロッティング解析した図面に替わる写真である。  [FIG. 3] 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.
[図 4]図 4は， 実施例 6における p 44Z42MAPKリン酸化に対するエフ リン B2， 配列番号 1のペプチド， 配列番号 2のペプチド， 及びそれらの併 用剤の結果を示すウェスタンブロッテイング解析した図面に替わる写真であ る。 [FIG. 4] 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
[図 5]図 5は， 実施例 7における p 4 4 Z 4 2 M A P Kリン酸化に対するエフ リン B 2， 配列番号 1のペプチド， 配列番号 2のペプチド， 及びそれらの併 用剤の結果を示すウェスタンブロッテイング解析した図面に替わる写真であ る。  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.
[図 6A]図 6は， 配列番号 4に係るぺプチドのアミノ酸配列及びこのべプチド をコードする遺伝子の塩基配列を示す図であり， 図 6 Aは配列番号 4に示さ れるアミノ酸の _ 2 5番目から 1 6 7番目までのアミノ酸を示す。 図中， 四 角で囲った部分は配列番号 1で示されるぺプチド部分である。  [FIG. 6A] 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. In the figure, the boxed part is the peptide part shown in SEQ ID NO: 1.
[図 6B]図 6 Bは， 図 6 Aの続きを示す。  [Figure 6B] Figure 6B shows the continuation of Figure 6A.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0049] [本発明のペプチド， 剤及び補助剤]  [0049] [Peptides, Agents and Adjuvants of the Present Invention]
本発明の第 1の側面は， 基本的には， "配列番号 1で表されるアミノ酸配 列からなるペプチド" ， "配列番号 1において， 1又は 2個のアミノ酸が欠 失， 置換， 挿入若しくは付加されたアミノ酸配列からなり，エフリン B 2の新 生血管の阻害活性を高めるぺプチド， 又はそれらの薬理学的に許容される塩 " ， "配列番号 2で表されるアミノ酸配列からなるペプチド， 又はその薬理 学的に許容される塩" ， "配列番号 2において， 1又は 2個のアミノ酸が欠 失， 置換， 挿入若しくは付加されたアミノ酸配列からなり，エフリン B 2の新 生血管の阻害活性を高めるぺプチド， 又はその薬理学的に許容される塩" ， 又は "配列番号 3で表されるアミノ酸配列を具備するペプチドであって， 当 該ペプチドに含まれるアミノ酸残基の数が 9以上， 1 0 0以下であるべプチ ド， 又はその薬理学的に許容される塩" に関する。 以下， これらをまとめて "本発明のペプチド" ともよぶ。 本発明のペプチドは， 自ら新生血管の阻害 能を有するほか， エフリン B 2の活性を高める働きを有する補助剤として機 能する。 そして， エフリン B 2は， 新生血管の発生を阻害するので， 本発明 のべプチドは， 新生血管が関与する疾患の治療又は予防用に有用である。  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 salt thereof ". Hereinafter, these are collectively referred to as “the peptide of the present invention”. 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.
[0050] 本発明のペプチドのうち， 配列番号 1において 1又は 2個のアミノ酸が欠 失， 置換， 挿入若しくは付加されたアミノ酸配列からなるペプチドは， 配列 番号 1で表されるぺプチドと同様の活性を有するものである。 このようなぺ プチドは， 容易に製造できるし， また， その活性も本明細書における実施例 と同様にして行うことで容易に確かめることができる。 配列番号 1で表され るアミノ酸配列において 2位から 1 0位の部分 （配列番号 3 ) が G _ Hルー プを構成する部位であると考えられ， しかもこの部位が作用部位であると推 測される。 従って， "配列番号 1において 1又は 2個のアミノ酸が欠失， 置 換， 挿入若しくは付加されたアミノ酸配列からなるペプチド" として， 配列 番号 1で表されるアミノ酸配列において 2位から 1 0位の部分以外の部位に おけるアミノ酸が 1又は 2個欠失， 置換， 挿入若しくは付加されたアミノ酸 配列からなるペプチドであることが好ましい。 また， "配列番号 2で表され るアミノ酸配列からなるペプチド， 又はその薬理学的に許容される塩" の有 効性は， 後述する実施例において実証されたとおりである。 そして， "配列 番号 2において， 1又は 2個のアミノ酸が欠失， 置換， 挿入若しくは付加さ れたァミノ酸配列からなり，エフリン B 2の新生血管の阻害活性を高めるぺプ チド， 又はその薬理学的に許容される塩" についても， 配列番号 3で示され るアミノ酸残基が保存され， それ以外の部位について 1又は 2個のアミノ酸 が欠失， 置換， 挿入若しくは付加されたアミノ酸配列からなるものが好まし い。 なお， 配列番号 1で表されるアミノ酸配列において 2位から 1 0位の部 分 （配列番号 3 ) を含めば， 適宜公知のアミノ酸配列が付加されてもよぐ 例えば 5から 1 0 0個のアミノ酸残基からなるものであっても， 1 0から 2 0個のアミノ酸残基からなるものであってもよい。 なお， このようなぺプチ ドは， 例えば， 配列番号 4で表されるエフリン B 2のアミノ酸配列から前記 した部位を含む部分ペプチド （例えば 5から 1 0 0個のアミノ酸残基， 好ま しくは 1 0から 3 0個のアミノ酸残基， より好ましくは 1 0から 2 0個のァ ミノ酸残基） を取り出すことによって得ることもできる。 さらに， そのよう にして取り出したエフリン B 2の部分ペプチドから， 1又は数個 （具体的に は， 2 , 3 , 4 ,又は 5個） のアミノ酸が欠失， 置換， 挿入若しくは付加された アミノ酸配列からなるぺプチドであって， 本発明のぺプチドと同様の活性を 有するものであってもよい。 「本発明のペプチドと同様の活性」 とは， 例え ば， 眼内の新生血管の抑制能や， エフリン B 2の補助剤としての能力などを 有するものを意味する。 なお， 配列番号 5で示される塩基配列は， 配列番号 4で示されるエフリン B 2をコードする DN Aの塩基配列である。 [0050] Among the peptides of the present invention, SEQ ID NO: 1 lacks 1 or 2 amino acids. The peptide consisting of the amino acid sequence deleted, substituted, inserted or added has the same activity as the peptide represented by SEQ ID NO: 1. Such a peptide can be easily produced, and its activity can be easily confirmed by carrying out in the same manner as in the examples in this specification. In the amino acid sequence represented by SEQ ID NO: 1, the portion from position 2 to 10 (SEQ ID NO: 3) is considered to be the site that constitutes the G_H loop, and it is estimated that this site is the site of action. Is done. Therefore, as “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. And “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. Regarding the “physically acceptable salt”, 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. In addition, if 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). In addition, 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.
[0051] 本発明のペプチド （ポリペプチド） は， 左端が N末端 （ァミノ末端） ， 右 端が C末端 （カルボキシル末端） として表記される。 本発明のペプチドは， C末端がカルボキシル基 （_COOH) ， カルボキシレート （_COO_) ， アミド （_CON H2) 又はエステル （一COOR) のいずれであってもよ い。 ここでエステルにおける Rとして， 例えば， メチル， エーテル， n—プ 口ピル， イソプロピル， n _ブチルなどの d-6アルキル基などがあげられる 。 本発明のペプチドが， C末端以外にカルボキシル基 （またはカルボキシレ —卜） を有している場合， そのカルボキシル基がアミド化またはエステル化 されていてもよい。 さらに， 本発明のペプチドは， N末端のアミノ酸残基の ァミノ基が保護基 （例えば， ホルミル基， ァセチル基などの アルカノィ ルなどの C _6ァシル基など） で保護されているものなどであってもよい。 [0051] 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. In the peptide of the present invention, the C-terminus may be any of a carboxyl group (_COOH), a carboxylate (_COO_), an amide (_CON H 2 ), or an ester (one COOR). Here, examples of R in the ester include d-6 alkyl groups such as methyl, ether, n-propyl, isopropyl, and n_butyl. When the peptide of the present invention has a carboxyl group (or carboxylate group) other than the C-terminus, the carboxyl group may be amidated or esterified. Furthermore, 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) May be.
[0052] 本発明のペプチドにおける "薬理学的に許容される塩" として， 無機酸 （ 例えば， 塩酸， リン酸， 臭化水素酸， 硫酸） との塩， 又は有機酸 （例えば， 酢酸， ギ酸， プロピオン酸， フマル酸， マレイン酸， コハク酸， 酒石酸， ク ェン酸， リンゴ酸， 蓚酸， 安息香酸， メタンスルホン酸， ベンゼンスルホン 酸） との塩などがあげられる。 [0052] As the "pharmacologically acceptable salt" in the peptide of the present invention, a salt with an inorganic acid (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or 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).
[0053] 本発明のペプチド， またはその塩は， ヒトゃ哺乳動物などの細胞または組 織から公知のタンパク質の精製方法によって製造することができる。 また， ペプチドをコードする DNAを含有する形質転換体を培養することによって も製造できる。 また， ペプチド合成法に準じて製造することもできる。 ヒト や哺乳動物の組織または細胞から本発明のぺプチドを製造する場合， 例えば ， 組織または細胞をホモジナイズした後， 酸などで抽出を行ない， 該抽出液 を逆相クロマ卜グラフィー， イオン交換クロマ卜グラフィーなどのクロマ卜 グラフィーを組み合わせることにより， 本発明のぺプチドまたはその塩を， 単離精製することができる。 [0053] 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. When the peptide of the present invention is produced from human or mammalian tissue or cells, for example, 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.
[0054] ペプチドの合成法として， 例えば， 固相合成法， 液相合成法 （泉屋信夫， 加藤哲夫， 青柳東彦， 脇 道典， 「ペプチド合成の基礎と実験」 1 9 8 5， 丸善 （株） ） があげられる。 また， 反応後は通常の精製法， 例えば， 溶媒抽 出 '蒸留 'からムクロマ卜グラフィー■液体クロマ卜グラフィーゃ再結晶な どを組み合わせることで， 本発明のペプチドを単離精製できる。 上記方法で 得られるぺプチドが遊離体である場合は， 公知の方法によって適当な塩に変 換することができるし， 逆に塩で得られた場合は， 公知の方法によって遊離 体または他の塩に変換することができる。  [0054] 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))). Further, after the reaction, 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. When the peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, the free form or other substances can be converted by a known method. Can be converted to salt.
[0055] 本発明のポリべプチドのうち配列番号 1で表されるぺプチドを固相合成法 で製造する場合， 例えば， アミノ酸配列の 2 0位の残基の， 保護アミノ酸残 基のカルボキシル基を直接 （又は場合によリスぺーサ一を介して， ） クロロ メチル基あるいはォキシメチル基を有する不溶性樹脂に結合させる。 その後 ， アミノ酸配列の 1 9位から 1位までの各保護アミノ酸を固相合成法に従つ て順次結合させる。 その後， 不溶性樹脂およびアミノ酸の保護基を脱離させ る。 このようにして， 本発明のポリペプチドを得ることができる。 前記不溶 性樹脂， スぺーサ一， 及び N—保護アミノ酸を不溶性樹脂に結合した N—保 護アミノ酸樹脂などは， 公知の方法で調製でき， また市販されるものを適宜 用いてもよい。  [0055] Among 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.
[0056] 不溶性樹脂は， C末端の N _保護アミノ酸のカルボキシル基と直接結合で きるか， 又はスぺーサーを介して結合でき， 前記カルボキシル基から脱離で きるものであれば特に限定されず， 公知のものを適宜用いることができる。 このような不溶性樹脂として， 例えば， B o c (t_ブチルォキシカルボニル ) ストラテジーによりペプチドを合成する場合は， クロロメチル樹脂 （クロ ロメチル化スチレン一ジビニルベンゼン共重合体） ， ォキシメチル樹脂， 又 はスぺーサーを導入した 4 _ォキシメチル _ P a m (フエ二ルァセタミドメ チル） 樹脂が好ましい。 一方， F m o c ( 9—フルォレニルメチルォキシカ ルポニル） ストラテジーによりペプチドを合成する場合は， ォキシメチルフ エノキシメチル （Wa n g) 樹脂が好ましい。 [0056] 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. As such 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. On the other hand, F moc (9-fluorenylmethyloxyca When peptides are synthesized using the (Luponyl) strategy, oxymethylphenoxymethyl (Wang) resin is preferred.
保護ァミノ酸とは， 官能基を公知の方法によリ保護基で保護したアミノ酸 を意味する。 保護アミノ酸として， 市販される公知の保護アミノ酸を適宜用 いることができる。 本発明のポリペプチドを合成する場合には， 以下に示す 保護基のいずれかを選択するのが好ましい。 すなわち， アミノ酸の Of _アミ ノ基の保護基として， Bo c ( t _ブチルォキシカルボニル） 又は Fmo c (9—フルォレニルメチルォキシカルボニル） が好ましい。 アルギニン （A r g) のグァニジノ基の保護基として， To s (トシル） ， N02 (ニトロ） ， M t r (4—メ トキシ一 2, 3, 6_トリメチル ベンゼンスルホニル） 又は Pmc (2, 2, 5, 7, 8 _ペンタメチルクロマン一 6 _スルホ ニル） が好ましい。 リジン （L y s) の 一アミノ基の保護基として， Z ( ベンジルォキシカルボニル） ， CI ■ Z (2_クロ口べンジルォキシカルボ二 ル） ， Bo c， 又は N p y s (3—ニトロ _ 2 _ピリジンスルフエニル） が 好ましい。 ヒスチジン （H i s) のイミダゾリル基の保護基として， To s ， Z, P a c (フエナシル） ， Bom (ベンジルォキシメチル） ， Dn p ( ジニトロフエニル） 又は T r t (トリチル） が好ましい。 システィン （Cy s) のメルカプト基の保護基として， B z I 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. When synthesizing the polypeptide of the present invention, it is preferable to select any of the protecting groups shown below. In other words, Bo c (t_butyloxycarbonyl) or Fmo c (9-fluorenylmethyloxycarbonyl) is preferred as the protecting group for the Of_amino group of amino acids. As protecting groups for the guanidino group of arginine (A rg), To s (tosyl), N0 2 (nitro), M tr (4-methoxy-1,2,3,6_trimethylbenzenesulfonyl) or Pmc (2, 2, 5, 7, 8_pentamethylchroman-6_sulfonyl). As a protecting group for the mono-amino group of lysine (L ys), Z (benzyloxycarbonyl), CI ■ Z (2_black benzyloxycarbonyl), Bo c, or N pys (3-nitro_ 2_pyridinesulfenyl) is preferred. The protecting group for the imidazolyl group of histidine (His) 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)
(ベンジル） ， MB z I (4—メ トキシベンジル） ， 4_Me B z I (4—メ チルベンジル） ， A c m (ァセタミドメチル） ， T r t， N p y s, t _B u  (Benzyl), MB z I (4-methoxybenzyl), 4_Me B z I (4-methylbenzyl), A cm (acetamidomethyl), T r t, N p ys, t _B u
(t_プチル） ， t_Bu S (t_プチルチオ） があげられ， これらの中では， MB z I , 4— Me B z I， T r t , A c m, 又は N p y sが好ましい。 チ 口シン （T y r ) の水酸基の保護基として， B z l， C I 2 ■ Β ζ I (2, 6—ジクロ口ベンジル） ， t _Buがあげられる。 一方， チロシン （T y r ) の水酸基は， 特に保護基を導入しなくてもよい。 トリブトファン （T r p ) のインドール基の保護基として， CHO (フオルミル基） があげられる。 一方， トリブトファン （T r p) のインドール基は， 特に保護基を導入しな くてもよい。 メチォニン （Me t) のチオメチル基の保護基として， メチル スルホキシド基があげられるが， 特に保護基を導入しなくてもよい。 セリン(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. Examples of protecting groups for the hydroxyl group of thiocin (T yr) include B zl, CI 2 ■ Β ζ I (2, 6-dichloromouth benzyl), and t_Bu. On the other hand, the hydroxyl group of tyrosine (T yr) does not need to introduce a protective group. CHO (formyl group) is a protecting group for the indole group of tribtophan (T rp). On the other hand, the indole group of tribtophan (T rp) 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. Serine
(S e r ) および卜レオニン （T h r) の水酸基の保護基として， B z I又 は t _Bu基があげられる。 ァスパラギン酸 （As p) およびグルタミン酸Examples of the protecting group for the hydroxyl group of (S e r) and 卜 leonine (T h r) include the B z I or t _Bu group. Aspartic acid (As p) and glutamic acid
(G I u) のカルボキシル基の保護基として， OB z I (ベンジルエステル ) ， O t Bu (t _ブチルエステル） ， Oc H e x (シクロへキシルエステ ル） ， 又は OPa c (フエナシルエステル） が好ましい。 ァスパラギン （A s n) およびグルタミン （G i n) のカルバミド基の保護基として， T r t 又は Xa n (キサンチル基） が好ましい。 As the 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. . As a protecting group for the carbamide group of asparagine (A sn) and glutamine (G in), T r t or Xan (xanthyl group) is preferable.
保護アミノ酸の結合は， 通常の縮合法， 例えば， DCC (ジシクロへキシ ルカルポジイミド） 法， D I PCD I (ジイソプロピルカルポジイミド） 法 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.
[Ta r t a r. Aら ； J. O r . C h e m. ， 44， 5000 (1 97 9) ] ， 活性エステル法， 混合又は対称酸無水物法， カルボニルジイミダゾ ール法， DCC— HOB t (1—ヒドロキシベンゾトリァゾール） 法 [Ke o n i g ， Wら ； Ch em. Be r. ， 1 03， 788 ， p p. 2024— 2034 (1 970) ] ， ジフエ二ルホスホリルアジド法， BOP試薬 （ベ ンゾトリァゾリル一 N—ヒドロキシトリスジメチルァミノホスホニゥムへキ サフルォロリン化物塩） を用いる BOP— HOB t法 （H u d s o n, D . ， J . O r . Ch em. ， 53， 61 7 (1 988) ), HBT U (2- ( 1 H) —ベンゾトリアゾール 1—ィル）_ 1， 1， 3， 3—テトラ メチルゥロニゥム へキサフルォロホスフエ一卜） 一HOB t法 （Kn o r r， R. ら， T e t r a h e d r o n L e t t. , 30， 1 927[Ta rta r. A et al .; J. Or. Chem., 44, 5000 (1997)], active ester method, mixed or symmetric anhydride method, carbonyl diimidazole method, DCC—HOB t (1-Hydroxybenzotriazole) method [Keonig, W et al .; Chem. Ber., 103, 788, p. 2024-2034 (1 970)], diphenylphosphoryl azide method, BOP reagent BOP—HOB t method (Hudson, D., J. Or.Chem., 53, 61 7 (1 988) using benzotriazolyl mono N-hydroxytrisdimethylaminophosphonium hexafluorophosphate salt) )), HBT U (2- (1 H) —benzotriazole 1-yl) _ 1, 1, 3, 3-tetramethyluronium hexafluorphosphine) One HOB t method (Kn orr, R Et al., T etrahedron L et t., 30, 1 927
(1 989) ) ， TBTU (2- ( 1 H) —ベンゾトリアゾール 1—ィル）一 1， 1， 3， 3—テトラメチルゥロニゥム テトラフルォロボレイト） 一H OB t法 (Kn o r r, R. , T e t r a h e d r o n L e t t. ， 3 0， 1 927 (1 989) ) 等に従って行なうことができる。 これらのうち ， DCC法， DCC_HOB t法， ΒΟΡ— HOB t法， HBTU— HOB t法， 又は対称酸無水物法が好ましい。 [0059] これらの縮合反応は， たとえば， ジクロロメタン， ジメチルホルムアミド (DMF) ， N_メチルピロリ ドン （NMP) 等の有機溶媒又はそれらの混 合溶媒中で行なわれる。 Of _アミノ基の保護基の脱離試薬としては， トリフ ルォロ酢酸 Zジクロロメタン， HCI Zジォキサン， ピぺリジン ZDMF又 はピぺリジン Z N M P等が用いられ， 該保護基の種類によリ適宜選択する。 また， 合成の各段階における縮合反応の進行の程度は E. カイザーらの方法 (1 989)), TBTU (2- (1 H) —benzotriazole 1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate) One H OB t method (Kn orr, R., T etrahedron L et t., 30, 1 927 (1 989))). Of these, DCC method, DCC_HOB t method, ΒΟΡ-HOB t method, HBTU-HOB t method, or symmetric acid anhydride method are preferable. [0059] These condensation reactions are performed, for example, in an organic solvent such as dichloromethane, dimethylformamide (DMF), N_methylpyrrolidone (NMP), or a mixed solvent thereof. 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.
[An a I . B i o c h em. ， 34， 595 (1 970) ] (二 ンヒドリン反応法） によって検査されればよい。 以上のようにして， 所望の ァミノ酸配列を有する保護べプチド樹脂を得ることができる。  [An a I. Biochem., 34, 595 (1 970)] (Ninhydrin reaction method). As described above, a protected peptide resin having a desired amino acid sequence can be obtained.
[0060] 保護ペプチド樹脂は， フッ化水素， T FMSA (トリフルォロメタンスル ホン酸) [A a d e m i c P r e s s 発行， E. G o s s編集, H Ya j i maら； " T h e P e p t i d e s " 5 , 65 (1 98 3) ] ， TMSOTf (卜リメチルシリルトリフラー卜） ， TMSBr (トリ メチルシリルブロミド） [F u j i i . N ら ； Ch em. P h a rm. B u I I . , 35, 3880 (1 987) ] , またはトリフルォロ酢酸などで処 理することにより， 該樹脂および保護基を同時に脱離させることができる。 上記の脱離試薬は， 該ストラテジー （BO C又はFm O C)， 該樹脂， 該保護 基の種類によリ適宜選択する。 [0060] 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.
[0061] このようにして得られたペプチドは， 公知の方法， 例えば， 抽出， 再結晶 ， 各種クロマトグラフィー （ゲルろ過， イオン交換， 分配， 吸着， 逆相） ， 電気泳動， 向流分配等により単離精製することができる。 これらの中では， 逆相高速液体クロマ卜グラフィーを用いて精製する方法が好ましい。 上記方 法で得られるぺプチドが遊離体である場合は， 公知の方法によって適当な塩 に変換できる。 また， ペプチドが塩の形態で得られた場合は， 公知の方法に よつて遊離体または他の塩に変換することができる。  [0061] 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.
[0062] 後述する実施例で示されるように， エフリン B 2は， 内皮細胞 （EC) の DNA合成； ECにおける細胞外シグナル制御キナーゼ （ERK) リン酸化 ； VEG Fおよび b FG Fの両方で誘導される p 44Zp 42MAPキナー ゼ活性化； EC脈管形成； b FG Fで誘導された角膜血管形成；及び網膜か ら硝子体腔内に伸びる病的な新生血管を阻害する。 [0062] 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. P 44Zp 42MAP Kinner EC angiogenesis; b FG F-induced corneal angiogenesis; and inhibits pathological new blood vessels that extend from the retina into the vitreous cavity.
[0063] そして， 本発明のペプチドは， 後述する実施例で示されるように， それ自 体 M A P Kリン酸化活性阻害能を有するので， 公知の製剤化のための方法を 適宜採用することで， 本発明のぺプチドを含有する新生血管阻害剤に用いる ことができる。 この新生血管阻害剤は， 例えば， 本発明のペプチドと薬理学 的に許容される担体などを含有する剤であり， 眼内の新生血管阻害剤である ことが好ましく， 眼内の新生血管に関連する疾患の治療剤又は予防剤が好ま しい。 本発明のペプチドは， エフリン B 2に比べて短いので， 容易に製造で きる。 よって， 本発明のペプチドを含む新生血管阻害剤は， 安定であり， 比 較的安価に製造することができるという利点もある。  [0063] Since the peptide of the present invention itself has the ability to inhibit MAPK phosphorylation activity as shown in the examples described later, 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.
[0064] また， 本発明のペプチドは， エフリン B 2の活性を高めるので， 眼内の新 生血管阻害補助剤， 眼内の静脈内皮細胞における D N A合成阻害用補助剤及 び眼内の静脈内皮細胞における p 44Zp 42MAPKリン酸化活性阻害用 補助剤を提供できる。 これらの補助剤の好ましい態様は， 主剤としてエフリ ン B 2を含有する剤を用いるものである。  [0064] Furthermore, since 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. In a preferred embodiment of these adjuvants, an agent containing ephrin B 2 is used as the main agent.
[0065] 血管内皮細胞を VEG Fで刺激すると受容体下流のシグナル伝達系により MAPKが活性化され， リン酸化された MAP Kの上昇が認められる （Ab e d i， H. a n d Z a c h a r y , I . ， J. B i o l . C h e m. ， 272, 1 5442- 1 5451 ( 1 997 ) ) 。 M A P Kの活性化は血管 新生における血管内皮細胞の増殖に重要な役割を担うことが知られている （ Me r e nm i e s, J. e t a I . , Ce l l G r ow t h & D i f f e r. , 83— 1 0 (1 997) ； F e r r a r a, N. a n d Da v i s— Smy t h, T. En d o c r. Re v. , 1 8， 4— 25 (1 997) ) 。 従って， 本発明のペプチドは， 血管新生抑 制作用又はエフリン B 2によリ血管新生抑制作用の補助活性を有するといえ る。 病態部位における血管新生は， 眼内以外では主として， 腫瘍， 慢性関節 リウマチ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫のような疾患， 並び に固形癌の転移と深く結びついている （F o r kma n， J. N a t u r e Me d. 1 : 27-31 (1 995) , B i c k n e l l , R. , H a r r i s, A. L . C u r r . Op i n. On c o l . [0065] 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)). Therefore, it can be said that 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.
8 ： 60— 65 (1 996) ) 。 従って， 本発明のぺプチドは， 腫瘍， 慢性関節リウマチ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫のような疾 患， 並びに固形癌の転移， その他の血管形成または血管新生に関連する疾患 または障害の治療に用いることができ， 本発明はそのような治療剤をも提供 しうる。  8: 60-65 (1 996)). Thus, 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.
[0066] これらの剤や補助剤は， 本発明のペプチドを有効量含有するものがあげら れる。 「有効量」 とは， 所望の目的を達成するために十分な量を意味する。 眼内の新生血管阻害用補助剤における 「有効量」 とは， 例えば， エフリン B 2などの眼内の新生血管阻害剤の新生血管阻害作用を高めるために十分な量 を意味する。 具体的な， 本発明のぺプチドの投与量は， 対象疾患， 投与対象 ， 投与ルートなどにより適宜調整すればよい。 本発明のペプチドの投与量と して， 例えば， 目に硝子体注射投与する場合， 一般的に成人 （体重 60 k g として） においては， 一眼あたリ 1 X 1 02 n g〜 1 m g， 好ましくは 1 X 1 02n g〜1 g， より好ましくは I x l 02n g〜7 x l 02n gがあげられ る。 投与回数は適宜調整すればよぐ 例えば， 一日一回主剤であるエフリン B 2を含有する剤とともに投与するものなどがあげられる。 また， 補助剤を 経口投与する場合， 一般的に成人 （体重 6 O k gとして） においては， 一回 にっき 2 g〜 1 X 1 02m g， 好ましくは 2 g〜1 m g， より好ましくは 1 X 1 02 n g〜2 g， より好ましくは 2 g〜1 X 1 02 gがあげられ る。 これらの量は， 患者の体重， 性別などを適宜考慮して， 調整すればよい 。 ヒト以外の哺乳動物に上記の剤又は補助剤を投与する場合も， 体重 6 O k g当たりに換算した量を適宜投与すればよい。 [0066] These 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. Specifically, 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. Further, when 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. These amounts should be adjusted with appropriate consideration of the patient's weight and gender. When administering the above-mentioned agents or adjuvants to mammals other than humans, the amount converted per 6 O kg body weight may be administered as appropriate.
[0067] 本発明のペプチドは， それ自体， 又は医薬組成物 （補助剤） として投与す ることができる。 本発明のペプチドは， たとえば薬理学的に許容され得る担 体， 希釈剤又は賦形剤を含むものがあげられる。 本発明のぺプチドを含有す る補助剤は， 投与形態に適する剤形として調整されればよい。 [0067] The peptide of the present invention can be administered per se or as a pharmaceutical composition (adjuvant). Examples of the peptide of the present invention include those containing a pharmacologically acceptable carrier, diluent or excipient. Contains the peptide of the present invention Adjuvants should be adjusted as dosage forms suitable for the dosage form.
[0068] たとえば， 本発明のぺプチド又は補助剤を点眼剤又は注射剤として用いる 場合， 有効量の本発明のペプチドと， 公知の希釈剤 （希釈剤は， 薬理学的に 許容され得る担体に含まれる） とを含む点眼剤又は注射剤であればよい。 希 釈剤として， 滅菌水， 純水， 蒸留水などの水；生理食塩水； ブドウ糖溶液； エタノールなどのアルコール； グリセロール， プロピレングリコール， ポリ エチレングリコールなどのポリアルコール；滅菌有機溶媒；又は水性デンプ ン； P B Sのいずれか 1種又は 2種以上の混合物などがあげられる。  [0068] For example, when the peptide or adjuvant of the present invention is 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.
[0069] 本発明のペプチドを含有する点眼剤又は注射剤は， ェピネフリン， 塩酸ェ ピネフリン， 塩酸エフェドリン， などの充血除去成分； メチル硫酸ネオスチ グミン， トロピカミドなどの眼調節薬成分；硫酸亜鉛， 乳酸亜鉛， アラント イン， ィプシロン一アミノカプロン酸， インドメタシン， 塩化リゾチームな どの抗炎症薬成分； ァシタザノラスト， アンレキサノクス， イブジラスト， トラニラス卜， 塩酸ジフェンヒドラミンなどの抗ヒスタミン薬成分又は抗ァ レルギ一薬成分； アミノ酸類；塩酸ォキシブプロ力イン， 塩酸コカイン， 塩 酸コルネカイン， 塩酸ジブ力インなどの局所麻酔薬成分； などを適宜含有し てもよい。 コンドロイチン硫酸ナトリウム， ヒアルロン酸ナトリウムなどの 増粘剤；塩化ベンザルコニゥムなどの界面活性剤；安息香酸ナ卜リゥム， ェ タノール， 塩化ベンザルコニゥムなどの防腐剤， 殺菌剤又は抗菌剤；塩酸， ホウ酸， 水酸化ナ卜リゥム， 炭酸水素ナ卜リゥムなどの p H調節剤；亜硫酸 水素ナ卜リゥム， 亜硫酸ナ卜リゥム， 塩化力リゥムなどの等張化剤； メント ール， カンフル， ハツ力油， ペパーミント油などの香料または清涼化剤； ク ェン酸緩衝剤などの緩衝剤などを適宜含有するものであってもよい。  [0069] 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. 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.
[0070] 本発明のペプチドを含む補助剤は， 錠剤， カプセル剤， 顆粒剤， 散剤， シロ ップ剤などの経口投与剤であってもよい。 この場合， 薬理学的に許容される 担体として， 賦形剤， 希釈剤， 滑沢剤， 結合剤， 崩壊剤， 安定剤， 及び矯味 矯臭剤から適宜選択されるものがあげられる。  [0070] The adjuvant containing the peptide of the present invention may be an orally administered agent such as a tablet, capsule, granule, powder, or syrup. In this case, pharmacologically acceptable carriers include those appropriately selected from excipients, diluents, lubricants, binders, disintegrants, stabilizers, and flavoring agents.
[0071 ] 本発明のペプチドを含む補助剤は， 公知の方法に従って製造できる。 点眼剤 又は注射剤は， 例えば， 希釈剤などに本発明のペプチドを添加し， アンプル などの容器に入れることにより製造できる。 錠剤は， 例えば， 本発明のぺプ チドを公知の担体と混合した医薬組成物を打錠機によリ打錠することにより 製造できる。 カプセル剤は， 例えば， カプセルなどの形態をしている担体中 に， 本発明のペプチドを封入することにより製造できる。 [0071] 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.
[0072] また， 本発明のペプチドは， エフリン B 2の活性を高めるので， 後述する ようにエフリン B 2を含む医療用組成物， 又はキッ卜などに適宜利用されう る。  [0072] Further, since 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.
[0073] [エフリン B 2及び本発明のぺプチドを含有する医薬用組成物， 及び治療 •予防剤]  [0073] [Pharmaceutical Composition Containing Ephrin B 2 and Peptide of the Present Invention, and Treatment and Prophylactic Agent]
本発明の第 2の側面は， 基本的には， エフリン B 2又はその薬理学的に許 容される塩と；本発明のぺプチド， もしくはそれらの薬理学的に許容される 塩と； を含有する医薬用組成物に関する。 これらの中では， エフリン B2又 はその薬理学的に許容される塩と， 配列番号 1で表されるアミノ酸配列から なるぺプチド， 配列番号 2で表されるァミノ酸配列からなるペプチド， もし <はそれらの薬理学的に許容される塩を含有する医薬用組成物が好ましい。  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. Among these, 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.
[0074] WO2006Z006079号公報 （特許文献 1 ) に開示されるとおり， エフリン B 2は， 眼内における動脈及び静脈内皮細胞の DN A合成の阻害作 用；眼内における動脈及び静脈内皮細胞における p 44Zp 42MAPK活 性化の阻害作用；眼内における動脈及び静脈内皮細胞における管形成の阻害 作用を有する。 そして， 同文献における生体内実験で実証されたように， ェ フリン B 2は， 眼内の脈絡膜における新生血管を抑制し， 加齢黄斑変性 （A MD) など， 眼内の新生血管に関連する疾患の治療に有効である。  [0074] As disclosed in WO2006Z006079 (Patent Document 1), 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. And as demonstrated in the in vivo experiments in the same document, 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.
[0075] 一方， 後述する実施例によって実証されたとおり， 本発明のペプチドは， エフリン B 2と併用することでエフリン B 2の MAP Kリン酸化阻害能を飛 躍的に高める作用を有する。 このことは， エフリン B 2が有する， 眼内の静 脈 （動脈） 内皮細胞における新生血管の抑制能を高めることを意味する。 よ つて， エフリン B 2と本発明のペプチドなどを含有する医薬用組成物は， 特 に眼内の新生血管の抑制など， 眼内の新生血管に関連する疾患の治療に特に 有効であると考えられる。 本発明の医薬用組成物は， 有効量のエフリン B 2 を含有すればよい。 [0075] On the other hand, as demonstrated by the examples described later, 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. This means that 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.
[0076] 本発明の医薬用組成物に用いられるエフリン B 2は， アナログおよび改変 体を含む任意のエフリン B 2であってもよい。 なお， エフリン B 2は， 可溶 性エフリン B 2 (通常， 細胞外ドメインを含むが， 細胞質ドメインを含まな い） であってもよい。 エフリン B 2は， 天然に存在するタンパク質を単離精 製したものであっても， 遺伝子組換えにより微生物などから生成されたもの であってもよい。 例えば， 米国特許第 6， 3 0 3， 7 6 9号明細書又は M o [0076] 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. For example, US Pat. No. 6,300,769 or M o
1 I mm u n o に 1 9 9 5 N o v ； 3 2 ( 1 6 ) ： 1 1 9 7 - 2 0 5 に記載されるヒ卜エフリン B 2の c D N Aの配列を参照して， 慣用の技術を 用いて， 全長タンパク質， 細胞外ドメインを含むタンパク質などを作製して もよい。 当業者が通常用いる技術を用いることにより， 天然のエフリン B 2 を好ましく改変することもできる。 また， エフリン B 2は， 医薬品や試薬と して市販されているいずれを用いてもよい。 1 Immuno 1 99 5 N ov; 3 2 (1 6): 1 1 9 7-20 5, refer to the cDNA sequence of hyephrine B 2 It may be used to produce full-length proteins, proteins containing extracellular domains, and the like. Natural ephrin B 2 can also be preferably modified by using techniques commonly used by those skilled in the art. Ephrin B 2 may be any commercially available drug or reagent.
[0077] エフリン B 2は， 全長であってもよい。 エフリン B 2の好ましい態様は， 天然のエフリン B 2の細胞外ドメィンを含み， 細胞質ドメィンを含まないも のである。 天然のエフリン B 2の細胞外ドメインを含み， 細胞質ドメインを 含まない領域が， E p h B 4のファーマコフォアに作用する部位であると考 えられるので， そのような部位を含むエフリン B 2であれば， 全長でなくて も新生血管の抑制能を有すると考えられる。 そのようなエフリン B 2として ， 配列番号 4のうち一 2 5〜 3 0 8位のアミノ酸残基からなるもの， 配列番 号 4のうち 1〜3 0 8位のアミノ酸残基からなるもの， 配列番号 4のうち一 [0077] 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. As such 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, One of four
2 5〜 1 9 9位のァミノ酸残基からなるもの， 配列番号 4のうち 1〜 1 9 9 位のアミノ酸残基からなるもの， 及びそれらと 9 0 %以上の相同性を有する もの （好ましくは 9 5 %以上相同性を有するもの， 又は配列番号 3で示され る部位を含みつつ 9 0 %以上の相同性を有するもの） であって， エフリン B 2と同様の活性を有するものがあげられる。 なお， 相同性は， 例えば適宜パ ラメ一タを最適化した G A Pコンピュータプログラムのバージョン 6を用い て求めればよい。 なお， 配列番号 4で示されるペプチドは， エフリン B 2の N末端シグナルべプチド （配列番号 4のアミノ酸一 2 5から 1 ) ， 細胞外ド メイン （アミノ酸 1から 1 9 9 ) ， 膜貫通領域 （アミノ酸 2 0 0から 2 2 5 ) ， および細胞質ドメイン （アミノ酸 2 2 6から 3 0 8 ) からなるペプチド である （なお， 配列番号 4に示されるアミノ酸の番号については配列番号 4 に係るぺプチドのアミノ酸配列及びこのべプチドをコ一ドする遺伝子の塩基 配列を図 6に示す。 ） 。 2 consisting of an amino acid residue at positions 5 to 199, 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.
[0078] また， エフリン B 2が， ヒト F cとエフリン B 2の融合タンパク質であつ てもよい。 エフリン B 2が新生血管の抑制能を発揮するためには， エフリン B 2が二量体化していることが望ましいと考えられ， そのためヒ卜 F cとェ フリン B 2との融合タンパク質であれば， エフリン B 2が二量化し， 高い新 生血管の抑制能を発揮しうる。 この融合タンパク質に用いられるエフリン B 2は， エフリン B 2の細胞外ドメイン， 又は細胞外ドメインを含み， 細胞質 ドメインを含まないものが好ましい。 N末端シグナルべプチド及び膜貫通領 域については， それら全てが含まれてもよいし含まれなくてもよぐ またそ れらのうち一部のみが含まれていてもよい。 エフリン B 2とヒ卜 F cの融合 タンパク質は， たとえば， 特表平 1 0— 5 0 2 8 1 0号公報 （特許文献 3 ) に記載される方法により作出できる。  [0078] Further, ephrin B2 may be a fusion protein of human Fc and ephrin B2. 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 融合 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. The 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).
[0079] 本発明の第 2の側面の好ましい態様は， 新生血管阻害， 特に眼内の新生血 管阻害剤である上記いずれかに記載の医薬用組成物に関する。 本発明の第 2 の側面の好ましい態様は， 眼内の脈絡膜における新生血管阻害剤である上記 いずれかに記載の医薬用組成物に関する。 これら医薬組成物は， 公知の薬理 学的に許容される担体などを適宜含んでもよい。 このような担体として， 先 に説明したものを適宜用いることができる。 また， 新生血管阻害剤などとし て， 先に説明した補助剤と同様の剤形のものを同様にして製造できる。  [0079] 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. In addition, a neovascular inhibitor can be produced in the same manner as the adjuvant described above.
[0080] 本発明の第 2の側面の好ましい態様は， 眼内の新生血管に関連する疾患の 治療■予防剤である上記いずれかに記載の医薬用組成物に関する。 より具体 的に説明すると， 本発明の第 2の側面の好ましい態様は， "加齢黄斑変性， 虚血性網膜症， 眼内血管新生， 角膜血管新生， 網膜血管新生， 脈絡膜血管新 生， 糖尿病性黄斑浮腫， 糖尿病性網膜虚血， 糖尿病性網膜浮腫， 糖尿病性網 膜症" からなる群から選択される， 1又は 2種以上の疾患の治療，予防剤で ある上記いずれかに記載の医薬用組成物に関する。 特に好ましい態様は， 加 齢黄斑変性及び糖尿病性網膜症のいずれか又は両方の疾患の治療■予防剤で ある。 また， 先に説明したとおり， 本発明のペプチドは， 血管新生抑制作用 又はエフリン B 2により血管新生抑制作用の補助活性を有するといえる。 病 態部位における血管新生は， 眼内以外では主として， 腫瘍， 慢性関節リウマ チ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫のような疾患， 並びに固形 癌の転移と深く結びついている （F o r kma n， J. N a t u r e Me d. 1 ： 27 -3 1 ( 1 995) ； B i c k n e l l , R. , H a r r i s, A. I_. C u r r . O p i n. O n c o l . 8 ： 60-65 ( 1 996) ) 。 従って， 本発明の第 2の側面の好ましい態様は ， 腫瘍， 慢性関節リウマチ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫， 又は固形癌の転移の治療剤である医薬用組成物に関する。 [0080] 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. In addition, as described above, it can be said that 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. Oncol. 8: 60-65 ( 1 996)). Accordingly, 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.
[0081] すなわち， 本発明の医薬組成物は， 既に罹患している眼内の新生血管に関 連する疾患の治療剤として用いることができる他， これらの疾患を予防する ため， 又はこれらの疾患が発生する事態を遅延させるための予防剤としても 使用しうる。 [0081] That is, 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
[0082] 本発明の第 2の側面に係る医薬用組成物又は治療■予防剤に含まれるエフ リン B 2と本発明のペプチドの割合 （モル比） は適宜調整すればよい。 しか しながら， 後述する実施例によって実証されたとおり， エフリン B 2と本発 明のペプチドのモル比として 1 : 1 X 1 0_3〜 1 : 1 X 1 03があげられ， 1 : 1 X 1 0- 2〜 1 : 1 X 1 02が好ましぐ 1 : 1 X 1 0 -1〜 1 : 1 X 1 0が 好ましぐ 1 : 1〜 1 : 1 X 1 03が好ましぐ 1 : 1〜 1 : 1 X 1 02が好 ましぐ 1 : 1〜 1 : 1 X 1 0が好ましぐ 1 : 1〜 1 : 5 X 1 0が好まし < , 1 ： 1 X 1 0〜 1 : 5 X 1 0がよリ好ましい。 [0083] [医療用キット] [0082] 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. However, as demonstrated by the examples described later, 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. [0083] [Medical kit]
本発明の第 3の側面は， 基本的には， エフリン B 2又はその薬理学的に許 容される塩， 及び薬理学的に許容される担体を含有する第 1の組成物と；本 発明のペプチド， もしくはそれらの薬理学的に許容される塩， 及び薬理学的 に許容される担体を含有する第 2の組成物を含有する医療用キッ卜 （以下， 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”).
「本発明の医療用キット」 ともよぶ。 ） に関する。 すなわち， エフリン B 2 などを含有する第 1の組成物と， 本発明のぺプチドなどを含有する第 2の組 成物とを含む医療用キットに関する。 本発明のペプチドとして， 配列番号 1 で表されるァミノ酸配列からなるぺプチド， 配列番号 2で表されるアミノ酸 配列からなるぺプチド， もしくはそれらの薬理学的に許容される塩が好まし い。 Also referred to as “medical kit of the present invention”. ) That is, 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. As 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. .
[0084] 上記の医療用キッ卜は， 基本的には， 新生血管の抑制， 特に眼内の新生血 管の抑制のための第 1の組成物を主剤とし， 第 2の組成物を補助剤として含 むものである。 後述する実施例によって実証されたとおり， 本発明のぺプチ ドは， エフリン B 2と併用することでエフリン B 2の M A P Kリン酸化阻害 能を飛躍的に高める作用を有する。 このことは， エフリン B 2が有する， 眼 内の静脈 （動脈） 内皮細胞における新生血管の抑制能を高めることを意味す る。 さらに， 後述する実施例によって実証されたとおり， 第 1の組成物を投 与した後に第 2の組成物を投与すると M A P Kの活性を著しく阻害すること が示された。 よって， 本発明のキットは， 特にそのような投与方法に有効に 利用されうる。  [0084] 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. As demonstrated by the examples described later, 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. Furthermore, as demonstrated by the examples described below, it was shown that 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.
[0085] 本発明の医療用キットに含まれるエフリン B 2は， 先に説明したものを適 宜用いることができる。  [0085] As the ephrin B2 contained in the medical kit of the present invention, those described above can be suitably used.
[0086] 本発明の医療用キットの好ましい態様は， 新生血管阻害用， 特に眼内の新 生血管阻害用に用いられるものであり， さらに好ましい態様は， 眼内の脈絡 膜における新生血管阻害用に用いられるものである。 本発明の第 3の側面の 好ましい態様は， 眼内の新生血管に関連する疾患の治療又は予防用に用いら れる上記いずれかに記載のキッ卜に関する。 本発明の第 3の側面の好ましい 態様は， 加齢黄斑変性， 虚血性網膜症， 眼内血管新生， 角膜血管新生， 網膜 血管新生， 脈絡膜血管新生， 糖尿病性黄斑浮腫， 糖尿病性網膜虚血， 糖尿病 性網膜浮腫， 糖尿病性網膜症" からなる群から選択される， 1又は 2種以上 の疾患の治療用又は予防用に用いられる上記いずれかに記載のキッ卜に関す る。 本発明の第 3の側面の好ましい態様は， 加齢黄斑変性及び糖尿病性網膜 症のいずれか又は両方の疾患の治療用又は予防用に用いられる上記いずれか に記載のキッ卜に関する。 [0086] 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. Preferred of the third aspect of the present invention 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.
[0087] 先に説明したとおり， 本発明のペプチドは， 血管新生抑制作用又はエフリ ン B 2により血管新生抑制作用の補助活性を有するといえる。 病態部位にお ける血管新生は， 眼内以外では主として， 腫瘍， 慢性関節リウマチ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫のような疾患， 並びに固形癌の転移と 深く結びついている （F o r kma n， J. N a t u r e Me d. 1 : 27 -3 1 ( 1 995) ； B i c k n e l l , R. , H a r r i s, A. L. C u r r . O p i n. O n c o に 8 ： 60— 6 5 ( 1 996) ) 。 従って， 本発明の第 3の側面の好ましい態様は， 腫瘍， 慢性関節リウマチ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫， 又は固形 癌の転移の治療に用いられる上記いずれかに記載のキッ卜に関する。 すなわ ち， 本発明の第 3の側面の好ましい態様は， 腫瘍， 慢性関節リウマチ， 乾癬 ， ァテローム性動脈硬化症， 力ポジ肉腫， 又は固形癌の転移の治療用キット に関する。 [0087] As described above, it can be said that 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 )). Therefore, 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. About. That is, 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.
[0088] 第 1の組成物は眼内の新生血管など新生血管に関連する疾患の治療剤又は 予防剤であり， 第 2の組成物は前記第 1の組成物の補助剤であってもよい。 これらの組成物， 治療剤， 予防剤及び補助剤は， 先に説明したものを適宜用 いることができる。 そして， これらに含まれるエフリン B 2や本発明のぺプ チドの含有量も上記したものを適宜用いることができる。  [0088] The first composition may be a therapeutic or prophylactic agent for a disease related to neovascularization such as neovascularization in the eye, and the second composition may be an adjuvant of the first composition. . As these compositions, therapeutic agents, preventive agents and adjuvants, those described above can be used as appropriate. In addition, the contents of ephrin B 2 and the peptide of the present invention contained therein can be appropriately used.
[0089] 後述する実施例によって実証されたとおり， エフリン B 2と本発明のぺプ チドを同時に投与した場合に比べ， エフリン B 2を投与した後に， 本発明の ぺプチドを投与した場合のほうが， MAPK活性化を抑えることができる。 したがって， 本発明の第 3の側面の好ましい態様は， 前記第 1の組成物が投 与された後に， 前記第 2の組成物が投与される上記いずれかに記載のキッ卜 に関する。 前記第 1の組成物が投与された後， 30秒後〜 24時間後， 好ま しくは 1分後〜 1 2時間後， より好ましくは 1分後〜 1 0分後に前記第 2の 組成物が投与される上記いずれかに記載のキッ卜に関する。 また， 本発明の ぺプチドを投与した後にエフリン B 2を投与しても， エフリン B 2と本発明 のべプチドを同時に投与した場合に比べ MAP K活性化を抑えることができ る。 したがって， 本発明の第 3の側面の好ましい態様は， 前記第 2の組成物 が投与された後に， 前記第 1の組成物が投与される上記いずれかに記載のキ ッ卜に関する。 前記第 2の組成物が投与された後， 30秒後〜 24時間後， 好ましくは 1分後〜 1 2時間後， よリ好ましくは 1分後〜 1 0分後に前記第 1の組成物が投与される上記いずれかに記載のキッ卜に関する。 [0089] As demonstrated by the examples described below, the administration of the peptide of the present invention after administration of ephrin B 2 is more effective than the administration of ephrin B 2 and the peptide of the present invention at the same time. , MAPK activation can be suppressed. Therefore, 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. Moreover, even if ephrin B 2 is administered after the administration of the peptide of the present invention, MAPK activation can be suppressed as compared with the case where ephrin B 2 and the peptide of the present invention are administered simultaneously. Therefore, 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.
[0090] [CNVモデルに対する本発明のぺプチド及びエフリン B 2の評価]  [0090] [Evaluation of peptides and ephrin B 2 of the present invention against CNV model]
本発明のペプチド及びエフリン B 2が， AMDなど， 眼内の血管形成また は血管新生に関連する疾患または障害の治療剤として有効であることは， た とえば， レーザー誘導性脈絡膜血管新生 （CNV) モデルを用いて実証でき る。 レーザー誘導性 CNVモデルは， AMDの生態モデルと考えられる。 以 下， CNVモデルのプロトコル例について説明する。  The fact that the 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.
[0091] たとえば， 6匹の力二クイザル （ォス 3〜6 k g) 由来の 1 2眼を用いて CNVモデル実験を行う。 これらの動物は全て， 苦痛のないように無病原状 態で収容する。 そのため， 実験は， 塩酸ケタミンで全身麻酔を施した状態で 行う。  [0091] For example, 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.
[0092] —実験的脈絡膜血管新生 （CNV) の誘導—  [0092] —Induction of experimental choroidal neovascularization (CNV) —
上記サルの眼の眼底部の後部極において， 眼科レーザー用レンズ （3 -M I n f a n t La s e r O G 3M I A, Oc u l a r I n s t r u me n t s, I n c. ) を使用し， 眼底後極部をクリプトンレーザー （マ ルチカラ一レーザー光凝固装置 MC— 300, 株式会社ニデック， スリツ トランプ 900BQ, H a a g S t r e i t) で光凝固する。 レーザー 光の強度はたとえば， 約 700mWとする。 その際， 凝固サイズを 1 00 m， 凝固時間を 0. 1秒とし， 1眼あたり 8発の凝固を行えばよい。 At the posterior pole of the fundus of the above monkey eye, 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. (Multi-laser photocoagulator MC-300, Nidec Corporation, slit lamp 900BQ, Haag S treit). laser For example, the light intensity is about 700 mW. At that time, the coagulation size should be 100 m, the coagulation time should be 0.1 sec, and 8 coagulations per eye should be performed.
—本発明のぺプチド及びエフリン B 2投与方法と濃度一）  —Peptide and ephrin B 2 administration method and concentration of the present invention 1)
投与方法と濃度の例を下記表 1に示す。 表中， 被験物質及び対照物質の欄 中， 矢印は， あるペプチドを投与した後に次のペプチドを投与することを意 味する。 レーザー照射直後と照射後 5曰の合計 2回， 下記の硝子体内濃度の PBS, エフリン B2， 本発明のペプチドを硝子体内に投与する。 1投与あ たり 0. 1 mLずつ， 30G針を用いて毛様体扁平部から眼内に注射する。 ネガティブコントロールとして， 0. 1 mLの PBSを注射する。 ポジティ ブコントロールとして， 1. 66 nMのエフリン B2を用いる。 動物番号 3 〜5は， 3分差で投与を行う。 例えば， 動物番号 3 (右眼） は， エフリン B 2 (1 00 L) を投与した 3分後に配列番号 1のペプチド （1 00 L) を投与するので， 2種類の被験物質を各 1 00 Lの計 200 L投与する ことになる。 Examples of administration methods and concentrations are shown in Table 1 below. In the table, in the column of test substance and control substance, 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. As a negative control, inject 0.1 mL of PBS. As a positive control, 1.66 nM ephrin B2 is used. Animal numbers 3 to 5 should be administered at 3 minute intervals. For example, 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.
[表 1] [table 1]
表 1. CNVモデルにおける被験物質の投与方法及び投½—.量 Table 1. Test substance administration method and dose in CNV model
Figure imgf000033_0001
Figure imgf000033_0001
[0095] —CNVの評価—  [0095] —CNV evaluation—
CNVモデル実験は， 例えば， 以下のようにして評価する。 レーザー照射 後 10日目に蛍光眼底造影検査を行って， 脈絡膜新生血管を評価する。 検査 は両眼で時間をずらして実施する。 両眼ともにフルォレセイン （蛍光眼底造 影剤フルォレサイト （登録商標） 注射液 1号， 日本アルコン株式会社， 0. I mLZk g) 投与後 5分及び 10分に， それぞれ右， 左の順に連続して 2 枚ずつ撮影を行う。 撮影された写真に基づいて， 眼科専門医が評価を行う。  For example, 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.
[0096] その後， ペントバルビタールナトリウム （東京化成工業株式会社） 水溶液  [0096] Then, sodium pentobarbital (Tokyo Chemical Industry Co., Ltd.)
(64. 8mgZmL， 0. 4 m LZ k g ) の橈側皮静脈内投与により試験 動物に麻酔を行い， 体重を測定後， 放血安楽死させ， 外表， 内部器官及び組 織を肉眼的に観察する。 左右眼球 （視神経を含む） について， 摘出後 3%グ ルタールアルデヒド■ 2%パラホルムアルデヒド混合固定液で浸漬固定する 。 固定後の眼球のレーザー照射部位 （8部位） を細切し， パラフィン包埋を 行い， パラフィン切片 （1 0切片） を作製した後， H. E. 染色 （1切片の み） を施す。 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. Fix the left and right eyeballs (including the optic nerve) by immersion with 3% glutaraldehyde 2% paraformaldehyde mixed fixative after removal . After fixation, the laser-irradiated sites (8 sites) of the eyeball are cut into small pieces, embedded in paraffin, and paraffin sections (10 sections) are prepared, followed by HE staining (only one section).
[0097] 以下， 実施例に基づいて本発明を具体的に説明する。 しかしながら， 本発 明は， 以下の実施例によって限定されない。  [0097] Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited by the following examples.
[0098] —配列番号 1で表されるポリべプチドの合成— [0098] —Synthesis of Polypeptide represented by SEQ ID NO: 1—
配列番号 1 (E F S P N LWG L E FQKN KD Y Y I I ) で表されるポ リペプチドを以下のようにして合成した。 すなわち， マルチシンテック （M u I t i S y n t e c h) 社の自動べプチド合成装置 "シロ I I (S y r o I I ) " を用い， Fmo c (9 _フルォレニルメチルォキシカルボニル） ストラテジーによる固相合成法により調整した。 合成には， HBTU (2- ( 1 H) —ベンゾトリアゾール 1 _ィル） _ 1， 1， 3， 3—テトラメチル ゥロニゥムへキサフルォロホスフエ一卜） 一HOB t (1—ヒドロキシベンゾ 卜リアゾール）法 （Kn o r r, R.ら， T e t r a h e d r o n L e t t . , 1 927 ( 1 989) ) を用いた。 合成スケールは， 25マイクロモル であった。 なお， 用いたレジン及びアミノ酸は， それぞれ表 3と表 4とに示 すとおりであった。  A polypeptide represented by SEQ ID NO: 1 (EFSPN LWGLE FQKNKDYYII) 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.
[0099] (1 ) Fmo c_アミノ酸樹脂を反応ベッセル及び Fmo cアミノ酸用カー 卜リッジにとり， そのベッセル及びカートリッジを， 上記した自動ペプチド 合成装置に装着し， Fa s t Mo cプログラムにより自動合成操作を行った  [0099] (1) Take the Fmo c_amino acid resin in the reaction vessel and the cartridge for Fmo c amino acid, attach the vessel and cartridge to the above-mentioned automatic peptide synthesizer, and perform the automatic synthesis operation by the Fast Moc program. went
(2) ペプチドの自動合成に際し， まず DCM (ジクロロメタン） にて 5分 間樹脂を潤滑させた。 その後， NMP (N—メチル _2_ピロリドン）にて 5 分間処理し， 洗浄した。 その後， 以下のプロトコルを繰返し行った。 1サイ クルの合成反応は， 下表 2のとおリである。 (2) In the automatic peptide synthesis, the resin was first lubricated with DCM (dichloromethane) for 5 minutes. After that, it was treated with NMP (N-methyl_2_pyrrolidone) for 5 minutes and washed. After that, the following protocol was repeated. The synthesis reaction for one cycle is shown in Table 2 below.
[0100] [表 2] [0100] [Table 2]
表 2 ぺプチドの合成サイクル Table 2 Synthesis cycle of peptides
Figure imgf000035_0001
Figure imgf000035_0001
表 2中， DIEAは， Ν,Ν' -ジィ ソプロピルェチルァミ ンを意味する。  In Table 2, DIEA means Ν, Ν'-disopropylethylamine.
[0101] (3) 脱保護基及び脱樹脂操作 [0101] (3) Deprotection group and resin removal operation
上記のようにして得られた合成ペプチドを T F Α (トリフルォロ酢酸） Z水 /卜リエチルシラン （T r i e t h y l s i l a n) [90:5:5] を用い ， 室温にて 2時間反応させた。 反応混合液から樹脂をろ別し， トリフルォロ 酢酸 1 m Iにて 2回洗浄し， ろ液及び洗液をあわせたものに氷冷下エーテル 1 00m l を加え， 生じた沈殿物を遠心分離し， 残渣をデカンテ一シヨンに より上清から分離した。  The synthetic peptide obtained as described above was reacted at room temperature for 2 hours using TFΑ (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.
[0102] (4) 精製  [0102] (4) Purification
得られた残渣を， 氷冷下エーテルにて洗浄した後， 遠心分離し， 上清を逆 相高速液体クロマ卜グラフィ一により精製した。 逆相高速液体クロマトグラ フィ一は， 島津社製 H P LC "LC 1 0AD" を用い， カラムはクロマシル (K r oma s i I ) KR "1 00_ 1 0C 1 8" の 250 x 3 Ommのも のを用いた。 逆相高速液体クロマトグラフィーの条件は， 溶離液として， 0 . 1 %T F A水溶液 （A液） 及び 80%ァセトニトリルを含む 0. 1 %T F A水溶液 （B液） の 2液グラジェン卜で， 当初 B液 5%， 30分後に B液 8 0%, 検出波長 21 0 nmであった。 精製されたべプチドを含む画分を回収 後， 凍結乾燥した。  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. Was used. 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.
[0103] (5) ポリべプチドの分析  [0103] (5) Polypeptide analysis
最終的に精製されたペプチドを， 島津社製 H P LC "LC 1 0AD" 及び ， クロマシル （K r oma s i I ) KR "1 00_ 1 0C 1 8" の 250 x 4 (又は 6) mmのカラムを用いた高速液体クロマトグラフィーにより確認 した。 また， 最終的に精製されたべプチドを， ブル力一 （B r u k e r) 社 製 MA L D I— TO F質量分析装置 "リフレックス （Re f l e x) I I " によっても確認した。 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.
[表 3] [Table 3]
表 3 実施例 1.で用いた F m o c レジン · o  Table 3 F m o c resin used in Example 1 o
：ト : G
； ;
Figure imgf000036_0001
[表 4]
Figure imgf000036_0001
[Table 4]
表 4 ¾施例 1で用いた Fmoc アミ ノ酸を示す  Table 4 ¾Shows the Fmoc amino acids used in Example 1
レジン M. W.  Resin M. W.
Λ Fmoc-Ala-0H 311.3  Λ Fmoc-Ala-0H 311.3
R Fmoc-Arg(Pbf)-0H 648.8  R Fmoc-Arg (Pbf) -0H 648.8
C Fmoc-Cys (Trt) -OH 585.7  C Fmoc-Cys (Trt) -OH 585.7
Q Fmoc- Gin (Trt)- OH 610.7  Q Fmoc- Gin (Trt)-OH 610.7
E Pmoc-Glu(otBu)-0H 425.5  E Pmoc-Glu (otBu) -0H 425.5
G ト' moc - G〗y - OH 297.3  G 'moc-G〗 y-OH 297.3
H Pmoc-His (Trt)-()H  H Pmoc-His (Trt)-() H
T 353.4  T 353.4
L Fmoc■■■■ Leu Oil 353.4  L Fmoc ■■■■ Leu Oil 353.4
K K
M Fmoc '■ Met - Oil M Fmoc '■ Met-Oil
F Fmoc - Phe-OII 387.4  F Fmoc-Phe-OII 387.4
P Fmoc - Pro - OH  P Fmoc-Pro-OH
S Fmoc Ser (tBu) - OH  S Fmoc Ser (tBu)-OH
T Fmoc-Thr (tBu)-OH 397.5  T Fmoc-Thr (tBu) -OH 397.5
W Fmoc-Trp (Boc) -OH 526.6  W Fmoc-Trp (Boc) -OH 526.6
Y Fmoc-Tyr (tBu)-OH 459.6  Y Fmoc-Tyr (tBu) -OH 459.6
V Fmoc-Val - OH 339.4  V Fmoc-Val-OH 339.4
Fmoc-Asn (Trt) -OH 596.7  Fmoc-Asn (Trt) -OH 596.7
D Fmoc - Asp (OtBu) - OH 411.5 実施例 2 D Fmoc-Asp (OtBu)-OH 411.5 Example 2
[0106] 一細胞培養一  [0106] One cell culture
本実施例は， HUVEC (人臍帯静脈内皮細胞） を用いて行った。 細胞株 は 37 °Cの恒温槽で解凍し， 1 0m lの血管内皮細胞培地 （H ume d i a ， EG_2キット， クラボウ製） に溶解後， タイプ Iコラーゲンコート 1 0 cm培養皿 （ I WAK I製） 1枚に全量をプレーティングした。 その後， 3 7°C, C02Za i r =6%Z94%で H U VECを 24時間培養した。 DM SOを除去するため， EG— 2培地 1 Om Iに培地の交換を行った。 80〜 90%コンフルェント （c o n f l u e n t) の状態になったら継代培養を 行った。 この時の細胞濃度は， およそ 5 X 1 06個 ZD i s h程度であった。 In this example, HUVEC (human umbilical vein endothelial cells) was used. 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) ) The whole amount was plated on one sheet. After that, HU VEC was cultured for 24 hours at 37 ° C, C0 2 Zair = 6% Z94%. To remove DMSO, 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.
72時間程でコンフルェントになった。  It became confluent in about 72 hours.
[0107] コラーゲンコート 6ゥエルプレート （ IWAK I製） に 5 X 1 05個 Zwe [0107] Collagen coated 6well plate (made by IWAK I) with 5 X 1 0 5 pieces Zwe
I I程度でプレーティングを行った。 培養を続け， コンフルェントになった ら血管内皮細胞培地 （H ume d i a, EG— 2キット， クラボウ製） から 細胞増殖因子 （h EG F, h FG F— B) を除いた培地 （6 we I Iの場合は 2m l ) に培地交換して 24時間培養した （s t a r V a t i o n) 。 培地 を除き， 血管内皮細胞培地 （H ume d i a， EG— 2キット， クラボウ製 ) から細胞増殖因子を除いた培地にぺプチド分解酵素の阻害剤バシトラシン（ 最終濃度 1 00マイクロ gZm I )を加えたものをコントロールとして加えた 。 他の処理群は， このトンコロール培地でペプチド及びエフリン B 2を希釈 して細胞に加え， 1 0分後 （もしくは適当な時間を設定） に PBSで 2回洗 浄した後， ライシスバッファ （L y s i s Bu f f e r) (6 w e I Iの場 合は 0. 2m l Zwe l I ) を用いて細胞の全細胞ライセートを回収した。 実施例 3  I plated at about I. When the culture is continued and confluent, 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 In some cases, 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. In the other treatment group, 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
[0108] _ p 44Z42 M A P Kリン酸化に対する配列番号 1で表されるアミノ酸 配列を有するぺプチドの効果一  [0108] _p 44Z42 Effect of peptide having amino acid sequence represented by SEQ ID NO: 1 on M A P K phosphorylation
p 44Z42MAP Kリン酸化に対するエフリン B 2， 実施例 1で得られ たべプチド， 及びそれらの併用剤の効果を以下のように実証した。 p 44Z42MAP K phosphorylation ephrin B 2, obtained in Example 1 The effects of the peptide and their combination were demonstrated as follows.
血管内皮細胞を， 血管内皮細胞培地 （H ume d i a EG_2キット， ク ラボゥ製） から細胞増殖因子を除いた培地にぺプチド分解酵素の阻害剤バシ トラシン （最終濃度 1 00 gZm I ) を加えたものをコントロールとし， 実施例 1で得られたぺプチド （配列番号 1で表されるぺプチドである。 以下 ， 「配列番号 1のペプチド」 ともよぶ） とエフリン B 2 (エフリン B 2の細 胞外ドメインと F cとの融合タンパク質である） を混合したものなどを用い て 1 0分間処理した。 ドデシル硫酸ナトリウム—ポリアクリルアミド電気泳 動 （SDS— PAGE) は 1 0%のアクリルアミドを用し、， 200ポルトに て 40分間の条件で分離し， ゥエツ卜方式で 1 00ポル卜の条件下一時間 P VD F膜に転写した。 3%スキムミルクで 1時間ブロッキングした後， 膜を 一次抗体としてリン酸化 P 44/42MAPKに対する抗体 （Ce l l S i g n a I I n g t e c h n o l o g y子ェ， D a n v e r s , A, U t> A) を用い， これとともに 4°Cにてー晚インキュベートした （1 ： 2， 0 00) 。 TBS_ tで 1 0分間 X 4回の洗浄後， 膜を西洋ヮサビペルォキシ ダーゼ標識 2次抗体 （B i o_Ra d, R i c hmo n d, C A, USA ) (1 : 20， 000)と共に室温にて 40分間インキュベートした。 可視化を 製造業者の指示に従ってアマシャム社製増強化学発光 （ECL) 検出システ ムを用いて行った。  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). 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). After washing with TBS_t for 10 minutes x 4 times, 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.
図 1は， 実施例 3における p 44Z42MAPKリン酸化に対するエフリ ン B2， 配列番号 1のペプチド， 及びそれらの併用剤の結果を示すウェスタ ンブロッテイングの写真である。 図 1中， 第 1 レーンはコントロールを示し ；第 2レーンは， 配列番号 1の 1 00 nMのぺプチドで処理したものを示し ；第 3レーンは， 3 n Mのエフリン B 2で処理したものを示し；第 4レーン は， 200 η Μの配列番号 1のぺプチドと 6 η Μのエフリン Β 2で同時に処 理したものを示し；第 5レーンは， 200 nMの配列番号 1のぺプチドで処 理し， 3分後に 6 nMのエフリン B 2で処理したものを示し；第 6レーンは ， 6 n Mのエフリン B 2で処理し， 3分後に 200 n Mの配列番号 1のぺプ チドで処理したものを示す。 なお， 処理開始から 1 0分後の全細胞ライセー 卜回収時における第 4， 第 5および第 6レーンにおける最終濃度は， それぞ れ配列番号 1のぺプチドが 1 00 nM， エフリン B 2が 3 nMであった。 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. In Figure 1, 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 η Μ peptide with SEQ ID NO: 1 and 6 η エ ephrin Β 2 processed simultaneously; Lane 5 shows 200 nM with SEQ ID NO: 1 peptide. Treated and treated with 6 nM ephrin B 2 after 3 minutes; Lane 6 is treated with 6 nM ephrin B 2 and after 3 minutes 200 nM SEQ ID NO: 1 peptide The one treated with chido. 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 SEQ ID NO: 1 and 3 for ephrin B 2 respectively. nM.
[0110] また， 図 1中， 第 7レーンはコントロールを示し；第 8レーンは， 1 0 η Μの配列番号 1のぺプチドで処理したものを示し；第 9レーンは， 3 nMの エフリン B 2で処理したものを示し；第 1 0レーンは， 20 η Μの配列番号 1のぺプチドと 6 nMのエフリン Β 2で同時に処理したものを示し；第 1 1 レーンは， 20 n Mの配列番号 1のペプチドで処理し， 3分後に 6 nMのェ フリン B 2で処理したものを示し；第 1 2レーンは， 6 n Mのエフリン B 2 で処理し， 3分後に 20 nMの配列番号 1のぺプチドで処理したものを示す 。 なお， 処理開始から 1 0分後の全細胞ライセート回収時における第 1 0， 第 1 1及び第 1 2レーンにおける最終濃度は， それぞれ配列番号 1のべプチ ドが 1 0 nM， エフリン B 2が 3 n Mであった。  [0110] Also, in Figure 1, lane 7 shows control; lane 8 shows that treated with the peptide of SEQ ID NO: 1 of 10 ηΜ; lane 9 shows 3 nM ephrin B The first lane shows the 20 η ぺ peptide of SEQ ID NO: 1 and 6 nM ephrin Β 2 treated simultaneously; the first lane shows the 20 nM sequence. Treatment with peptide number 1 and treatment with 6 nM ephrin B 2 after 3 minutes; Lane 12 shows treatment with 6 nM ephrin B 2 and 3 minutes later with 20 nM SEQ ID NO: Shown is processed with 1 peptide. 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.
[0111] また， 図 1に， 一次抗体としてリン酸化■脱リン酸化型を問わずすべての p 44Z42MAPKを認識する抗体， または， Q?—チューブリンを認識する 抗体を用いてウェスタンブロッテイング解析した写真を示す。  [0111] In addition, 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.
[0112] 第 1 レーンと第 3レーンを比較すると， エフリン B2は p 44Z42MA PKリン酸化活性を阻害することがわかる。 第 7レーンと第 9レーンとを比 較しても同様のことがいえる。 また， 第 1 レーンと第 2レーンを比較すると ， 配列番号 1のべプチドは血清による p 44Z42MAPKリン酸化活性を 阻害することがわかる。 第 7レーンと第 8レーンとを比較しても同様のこと がいえる。  [0112] 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. In addition, comparing the first and second lanes, it can be seen that 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.
[0113] ただし， 第 2レーンと第 3レーンを比較すると， 配列番号 1のペプチドは ， エフリン B 2に比べて血清による p 44Z42MAPKリン酸化活性の阻 害能が高くない可能性がわかる。 第 8レーンと第 9レーンとを比較しても同 様のことがいえる。 しかしながら， 第 3レーンと， 第 5レーン及び第 6レー ンを比較すると， エフリン B 2を単独で投与する場合に比べて時間差をおい てエフリン B 2と配列番号 1のべプチドを投与する併用剤は， 血清による p 44Z42MAPKリン酸化活性の高い阻害能を有することがわかる。 特に 第 6レーンによって示されるとおり， エフリン B 2で処理した後に， 配列番 号 1のぺプチドで処理することで， 血清による p 44Z42MAPKリン酸 化活性の極めて高い阻害能を有することがわかる。 これらの結果は， 第 9レ ーンと， 第 1 1 レーン及び第 1 2レーンによっても同様のことがいえる。 実施例 4 [0113] However, comparing the second lane and the third lane, it is clear that the peptide of SEQ ID NO: 1 may not have a high ability to inhibit p44Z42MAPK phosphorylation activity by serum compared to ephrin B2. The same can be said for the 8th and 9th lanes. However, comparing the third lane with the fifth lane and the sixth lane, a concomitant drug that administers ephrin B 2 and the peptide of SEQ ID NO: 1 with a time lag compared to when ephrin B 2 is administered alone. P by serum It can be seen that 44Z42MAPK phosphorylation activity has high inhibitory ability. In particular, as shown by the 6th lane, treatment with ephrin B 2 followed by treatment with the peptide of SEQ ID NO: 1 reveals that it has an extremely high ability to inhibit p44Z42MAPK phosphorylation activity by serum. These results are the same for the ninth lane and the first and second lanes. Example 4
[0114] エフリン B 2及び配列番号 1のペプチドの量を変えた以外は， 実施例 3と 同様にしてウェスタンプロットを行った。 図 2は， 実施例 4における p 44 Z42MAPKリン酸化に対するエフリン B 2を投与した結果を示すウェス タンブロッテイング解析した写真である。 図 2中， 第 1及び第 6レーンはコ ントロールを示し， 第 2レーンは本発明の 80 n Mのエフリン B 2で処理後 1 0分が経過したものを示し， 第 3レーンは本発明の 30 nMのエフリン B 2で処理後 1 0分が経過したものを示し， 第 4レーンは本発明の 9 nMのェ フリン B 2で処理後 1 0分が経過したものを示し， 第 5レーンは本発明の 3 门|\|のェフリン82で処理後1 0分後が経過したものを示す。 第 7レーンは 本発明の 80 nMのエフリン B 2で処理後 30分後が経過したものを示し， 第 8レーンは本発明の 30 nMのエフリン B 2で処理後 30分後が経過した ものを示し， 第 9レーンは本発明の 9 n Mのエフリン B 2で処理後 30分後 が経過したものを示し， 第 1 0レーンは本発明の 3 nMのエフリン B 2で処 理後 30分後が経過したものを示す。  [0114] Western plotting was performed in the same manner as in Example 3 except that the amounts of ephrin B 2 and the peptide of SEQ ID NO: 1 were changed. 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. In Fig. 2, 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, and 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 処理 | \ | 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.
また， 図 2に， 上記と同じ全細胞ライセ一トを， リン酸化■脱リン酸化型 を問わず P 44Z42MAPKを認識する抗体， または， Q?—チューブリン を認識する抗体を用いてウェスタンブロッテイング解析した写真を示す。 第 1 レーンと第 2， 3， 4， 5レーンを比較すると， 3门1\1から80门1\1まで のエフリン B 2は MAP Kリン酸化活性を阻害することが分かる。 第 7レー ンと第 8， 9, 1 0， 1 1， 1 2レーンを比較しても同様のことがいえる。 実施例 5  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 门 1 \ 1 to 80 门 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
[0115] エフリン B 2及び配列番号 1のペプチドの量を変えた以外は， 実施例 3と 同様にしてウェスタンプロットを行った。 図 3は， 実施例 5における p 44[0115] 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.
Z42MA P Kリン酸化に対するエフリン B 2， 配列番号 1のペプチド， 及 びそれらの併用剤の結果を示すウェスタンブロッテイング解析した写真であ る。 図 3中， 第 1及び第 7レーンはコントロールを示し， 第 2レーンは 1 0 0 Mの配列番号 1のペプチドで処理後 1 0分が経過したものを示し， 第 3 レーンは 3 n Mの本発明のエフリン B 2で処理後 1 0分が経過したものを示 し， 第 4レーンは 200 Mの配列番号 1のぺプチドと 6 n Mのエフリン B 2で同時に処理後 1 0分後が経過したものを示し， 第 5レーンは 200 M の配列番号 1のぺプチドで処理し， 3分後に 6 n Mの本発明のエフリン B 2 で処理後 1 0分後が経過したものを示し， 第 6レーンは 6 n Mの本発明のェ フリン B 2で処理し， 3分後に 200 Mの配列番号 1のぺプチドで処理後 1 0分後が経過したものを示す。 第 8レーンは 1 Mの配列番号 1のべプチ ドで処理後 1 0分後が経過したものを示し， 第 9レーンは 3 n Mのエフリン B 2で処理後 1 0分後が経過したものを示し， 第 1 0レーンは 2 Mの配列 番号 1のぺプチドと 6 n Mの本発明のエフリン B 2で同時に処理後 1 0分後 が経過したものを示し， 第 1 1 レーンは 2 Mの配列番号 1のペプチドで処 理し， 3分後に 6 n Mの本発明のエフリン B 2で処理後 1 0分後が経過した ものを示し， 第 1 2レーンは 6 n Mの本発明のエフリン B 2で処理し， 3分 後に 2 Mの配列番号 1のべプチドで処理後 1 0分後が経過したものを示す 。 なお， 処理開始から 1 0分後の全細胞ライセート回収時における第 4， 第 5及び第 6レーンにおける最終濃度は， それぞれ配列番号 1のべプチドが 1 Ο Ο β Μ, エフリン Β 2が 3 η Μであった。 また， 処理開始から 1 0分後の 全細胞ライセート回収時における第 1 0， 第 1 1及び第 1 2レーンにおける 最終濃度は， それぞれ配列番号 1のペプチドが 1 Μ， エフリン Β 2が 3 η Μでめった。 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. In Fig. 3, the first and seventh lanes show controls, the second lane shows 10 minutes after treatment with 100 M peptide of SEQ ID NO: 1, and the third lane shows 3 nM. 10 minutes after treatment with ephrin B 2 of the present invention is shown, and 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, and 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 Ο Ο β Μ for SEQ ID NO: 1 and 3 η for ephrin Β 2 respectively. It was a spear. In addition, 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 が for the peptide of SEQ ID NO: 1 and 3 ηΜ for ephrin Β2, respectively. It was rare.
また， 図 3に， 一次抗体としてリン酸化■脱リン酸化型を問わずすべての ρ 44Ζ42ΜΑ Ρ Κを認識する抗体， または， Q?—チューブリンを認識す る抗体を用いてウェスタンブロッテイング解析した写真を示す。 第 1 レーン と第 3レーンを比較すると， エフリン B 2は MAP Kリン酸化活性を阻害す ることがわかる。 第 7レーンと第 9レーンとを比較しても同様のことがいえ る。 また， 第 1 レーンと第 2レーンを比較すると， 配列番号 1のペプチドは MAP Kリン酸化活性を阻害することがわかる。 第 7レーンと第 8レーンと を比較しても同様のことがいえる。 Figure 3 also shows Western blotting analysis using antibodies that recognize all ρ 44Ζ42ΜΑ Κ ず 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.
ただし， 第 2レーンと第 3レーンを比較すると， 配列番号 1のペプチドは ， エフリン B 2に比べて M A P Kリン酸化活性の阻害能が高くない可能性が わかる。 第 8レーンと第 9レーンとを比較しても同様のことがいえる。 しか しながら， 第 3レーンと， 第 5レーン及び第 6レーンを比較すると， エフリ ン B 2を単独で投与する場合に比べて時間差をおいてエフリン B 2と配列番 号 1のべプチドを投与する併用剤は， 高い MAP Kリン酸化活性の阻害能を 有することがわかる。 特に第 6レーンによって示されるとおり， エフリン B 2で処理した後に， 配列番号 1のペプチドで処理することで， 極めて高い M APKリン酸化活性の阻害能を有することがわかる。 これらの結果は， 第 9 レーンと， 第 1 1 レーン及び第 1 2レーンによっても同様のことがいえる。 実施例 6  However, comparing the 2nd and 3rd lanes, it is clear that the peptide of SEQ ID NO: 1 may not have a higher ability to inhibit the MA PK phosphorylation activity than ephrin B2. The same can be said by comparing the 8th and 9th lanes. However, comparing the third lane with the fifth and sixth lanes, ephrin B 2 and SEQ ID NO: 1 peptide were administered with a time lag compared to when ephrin B 2 was administered alone. It can be seen that the concomitant drug has high MAP K phosphorylation inhibitory activity. In particular, as shown by the 6th lane, treatment with ephrin B 2 followed by treatment with the peptide of SEQ ID NO: 1 reveals that it has a very high ability to inhibit the phosphorylation activity of M APK. These results are the same for the 9th lane and the 11th and 1st lanes. Example 6
配列番号 2 (E F S P N LWG L E FQKN K) で表されるアミノ酸配列 からなるペプチドを実施例 1と同様にして得て （得られたペプチドを 「配列 番号 2のペプチド」 ともよぶ） ， 実施例 3と同様にしてウェスタンプロット を行った。 図 4は， 実施例 6における p 44Z42MAPKリン酸化に対す るエフリン B 2， 配列番号 2のペプチド， 及びそれらの併用剤の結果を示す ウェスタンブロッテイング解析した写真である。 図 4中， 第 1 レーンはコン トロールを示し， 第 2レーンは本発明の 1 00 nMの配列番号 1のぺプチド で処理後 1 3分が経過したものを示し， 第 3レーンは本発明の 1 OO nMの 配列番号 2のペプチドで処理後 1 3分が経過したものを示し， 第 4レーンは 本発明の 3 nMのエフリン B 2で処理後 1 3分が経過したものを示し， 第 5 レーンは本発明の 6 nMのエフリン B2で処理後 3分が経過しさらに本発明 の 200 nMの配列番号 1のぺプチドで処理後 1 0分が経過したものを示し ， 第 6レーンは本発明の 6 nMのエフリン B 2で処理後 3分が経過しさらに 本発明の 200 nMの配列番号 2のぺプチドで処理したものを示す。 なお， 処理開始から 1 3分後の全細胞ライセ一卜回収時における第 5， 第 6レーン における最終濃度は， それぞれ配列番号 1のべプチドが 1 00 n M， 配列番 号 2のぺプチドが 1 00 n Mおよびエフリン B 2が 3 n Mであった。 A peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (EFSPN LWG LE FQKNK) was obtained in the same manner as in Example 1 (the obtained peptide is also referred to as “peptide of SEQ ID NO: 2”). A Western plot was performed in the same manner. 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. In Fig. 4, 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, and the third lane shows the present invention. 1 1 3 minutes after treatment with OO nM peptide 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.
[0118] 第 1 レーンと第 4レーンを比較すると， エフリン B2は MAPKリン酸化 活性を阻害することがわかる。 また， 第 4レーンと第 6レーンを比較すると ， エフリン B 2を単独で投与する場合に比べて時間差をおいてエフリン B 2 と配列番号 2のべプチドを投与する併用剤は， 高い MAP Kリン酸化活性の 阻害能を有することがわかる。 [0118] Comparing the first and fourth lanes shows that ephrin B2 inhibits MAPK phosphorylation activity. In addition, when comparing lane 4 and lane 6, the combination of ephrin B 2 and SEQ ID NO: 2 peptide with a time difference compared to ephrin B 2 administered alone has a higher MAP K phosphorus It can be seen that it has the ability to inhibit oxidation activity.
実施例 7  Example 7
[0119] 実施例 3と同様にしてウェスタンプロットを行った。 図 5は， 実施例 7に おける P 44Z42MAPKリン酸化に対するエフリン B 2， 配列番号 2の ぺプチド， 及びそれらの併用剤の結果を示すウェスタンブロッテイング解析 した写真である。 図 7中， 第 1 レーンはコントロールを示し， 第 2レーンは 本発明の 1 00 η Μの配列番号 2のぺプチドで処理後 1 0分が経過したもの を示し， 第 3レーンは本発明の 3 nMのエフリン Β 2で処理後 1 0分が経過 したものを示し， 第 4レーンは本発明の 6 n Mのエフリン B2と 200 nM の配列番号 1のペプチドで同時に処理後 1 0分が経過したものを示し， 第 5 レーンは本発明の 200 n Mの配列番号 1のぺプチドで処理後 3分が経過し さらに本発明の 3 nMのエフリン B 2で処理したものを示す。 第 6レーンは 本発明の 6 nMのエフリン B2で処理後 3分が経過しさらに本発明の 200 nMの配列番号 1のぺプチドで処理したものを示す。  [0119] Western plotting was performed in the same manner as in Example 3. 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. In FIG. 7, 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 ηΜ of the present invention, and the third lane shows the present invention. Shows 10 minutes after treatment with 3 nM ephrin 示 し 2, and lane 4 shows 10 minutes after treatment with 6 nM ephrin B2 of the present invention and 200 nM peptide of SEQ ID NO: 1 simultaneously. 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.
なお， 処理開始から 1 0分後の全細胞ライセ一ト回収時における第 4， 第 5 ， 第 6レーンにおける最終濃度は， それぞれ配列番号 1のべプチドが 1 00 nMおよびエフリン B 2が 3 nMであった。 第 1 レーンと第 2レーンを比較 すると， 配列番号 2のべプチドは MAP Kリン酸化活性を阻害することがわ かる。 産業上の利用可能性 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. Industrial applicability
本発明のペプチド， 剤， 補助剤， 医薬組成物及び医療用キットは， 特に眼 内の新生血管に関する疾患の治療又は予防などに有効であるから， 医薬産業 などにおいて利用されうる。  Since the 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.

Claims

請求の範囲 The scope of the claims
[1 ] 配列番号 1で表されるアミノ酸配列からなるペプチド， 又はその薬理学的 に許容される塩。  [1] A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, or a pharmacologically acceptable salt thereof.
[2] 配列番号 1において， 1又は 2個のアミノ酸が欠失， 置換， 挿入若しくは 付加されたアミノ酸配列からなり，エフリン B 2の新生血管の阻害活性を高め るべプチド， 又はその薬理学的に許容される塩。  [2] A peptide 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 a peptide that enhances the inhibitory activity of ephrin B 2 on new blood vessels, or a pharmacological thereof Acceptable salt.
[3] 配列番号 2で表されるアミノ酸配列からなるペプチド， 又はその薬理学的 に許容される塩。  [3] A peptide comprising the amino acid sequence represented by SEQ ID NO: 2, or a pharmacologically acceptable salt thereof.
[4] 配列番号 2において， 1又は 2個のアミノ酸が欠失， 置換， 挿入若しくは 付加されたアミノ酸配列からなり，エフリン B 2の新生血管の阻害活性を高め るべプチド， 又はその薬理学的に許容される塩。  [4] In SEQ ID NO: 2, consisting of an amino acid sequence in which one or two amino acids are deleted, substituted, inserted or added, and a peptide that enhances the inhibitory activity of ephrin B 2 on neovascularization, or a pharmacological thereof Acceptable salt.
[5] 配列番号 3で表されるアミノ酸配列を具備するペプチドであって， 当該べ プチドに含まれるアミノ酸残基の数が 9以上， 1 0 0以下であるペプチド， 又はその薬理学的に許容される塩。 [5] A peptide having the amino acid sequence represented by SEQ ID NO: 3, wherein the number of amino acid residues contained in the peptide is 9 or more and 100 or less, or a pharmacologically acceptable peptide thereof Salt.
[6] 請求項 1から請求項 5のいずれかに記載のペプチド， 又はそれらの薬理学 的に許容される塩を含有する新生血管阻害用補助剤。 [6] An adjuvant for neovascularization containing the peptide according to any one of claims 1 to 5, or a pharmacologically acceptable salt thereof.
[7] 請求項 1から請求項 5のいずれかに記載のペプチド， 又はそれらの薬理学 的に許容される塩を含有する血管内皮細胞における D N A合成阻害用補助剤 [7] An adjuvant for inhibiting DNA synthesis in vascular endothelial cells, comprising the peptide according to any one of claims 1 to 5, or a pharmacologically acceptable salt thereof.
[8] 請求項 1から請求項 5のいずれかに記載のペプチド， 又はそれらの薬理学 的に許容される塩を含有する血管内皮細胞における P 4 4 Z p 4 2 M A P K リン酸化活性阻害用補助剤。 [8] Aid for inhibiting P 4 4 Z p 4 2 MAPK phosphorylation activity in vascular endothelial cells containing the peptide according to any one of claims 1 to 5 or a pharmacologically acceptable salt thereof Agent.
[9] 請求項 1から請求項 5のいずれかに記載のペプチド， 又はそれらの薬理学 的に許容される塩を含有する新生血管阻害剤。  [9] A neovascular inhibitor comprising the peptide according to any one of claims 1 to 5, or a pharmacologically acceptable salt thereof.
[10] 請求項 1から請求項 5のいずれかに記載のペプチド， 又はそれらの薬理学 的に許容される塩を含有する， "加齢黄斑変性， 虚血性網膜症， 眼内血管新 生， 角膜血管新生， 網膜血管新生， 脈絡膜血管新生， 糖尿病性黄斑浮腫， 糖 尿病性網膜虚血， 糖尿病性網膜浮腫， 糖尿病性網膜症" からなる群から選択 される， 1又は 2種以上の疾患の治療剤又は予防剤。 [10] The peptide according to any one of claims 1 to 5, or a pharmacologically acceptable salt thereof, “age-related macular degeneration, ischemic retinopathy, intraocular neovascularization, Corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diuretic retinal ischemia, diabetic retinal edema, diabetic retinopathy A therapeutic or prophylactic agent for one or more diseases.
[11 ] エフリン B 2又はその薬理学的に許容される塩と；  [11] Ephrin B 2 or a pharmacologically acceptable salt thereof;
( i )配列番号 1で表されるアミノ酸配列からなるぺプチド， もしくはその薬 理学的に許容される塩， 又は  (i) a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, or
( i i )配列番号 1において， 1又は 2個のアミノ酸が欠失， 置換， 挿入若し <は付加されたアミノ酸配列からなり，前記ェフリン B 2の新生血管の阻害活 性を高めるペプチド， もしくはそれらの薬理学的に許容される塩と； を含有する医薬用組成物。  (ii) A peptide consisting of an amino acid sequence in which 1 or 2 amino acids have been deleted, substituted, inserted or <added in SEQ ID NO: 1, and which enhances the inhibitory activity of the above-mentioned ephrin B 2 on new blood vessels, or they And a pharmacologically acceptable salt thereof.
[12] エフリン B 2又はその薬理学的に許容される塩と； [12] Ephrin B 2 or a pharmacologically acceptable salt thereof;
( i )配列番号 2で表されるアミノ酸配列からなるぺプチド， もしくはその薬 理学的に許容される塩， 又は  (i) a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, or
( i i )配列番号 2において， 1又は 2個のアミノ酸が欠失， 置換， 挿入若し <は付加されたアミノ酸配列からなり，前記ェフリン B 2の新生血管の阻害活 性を高めるペプチド， もしくはそれらの薬理学的に許容される塩と； を含有する医薬用組成物。  (ii) In SEQ ID NO: 2, a peptide consisting of an amino acid sequence in which 1 or 2 amino acids have been deleted, substituted, inserted or <added, and which enhances the inhibitory activity of ephrin B 2 on neovascularization, or And a pharmacologically acceptable salt thereof.
[13] エフリン B 2又はその薬理学的に許容される塩と； [13] Ephrin B 2 or a pharmacologically acceptable salt thereof;
配列番号 3で表されるアミノ酸配列を具備するべプチドであって， 当該べ プチドに含まれるアミノ酸残基の数が 9以上， 1 0 0以下であるペプチド， 又はその薬理学的に許容される塩と  A peptide having the amino acid sequence represented by SEQ ID NO: 3, wherein the number of amino acid residues contained in the peptide is 9 or more and 100 or less, or a pharmacologically acceptable peptide thereof With salt
を含有する医薬用組成物。  A pharmaceutical composition comprising
[14] 前記エフリン B 2が， 天然のエフリン B 2の細胞外ドメインを含み， 細胞 質ドメインを含まない， 請求項 1 1から請求項 1 3のいずれかに記載の医薬 用組成物。 14. The pharmaceutical composition according to any one of claims 11 to 13, wherein the ephrin B 2 contains an extracellular domain of natural ephrin B 2 and does not contain a cytoplasmic domain.
[15] 前記エフリン B 2が， ヒト F cとエフリン B 2の融合タンパク質である請 求項 1 1から請求項 1 3のいずれかに記載の医薬用組成物。  [15] The pharmaceutical composition according to any one of claims 11 to 13, wherein the ephrin B2 is a fusion protein of human Fc and ephrin B2.
[16] 新生血管阻害剤である請求項 1 1から請求項 1 3のいずれかに記載の医薬 用組成物。  [16] The pharmaceutical composition according to any one of claims 11 to 13, which is a neovascular inhibitor.
[17] 眼内の新生血管阻害剤である請求項 1 1から請求項 1 3のいずれかに記載 の医薬用組成物。 [17] The method according to any one of claims 11 to 13, which is an intraocular neovascular inhibitor. Pharmaceutical composition.
[18] 血管形成または血管新生に関連する疾患または障害の治療剤である請求項  [18] The therapeutic agent for a disease or disorder associated with angiogenesis or neovascularization
1 1から請求項 1 3のいずれかに記載の医薬用組成物。  11. The pharmaceutical composition according to any one of claims 1 to 13.
[19] 眼内の新生血管に関連する疾患の治療剤又は予防剤である請求項 1 1から 請求項 1 3のいずれかに記載の医薬用組成物。  [19] The pharmaceutical composition according to any one of claims 11 to 13, which is a therapeutic or prophylactic agent for a disease associated with neovascularization in the eye.
[20] "加齢黄斑変性， 虚血性網膜症， 眼内血管新生， 角膜血管新生， 網膜血管 新生， 脈絡膜血管新生， 糖尿病性黄斑浮腫， 糖尿病性網膜虚血， 糖尿病性網 膜浮腫， 糖尿病性網膜症" からなる群から選択される， 1又は 2種以上の疾 患の治療剤又は予防剤である請求項 1 1から請求項 1 3のいずれかに記載の 医薬用組成物。  [20] "Age-related macular degeneration, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retinal ischemia, diabetic retina edema, diabetic The pharmaceutical composition according to any one of claims 11 to 13, which is a therapeutic or prophylactic agent for one or more diseases selected from the group consisting of "retinopathy".
[21 ] 加齢黄斑変性及び糖尿病性網膜症のいずれか又は両方の疾患の治療剤又は 予防剤である請求項 1 1から請求項 1 3のいずれかに記載の医薬用組成物。  [21] The pharmaceutical composition according to any one of claims 11 to 13, which is a therapeutic or prophylactic agent for either or both of age-related macular degeneration and diabetic retinopathy.
[22] 腫瘍， 慢性関節リウマチ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫， 又は固形癌の転移の治療剤である請求項 1 1から請求項 1 3のいずれかに記 載の医薬用組成物。  [22] The pharmaceutical composition according to any one of claims 11 to 13, which is a therapeutic agent for metastasis of tumor, rheumatoid arthritis, psoriasis, atherosclerosis, force positive sarcoma, or solid cancer. object.
[23] エフリン B 2又はその薬理学的に許容される塩， 及び薬理学的に許容され る担体を含有する第 1の組成物と；  [23] a first composition comprising ephrin B2 or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable carrier;
( i )配列番号 1で表されるアミノ酸配列からなるぺプチド， もしくはその薬 理学的に許容される塩， 又は  (i) a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, or
( i i )配列番号 1において， 1又は 2個のアミノ酸が欠失， 置換， 挿入若し <は付加されたアミノ酸配列からなり，前記ェフリン B 2の新生血管の阻害活 性を高めるぺプチド， もしくはそれらの薬理学的に許容される塩， 及び薬理学的に許容される担体を含有する第 2の組成物と；  (ii) in SEQ ID NO: 1, consisting of an amino acid sequence in which 1 or 2 amino acids have been deleted, substituted, inserted, or <added, and a peptide that enhances the inhibitory activity of the ephrin B 2 on neovascularization, or A second composition comprising a pharmacologically acceptable salt thereof and a pharmacologically acceptable carrier;
を含有する医療用キット。  Containing a medical kit.
[24] エフリン B 2又はその薬理学的に許容される塩， 及び薬理学的に許容され る担体を含有する第 1の組成物と； [24] a first composition comprising ephrin B 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier;
( i )配列番号 2で表されるアミノ酸配列からなるぺプチド， もしくはその薬 理学的に許容される塩， 又は ( i i )配列番号 2において， 1又は 2個のアミノ酸が欠失， 置換， 挿入若し <は付加されたアミノ酸配列からなり，前記ェフリン B 2の新生血管の阻害活 性を高めるぺプチド， もしくはそれらの薬理学的に許容される塩， 及び薬理学的に許容される担体を含有する第 2の組成物と； (i) a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, or (ii) in SEQ ID NO: 2, consisting of an amino acid sequence in which 1 or 2 amino acids have been deleted, substituted, inserted, or <added, and a peptide that increases the inhibitory activity of the ephrin B 2 on neovascularization, or A second composition comprising a pharmacologically acceptable salt thereof and a pharmacologically acceptable carrier;
を含有する医療用キット。  Containing a medical kit.
[25] エフリン B 2又はその薬理学的に許容される塩， 及び薬理学的に許容され る担体を含有する第 1の組成物と；  [25] a first composition comprising ephrin B 2 or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable carrier;
配列番号 3で表されるアミノ酸配列を具備するべプチドであって， 当該べ プチドに含まれるアミノ酸残基の数が 9以上， 1 0 0以下であるペプチド， 又はその薬理学的に許容される塩及び薬理学的に許容される担体を含有する 第 2の組成物と；  A peptide having the amino acid sequence represented by SEQ ID NO: 3, wherein the number of amino acid residues contained in the peptide is 9 or more and 100 or less, or a pharmacologically acceptable peptide thereof A second composition comprising a salt and a pharmaceutically acceptable carrier;
を含有する医療用キット。  Containing a medical kit.
[26] 前記エフリン B 2が， 天然のエフリン B 2の細胞外ドメインを含み， 細胞 質ドメインを含まない， 請求項 2 3から請求項 2 5のいずれかに記載のキッ 卜  [26] The kit according to any one of claims 23 to 25, wherein the ephrin B 2 contains an extracellular domain of natural ephrin B 2 and does not contain a cytoplasmic domain.
[27] 前記エフリン B 2が， ヒト F cとエフリン B 2の融合タンパク質である請 求項 2 3から請求項 2 5のいずれかに記載のキッ卜。  [27] The kit according to any one of claims 23 to 25, wherein the ephrin B2 is a fusion protein of human Fc and ephrin B2.
[28] 血管形成または血管新生に関連する疾患または障害の治療用キッ卜である 請求項 2 3から請求項 2 5のいずれかに記載のキッ卜。 [28] The kit according to any one of claims 23 to 25, which is a kit for treating a disease or disorder associated with angiogenesis or angiogenesis.
[29] 眼内の新生血管に関連する疾患の治療又は予防用キッ卜である請求項 2 3 から請求項 2 5のいずれかに記載のキッ卜。 [29] The kit according to any one of [23] to [25], which is a kit for treating or preventing a disease associated with neovascularization in the eye.
[30] "加齢黄斑変性， 虚血性網膜症， 眼内血管新生， 角膜血管新生， 網膜血管新 生， 脈絡膜血管新生， 糖尿病性黄斑浮腫， 糖尿病性網膜虚血， 糖尿病性網膜 浮腫， 糖尿病性網膜症" からなる群から選択される， 1又は 2種以上の疾患 の治療又は予防用キッ卜である請求項 2 3から請求項 2 5のいずれかに記載 のキッ卜。 [30] "Age-related macular degeneration, ischemic retinopathy, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic macular edema, diabetic retinal ischemia, diabetic retinal edema, diabetic The kit according to any one of claims 23 to 25, which is a kit for treatment or prevention of one or more diseases selected from the group consisting of "retinopathy".
[31 ] 加齢黄斑変性及び糖尿病性網膜症のいずれか又は両方の疾患の治療用又は 予防用キッ卜である請求項 2 3から請求項 2 5のいずれかに記載のキッ卜。 [31] The kit according to any one of claims 23 to 25, which is a kit for treating or preventing one or both of age-related macular degeneration and diabetic retinopathy.
[32] 腫瘍， 慢性関節リウマチ， 乾癬， ァテローム性動脈硬化症， 力ポジ肉腫， 又は固形癌の転移の治療用キッ卜である請求項 2 3から請求項 2 5のいずれ かに記載のキッ卜。 [32] The kit according to any one of claims 23 to 25, which is a kit for treating tumor, rheumatoid arthritis, psoriasis, atherosclerosis, force positive sarcoma, or solid cancer metastasis. .
[33] 前記第 1の組成物が投与された後に， 前記第 2の組成物が投与される請求 項 2 3から請求項 2 5のいずれかに記載のキッ卜。  [33] The kit according to any one of claims 23 to 25, wherein the second composition is administered after the first composition is administered.
[34] 前記第 2の組成物が投与された後に， 前記第 1の組成物が投与される請求 項 2 3から請求項 2 5のいずれかに記載のキッ卜。 [34] The kit according to any one of claims 23 to 25, wherein the first composition is administered after the second composition is administered.
[35] 前記第 2の組成物が投与された後， 1分後〜 1 0分後に前記第 1の組成物 が投与される請求項 2 3から請求項 2 5のいずれかに記載のキッ卜。 [35] The kit according to any one of claims 23 to 25, wherein the first composition is administered 1 minute to 10 minutes after the second composition is administered. .
PCT/JP2007/000550 2006-05-24 2007-05-23 Peptide capable of enhancing the activity of ephrin-b2, salt thereof, composition for medical purposes, kit for therapy WO2007135781A1 (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002026827A1 (en) * 2000-09-29 2002-04-04 Novartis Ag Extracellular polypeptides of eph b receptors and ephrin b ligands and the corresponding nucleic acid molecules
GB2376016A (en) * 2001-05-30 2002-12-04 Nadir R Farid Thyrotropin receptor (TSHR) domain cleavage catalysed by the metalloprotease ADAM 10
WO2006006079A2 (en) * 2004-04-05 2006-01-19 Aqumen Biopharmaceuticals K.K. Methods for suppressing neovascularization using ephrinb2
JP2006507256A (en) * 2002-09-24 2006-03-02 ザ バーナム インスティチュート Novel drug that modulates Eph receptor activity
WO2006052409A2 (en) * 2004-10-23 2006-05-18 Case Western Reserve University Peptide and small molecule agonists of epha and their uses
WO2006081418A2 (en) * 2005-01-27 2006-08-03 The Burnham Institute Ephb receptor-binding peptides

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002026827A1 (en) * 2000-09-29 2002-04-04 Novartis Ag Extracellular polypeptides of eph b receptors and ephrin b ligands and the corresponding nucleic acid molecules
GB2376016A (en) * 2001-05-30 2002-12-04 Nadir R Farid Thyrotropin receptor (TSHR) domain cleavage catalysed by the metalloprotease ADAM 10
JP2006507256A (en) * 2002-09-24 2006-03-02 ザ バーナム インスティチュート Novel drug that modulates Eph receptor activity
WO2006006079A2 (en) * 2004-04-05 2006-01-19 Aqumen Biopharmaceuticals K.K. Methods for suppressing neovascularization using ephrinb2
WO2006052409A2 (en) * 2004-10-23 2006-05-18 Case Western Reserve University Peptide and small molecule agonists of epha and their uses
WO2006081418A2 (en) * 2005-01-27 2006-08-03 The Burnham Institute Ephb receptor-binding peptides

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