WO2007094193A1 - Anti-angiogenic agent, prophylactic or therapeutic agent for disease accompanied by angiogenesis, and food - Google Patents

Anti-angiogenic agent, prophylactic or therapeutic agent for disease accompanied by angiogenesis, and food Download PDF

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Publication number
WO2007094193A1
WO2007094193A1 PCT/JP2007/051955 JP2007051955W WO2007094193A1 WO 2007094193 A1 WO2007094193 A1 WO 2007094193A1 JP 2007051955 W JP2007051955 W JP 2007051955W WO 2007094193 A1 WO2007094193 A1 WO 2007094193A1
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Prior art keywords
angiogenesis
bilberry
inhibitor
therapeutic agent
extract
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PCT/JP2007/051955
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French (fr)
Japanese (ja)
Inventor
Hideaki Hara
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Wakasa Seikatsu Co., Ltd
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Priority to JP2008500445A priority Critical patent/JP5120848B2/en
Publication of WO2007094193A1 publication Critical patent/WO2007094193A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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
    • 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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives

Definitions

  • Angiogenesis inhibitors and preventive or therapeutic agents for diseases associated with angiogenesis are provided.
  • the present invention relates to an angiogenesis inhibitor comprising a Bilberry extract as an active ingredient, a preventive or therapeutic agent for diseases associated with angiogenesis, and a food for inhibiting angiogenesis.
  • Angiogenesis is a physiological phenomenon observed in an adult healthy human body and is "a process in which a new blood vessel is formed from a blood vessel originally in the living body".
  • angiogenesis is a physiological phenomenon necessary for the human body.
  • VEGF vascular endothelial growth factor
  • an anti-VEGF aptamer known as an angiogenesis-promoting factor
  • Bilberry extract is known for its various effects by anthocyanin, which is a component thereof, such as therapeutic effects on diabetes, retinopathy, arthritis, and the like. Is suggested.
  • angiogenesis inhibition there is no description regarding angiogenesis inhibition. From the description such as "vascular expansion action, prevention of thrombus formation, blood circulation promotion, strengthening of capillaries" ⁇ Therapeutic effect on retinopathy '' means that the blood vessels become brittle and bleeding It is thought to mean a therapeutic effect on blood. In other words, this document has nothing to do with angiogenesis inhibition, but merely describes the effect as a blood vessel enhancer.
  • Non-Patent Document 1 Handbook of functional food materials (from foods for specified health use to supplements 'health foods') (Pharmaceutical Daily, April 20, 2004, pages 385-386)
  • An object of the present invention is to provide a novel angiogenesis inhibitor, a preventive or therapeutic agent for a disease associated with angiogenesis, or a food, focusing on the new efficacy of Bilberry extract. .
  • the above-mentioned object is to provide signal transduction to ERK1 or ERK2 from PLC y Ka et al., Characterized in that it contains Bilberry extract, an angiogenesis inhibitor characterized by containing Bilberry extract.
  • Bilberry extract VEGF receptor strength Signaling inhibitor to Akt, Bilberry extract strength Bilberry ethanol extract or hydrous ethanol extract
  • a prophylactic or therapeutic agent for a disease associated with angiogenesis wherein the disease associated with angiogenesis is an ophthalmic disease, tumor or cancer disease, Or a disease associated with angiogenesis, such as chronic inflammation, and age-related macular degeneration, diabetic retinopathy, neovascular glaucoma, proliferative diabetic retinopathy, retinopathy of prematurity, Age-related macular degeneration, neovascular glaucoma, retinal vein occlusion, retinal artery occlusion, pterygium, rubeosis, corneal neovascular disease, solid tumor, myeloma, hemangioma, rheumatoid arthritis It is achieved by the anti-angiogenic food containing the above-mentioned disease preventive or therapeutic agent, which is gusset
  • the angiogenesis inhibitor and signal transduction inhibitor of the present invention significantly suppress angiogenesis in diseases such as proliferative diabetic retinopathy, and thus can be used as a so-called experimental research reagent.
  • Preventive or therapeutic agents for diseases with excessive angiogenesis, health foods, etc. Useful as an active ingredient.
  • a bilberry extract is used as an active ingredient as an active ingredient.
  • Pinoreberi 1 ⁇ (bilberry, whortleberry, huckleberry, Vaccinium myrtillus) is a kind of blueberry called mouth ⁇ bush 'blueberry, and belongs to the azalea family bilberry genus. Bilberry is widely grown in the United States, Canada, Scandinavia, etc.
  • the bilberry extract an extract mainly from bilberry fruits is preferably used.
  • the bilberry extract can be produced by crushing fruits and immersing them in the following extraction solvent. .
  • Extraction is performed by, for example, 100 kg of Bilberry, ethanol or 95% hydrous ethanol 500-2.
  • It can be produced by immersing in 000 liters for 5 to 20 hours, preferably 10 to 17 hours and filtering.
  • the Bilberry extract used in the present invention is used as an angiogenesis inhibitor, a signal transduction inhibitor, a preventive or therapeutic agent for angiogenesis-related diseases, or a food
  • the extract is used as it is.
  • it can also be used as a concentrate or a dry product.
  • the drying method include spray drying (spray drying), freeze drying, and hot air drying.
  • angiogenesis inhibitor the signal transduction inhibitor, the prophylactic or therapeutic agent for diseases associated with angiogenesis, or foods according to the present invention may be used as long as the inhibitory effect, the preventive or therapeutic effect is not inhibited.
  • a pharmaceutically acceptable carrier for example, as a pharmaceutically acceptable carrier
  • Excipients lubricants, binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption promoters, humectants, adsorbents, fillers, extenders It can be formulated by well-known methods using additives such as moisturizers and preservatives.
  • excipients include organic excipients and inorganic excipients.
  • the angiogenesis inhibitor, the signal transduction inhibitor, the prophylactic or therapeutic agent for diseases associated with angiogenesis, or the food of the present invention is a liquid, syrup, powder, granule, hard capsule, soft capsule. It can be used in various forms such as psel, tablets, powders, pills, troches, poultices, patches, and DDS preparations.
  • the angiogenesis inhibitor or signal transduction inhibitor of the present invention, a prophylactic or therapeutic agent for a disease associated with angiogenesis, or a food administration form includes intravenous administration such as intravenous injection, intramuscular administration, and transdermal administration.
  • intravenous administration such as intravenous injection, intramuscular administration, and transdermal administration.
  • Nasal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intrarectal administration, mucosal administration, inhalation, oral administration, etc. and are not particularly limited.
  • the preparation for topical ophthalmic administration comprises the aforementioned Bilberry extract as an active ingredient, and is a product for topical ophthalmic administration such as an injection for intravitreal administration, an eye drop or an eye ointment using a widely used technique. It can be manufactured by preparing an agent.
  • an injection for intravitreal administration can be produced by dissolving the Bilberry extract in distilled water for injection by a conventional method, and is appropriately prepared with mannitol, sodium chloride salt, Darcoel.
  • Tonicity agents such as sucrose, sorbit, glycerol, xylitol, fructose, maltose and mannose, stabilizers such as albumin, preservatives such as benzyl alcohol and methyl parahydroxybenzoate can be added to the preparation.
  • acids such as citrate and bases such as disoprono V-lamine can be added to the preparation as pH adjusters.
  • the injection for intravitreal administration may be a lyophilized preparation for dissolution at the time of use.
  • the lyophilized preparation can be produced by lyophilizing the Bilberry extract by a conventional method.
  • the above-mentioned tonicity agent, stabilizer, preservative, pH adjuster and the like are appropriately formulated. It can be added to the inside.
  • Eye drops can be produced by dissolving the Bilberry extract in distilled water by a conventional method, and mannitol, sodium chloride, glucose, sorbit, glycerol, xylitol, fructose, maltose are appropriately used.
  • Isotonic agents such as mannose and glycerin, stabilizers such as sodium edetate and albumin, preservatives such as benzyl alcohol and methyl parahydroxybenzoate, polyoxyethylene monooleate, polyoxyl 40 stearate, etc.
  • An activator or the like can be added to the formulation.
  • acids such as citrate and bases such as diisoprono V-lamine can be added to the preparation as pH adjusters.
  • hyaluronic acid or a salt thereof, and / or a polymer such as polycarbophil can be appropriately added in addition to the above-mentioned additives usually used for eye drops.
  • angiogenesis inhibitor, signal transduction inhibitor, or preventive or therapeutic agent for a disease associated with angiogenesis is a conventionally known ophthalmic disease, tumor or cancer disease, or prevention of chronic inflammation.
  • a combination with a therapeutic agent may be used.
  • angiogenesis inhibitor significantly suppresses angiogenesis, ophthalmic diseases and tumors associated with angiogenesis. It can be used effectively as a preventive or therapeutic agent for cancer diseases or chronic inflammation.
  • the ophthalmic diseases, tumors or cancer diseases associated with angiogenesis, or chronic inflammations to be used for the preventive or therapeutic agent of the present invention include age-related macular degeneration, diabetic retinopathy, neovascular glaucoma. , Proliferative diabetic retinopathy, retinopathy of prematurity, wet age-related macular degeneration, neovascular glaucoma, retinal vein occlusion, retinal artery occlusion, pterygium, rubeosis, corneal neovascularization, solid Type tumor, myeloma, hemangioma, rheumatoid arthritis, psoriasis, or osteoarthritis. Among them, it is particularly effective for diabetic retinopathy, neovascular glaucoma, proliferative diabetic retinopathy and the like.
  • the angiogenesis inhibitor, the signal transduction inhibitor of the present invention, and the prophylactic or therapeutic agent of the present invention are preferable because the safety is confirmed as a food ingredient and the Bilberry extract ingredient is used. .
  • the angiogenesis inhibitor, the signal transduction inhibitor of the present invention, and the prophylactic or therapeutic agent of the present invention are preferable because the safety is confirmed as a food ingredient and the Bilberry extract ingredient is used.
  • the Bilberry extract ingredient is used.
  • it when administered locally to the eye, it produces an effect at the affected area and is rapidly metabolized, so it is effective in preventing or treating diabetic retinopathy with high safety, especially proliferative diabetic retinopathy and other ophthalmic diseases. Can be used.
  • the dose of the angiogenesis inhibitor, the signal transduction inhibitor, or the prophylactic or therapeutic agent for a disease associated with angiogenesis according to the present invention should be appropriately selected depending on the patient's condition, age, weight, dosage form, and the like.
  • the Bilberry extract of the active ingredient is administered 0.01 to LOg, preferably 0.3 to 3 g per dose, once to several times a day.
  • the topical ocular containing 0.001 to 5% by weight of the active ingredient Bilberry extract is used.
  • the dosage formulation should be administered once or several times a day.
  • one to several drops of the preparation containing 0.001 to 1% by weight of the active ingredient Bilberry extract may be instilled once or several times a day.
  • an injection for intravitreal administration if the injection containing 0.01 to 1% by weight of the active ingredient Bilberry extract is administered once a day, 0.5 ml to: Lml. Good.
  • the food for inhibiting angiogenesis of the present invention can be produced by a conventional method by blending the above-mentioned angiogenesis inhibitor, signal transduction inhibitor and the like together with ingredients used as food.
  • Examples of the form include various forms such as liquid, powder, granule, paste, and jelly.
  • Antiangiogenic activity in vitro test using angiogenesis kit:
  • the angiogenesis inhibitory action of the therapeutic agent for diabetic retinopathy of the present invention was examined using an angiogenesis kit using vascular endothelial cell lumen formation as an index.
  • Bilberry extract 100 kg was immersed in 1000 liters of ethanol or 95% water-containing ethanol, filtered for 15 hours, filtered and dried by spray drying to produce a powdery bilberry extract.
  • angiogenesis kit (Kurabo) which is a co-culture system of human umbilical vein vascular endothelial cells and fibroblasts.
  • VEGF-A, angiogenesis medium-2, CD31 antibody was the one that came with this kit.
  • test was performed according to the instructions attached to the angiogenesis kit.
  • a normal group N
  • a VEGF administration group V
  • VEGF vascular endothelial growth factor
  • test component administration groups of various concentrations.
  • Reagents used for analgesics were prepared in lOOmM solution, diluted to a concentration of 0.1-: LOO / zM, and used for this test.
  • the normal group and the VEGF administration group were analyzed by Student's test.
  • the VEGF administration group and the group administered with VEGF and the test component were analyzed by Dunnett's multiple comparison test. The significance level for both tests was 5%.
  • the lumen formation diagram showing the angiogenesis inhibitory action is shown in FIGS.
  • Bilberry's ethanol extract significantly suppressed the angiogenesis-promoting effect of VEGF in a concentration-dependent manner.
  • no cell shape change was observed due to the effect of Bilberry ethanol extract, and the cytotoxicity of Bilberry ethanol extract was not confirmed.
  • Angiogenesis inhibitory action (in vivo test using mouse hyperoxia-loaded retinal neovascularization model): The angiogenesis inhibitory action of the angiogenesis inhibitor of the present invention was examined using a mouse hyperoxygen-loaded retinal neovascularization model.
  • Bilberry ethanol extract (powder) A powdery Bilberry extract produced in the same manner as in Test Example 1 was used.
  • mouse C57 / black6 (Japan SLC, Inc.) on the 18th day of pregnancy was purchased and the newborn was used.
  • mice on the 7th day of birth (postnatal day 7: P7) were raised to P12 in the animal chamber 1 under high oxygen conditions of 75 ⁇ 1%.
  • PROOX model 110 (Biospherix) was used.
  • mice were taken out of the animal chamber and kept under normal conditions until P17.
  • vitreous extract estimated concentration in the vitreous was calculated with a vitreous volume of about 10 L.
  • the flat-mounted retina was observed with a halogen lamp (U-UHL, OLYMPUS) under a microscope (BX50, OLYMPUS), and each fluorescent image was taken with a high-sensitivity cooled CCD camera (DP30BW, OLYMPUS).
  • the image acquired by the CCD camera was measured for the area of the new blood vessel using image analysis software (Metamorph, Molecular Devices).
  • image analysis software Metal, Molecular Devices. The smaller the area of the neovascular vessel, the more the angiogenesis is suppressed.
  • results are expressed as mean standard error (mean SEM.).
  • the specimen was also excluded from the specimens that did not stain the retinal blood vessels due to poor perfusion.
  • the paired t test was used to analyze the two groups of the solvent administration group and the bilberry extract administration group, and the significance level was 5% (* p ⁇ 0.05 vs. saline (paired t-test)).
  • Bilberry extract was evaluated using a mouse hyperoxic retinal neovascularization model. After high oxygen load, vehicle (Saline) and Bilberry extract were administered intravitreally.
  • FIGS. 9 and 10 Retinal angiograms of mice administered with a solvent after administration of a high oxygen load and mice administered with Bilberry extract are shown in FIGS. 9 and 10, respectively.
  • the bilberry extract administration group significantly suppressed retinal neovascularization (p ⁇ 0.05) compared to the solvent administration group (Fig. 11).
  • the angiogenesis inhibitor of the present invention significantly suppresses angiogenesis and suppresses angiogenesis such as ophthalmic diseases, tumors or cancer diseases, or chronic inflammation. It is clear that it can be used for the prevention or treatment of associated diseases.
  • a powdery Bilberry extract produced in the same manner as in Test Example 1 was used.
  • CCK-8 solution (manufactured by Dojindo Laboratories Co., Ltd.) used for the cell proliferation measurement kit was added to each cell at 10 / zl, reacted at 37 ° C for 3 hours, and absorbance at 492 nm (reference wavelength 660 nm).
  • a powdery Bilberry extract produced in the same manner as in Test Example 1 was used.
  • Cell culture insert (Betaton Dickinson Co., Ltd.) was used for the title test. HUVEC suspended in HuMedia-EB2 containing 0.1% BSA (usi serum albumin) was seeded in the upper chamber of the cell culture insert at a rate of 50000 cells per well. The bilberry extract was added to the upper chamber and the bilberry extract and Z or VEGF-A at the concentrations shown in the drawings were added to the lower chamber and cultured (final concentration: bilberry extract 3, 10,30 ⁇ g / ml, VEGF-A 10 ng / ml).
  • BSA usi serum albumin
  • non-migrated HUVECs were wiped off with a cotton swab to fix the migrating cells.
  • the cell culture insert membrane was cut off, stained with hematoxylin, and migrated cells were stained. The stained cells were photographed with a digital camera, and the number of stained cells was counted and measured.
  • Bilberry extract concentration-dependent and significantly suppressed the migration of HUVE C by VEGF-A induction (Fig. 13).
  • ERKl extracellular signal-regulated kinase 1 or 2
  • PL C y phospholipase C ⁇
  • Akt serine threonine protein kinase family protein k inase B
  • a powdery Bilberry extract produced in the same manner as in Test Example 1 was used.
  • the protein was subjected to 7.5% SDS-polyacrylamidamide gel electrophoresis, and the luminance of the band was analyzed using Western blotting.
  • Primary antibodies are anti-phosphorylated ERK1 / 2 antibody, anti-total ERK1 / 2 antibody, anti-phosphorylated PLC ⁇ antibody, anti-total PLC ⁇ antibody, anti-phosphorylated Akt antibody, anti-total Akt antibody (Cersignalin Technology) And anti-j8-actin antibody (Sigma Aldrich Co.).
  • Secondary antibodies include anti-ERK antibody, anti-PLC antibody, and anti-Akt antibody.
  • HRP horseradish peroxidase
  • 8-actin antibody goat anti-mouse HRP antibody was used.
  • Bilberry extract was found to suppress signal pathways downstream of PLCy and upstream of ERK1 or ERK2 in HUVEC proliferation induced by VEGF-VEGF (Figs. 14 and 15).
  • Bilberry extract was clearly able to suppress the signal pathway upstream of Akt during VEGF-A-induced HUVEC migration (Fig. 16).
  • error bars indicate standard errors. * Indicates a significance level of less than 0.05 for control, # indicates a significance level of less than 0.05 for VEGF alone, and indicates a significance level of less than 0.01 for VEGF alone. The number of examples is 4-7.
  • a powdered Bilberry extract is obtained in the same manner as in Test Example 1. Dissolve this powdered Bilberry extract (1 part by weight) in distilled water for injection (10 parts by weight or 100 parts by weight) and dispense 1 ml each to make the injections of Examples 1 and 2, respectively. .
  • a powdered Bilberry extract is obtained in the same manner as in Test Example 1. Dissolve this powdered bilberry extract (1 part by weight) in distilled water (10 parts by weight or 100 parts by weight) and dispense 10 ml each to make eye drops of Examples 3 and 4, respectively.
  • angiogenesis inhibitor and signal transduction inhibitor of the present invention significantly suppress angiogenesis, they can be used as so-called experimental and research reagents, as well as ophthalmic diseases and tumors associated with angiogenesis. It is also useful as an active ingredient for preventing or treating cancer diseases or chronic inflammation, or for health foods.
  • FIG. 1 is a graph showing an in vitro angiogenesis ( ⁇ 1> lumen area) inhibitory effect of the therapeutic agent of the present invention.
  • V represents VEGF.
  • FIG. 2 is a graph showing an in vitro angiogenesis ( ⁇ 2> lumen length (length)) inhibitory effect of the therapeutic agent of the present invention.
  • V represents VEGF.
  • FIG. 3 is a view showing an in vitro angiogenesis ( ⁇ 3> lumen branch point (joint)) inhibitory action of the therapeutic agent of the present invention.
  • V represents VEGF.
  • FIG. 4 is a graph showing the in vitro angiogenesis ( ⁇ 4> branch number (path)) inhibitory action of the therapeutic agent of the present invention.
  • V represents VEGF.
  • FIG. 5 is a luminal formation diagram showing the angiogenic action of VEGF in vitro.
  • FIG. 6 is a luminal formation diagram showing in vitro angiogenesis inhibitory action of the therapeutic agent of the present invention.
  • FIG. 7 is a luminal formation diagram showing in vitro angiogenesis inhibitory action of the therapeutic agent of the present invention.
  • FIG. 8 is a luminal formation diagram showing the in vitro angiogenesis inhibitory action of the therapeutic agent of the present invention.
  • FIG. 9 is an angiogram of the retina of a mouse administered with a solvent after being subjected to a high oxygen load.
  • FIG. 10 is an angiogram of the retina of a mouse administered with Bilberry extract after being exposed to high oxygen load.
  • FIG. 11 is a diagram showing an in vivo angiogenesis inhibitory effect of the therapeutic agent of the present invention.
  • FIG. 12 is a graph showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is caused by inhibiting the proliferation of HUVEC.
  • FIG. 13 is a graph showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is due to inhibition of HUVEC migration.
  • FIG. 14 is a graph showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is caused by suppression of HUVEC proliferation by suppressing a signal pathway upstream of ERK1 or ERK2.
  • FIG. 15 is a graph showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is due to inhibition of HUVEC proliferation by inhibiting a signal pathway downstream from PLC ⁇ .
  • FIG. 16 is a diagram showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is due to inhibition of HUVEC migration by suppressing a signal pathway downstream from the VEGF receptor and upstream from Akt.

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Abstract

[PROBLEMS] To provide: an anti-angiogenic agent comprising a bilberry extract as an active ingredient; a prophylactic or therapeutic agent for a disease accompanied by angiogenesis; and a food for inhibition of angiogenesis. [MEANS FOR SOLVING PROBLEMS] Disclosed is an anti-angiogenic agent, a prophylactic or therapeutic agent for a disease accompanied by angiogenesis, or a food for inhibition of angiogenesis which comprises a bilberry extract.

Description

血管新生阻害剤及び血管新生を伴う疾病に対する予防又は治療剤並び に食ロロ  Angiogenesis inhibitors and preventive or therapeutic agents for diseases associated with angiogenesis
技術分野  Technical field
[0001] 本発明は、ビルべリー抽出物を有効成分とする血管新生阻害剤，及び血管新生を伴 う疾病に対する予防又は治療剤，並びに血管新生阻害用の食品に関する。  [0001] The present invention relates to an angiogenesis inhibitor comprising a Bilberry extract as an active ingredient, a preventive or therapeutic agent for diseases associated with angiogenesis, and a food for inhibiting angiogenesis.
背景技術  Background art
[0002] 血管新生 (angiogenesis)とは、成人の健常人体にみられる生理的な現象であり、「生 体内に元々ある血管から、新し 、血管が形成されるプロセス」である。  [0002] Angiogenesis is a physiological phenomenon observed in an adult healthy human body and is "a process in which a new blood vessel is formed from a blood vessel originally in the living body".
つまり、血管新生は、人体に必要な生理現象である。  That is, angiogenesis is a physiological phenomenon necessary for the human body.
[0003] その一方、加齢性黄斑変性症 (ARMD) ,糖尿病性網膜症，癌，乾癬，慢性関節性リ ゥマチ，変形性関節症等に代表される、種々の眼科疾患，腫瘍又は癌疾患，あるい は慢性炎症その他の疾病において、腫瘍や癌の増殖'転移，組織の肥大化の際、癌 •腫瘍 ·肥大化組織への栄養供給のために血管新生が起こって 、ることが分力つて いる。  [0003] On the other hand, various ophthalmic diseases, tumors or cancer diseases represented by age-related macular degeneration (ARMD), diabetic retinopathy, cancer, psoriasis, rheumatoid arthritis, osteoarthritis, etc. , Or in chronic inflammation and other diseases, tumors and cancers grow and metastasize, and when the tissue enlarges, it is understood that angiogenesis occurs due to the supply of nutrients to the tumor and enlarged tissue. Powerful.
[0004] 従って、これらの疾病は、「血管新生」と「血管新生の抑制」のバランスを、健常人の 持つ適度な状態に戻すことによって、予防または治療できると考えられる。  Therefore, it is considered that these diseases can be prevented or treated by returning the balance between “angiogenesis” and “suppression of angiogenesis” to an appropriate state of a healthy person.
[0005] 近年、血管新生促成因子として知られている血管内皮増殖因子 (VEGF : vascular endothelial growth factor)の阻害剤（抗 VEGFァプタマ一），つまり一種の血 管新生阻害剤の、臨床応用が実現し、増殖糖尿病網膜症に対する治療剤としての 有効性が確認されている。 [0005] In recent years, clinical application of an inhibitor of vascular endothelial growth factor (VEGF), an anti-VEGF aptamer, known as an angiogenesis-promoting factor has been realized. Therefore, its effectiveness as a therapeutic agent for proliferative diabetic retinopathy has been confirmed.
[0006] 一方、ビルべリー抽出物は、非特許文献 1に記載されているように、その成分である アントシァニンによる効能が種々知られており、例えば、糖尿病や網膜症，関節炎等 に対する治療効果が示唆されて ヽる。 [0006] On the other hand, as described in Non-Patent Document 1, Bilberry extract is known for its various effects by anthocyanin, which is a component thereof, such as therapeutic effects on diabetes, retinopathy, arthritis, and the like. Is suggested.
[0007] しかしながら、この文献においては、血管新生阻害に関する記載は一切無ぐ「血管 拡張作用，血栓形成予防，血行促進，毛細血管を強くする」等の記載からは、ここで 示唆する「糖尿病性網膜症に対する治療効果」とは、血管が脆くなり出血する網膜出 血への治療効果を意味していると考えられる。つまり、この文献では、血管新生阻害 とは関係の無 、、血管増強剤としての効果が述べられて 、るに過ぎな 、。 [0007] However, in this document, there is no description regarding angiogenesis inhibition. From the description such as "vascular expansion action, prevention of thrombus formation, blood circulation promotion, strengthening of capillaries" `` Therapeutic effect on retinopathy '' means that the blood vessels become brittle and bleeding It is thought to mean a therapeutic effect on blood. In other words, this document has nothing to do with angiogenesis inhibition, but merely describes the effect as a blood vessel enhancer.
[0008] 非特許文献 1 :機能性食品素材便覧 (特定保健用食品からサプリメント '健康食品ま で）（薬事日報社， 2004年 4月 20日発行， P.385〜386)  [0008] Non-Patent Document 1: Handbook of functional food materials (from foods for specified health use to supplements 'health foods') (Pharmaceutical Daily, April 20, 2004, pages 385-386)
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
[0009] 本発明の目的は、ビルべリー抽出物の新たな効能に着目し、新規な血管新生阻害 剤，及び血管新生を伴う疾病に対する予防又は治療剤，あるいは食品を提供するこ とにある。 [0009] An object of the present invention is to provide a novel angiogenesis inhibitor, a preventive or therapeutic agent for a disease associated with angiogenesis, or a food, focusing on the new efficacy of Bilberry extract. .
課題を解決するための手段  Means for solving the problem
[0010] 上述の目的は、ビルべリー抽出物を含むことを特徴とする血管新生阻害剤，ビルベリ 一抽出物を含むことを特徴とする、 PLC yカゝら ERK1又は ERK2へのシグナル伝達 の阻害剤，ビルべリー抽出物を含むことを特徴とする、 VEGFレセプター力 Aktへの シグナル伝達の阻害剤，ビルべリー抽出物力 ビルべリーのエタノール抽出物又は 含水エタノール抽出物であることを特徴とする前記血管新生阻害剤，前記血管新生 阻害剤を有効成分として含むことを特徴とする、血管新生を伴う疾病に対する予防又 は治療剤，血管新生を伴う疾病が、眼科疾患，腫瘍又は癌疾患，あるいは慢性炎症 である、前記疾病予防又は治療剤，及び血管新生を伴う疾病が、加齢性黄斑変性 症，糖尿病性網膜症，新生血管緑内障，増殖性糖尿病網膜症，未熟児網膜症，滲 出型加齢黄斑変性症，血管新生緑内障，網膜静脈閉塞症，網膜動脈閉塞症，翼状 片，ルべォ一シス，角膜新生血管症，固型腫瘍，骨髄腫，血管腫，慢性関節性リウ マチ，乾癬，又は変形性関節症である、前記疾病予防又は治療剤，並びに前記血 管新生阻害剤を含む血管新生阻害用の食品によって達成される。 [0010] The above-mentioned object is to provide signal transduction to ERK1 or ERK2 from PLC y Ka et al., Characterized in that it contains Bilberry extract, an angiogenesis inhibitor characterized by containing Bilberry extract. Inhibitor, Bilberry extract, VEGF receptor strength Signaling inhibitor to Akt, Bilberry extract strength Bilberry ethanol extract or hydrous ethanol extract A prophylactic or therapeutic agent for a disease associated with angiogenesis, wherein the disease associated with angiogenesis is an ophthalmic disease, tumor or cancer disease, Or a disease associated with angiogenesis, such as chronic inflammation, and age-related macular degeneration, diabetic retinopathy, neovascular glaucoma, proliferative diabetic retinopathy, retinopathy of prematurity, Age-related macular degeneration, neovascular glaucoma, retinal vein occlusion, retinal artery occlusion, pterygium, rubeosis, corneal neovascular disease, solid tumor, myeloma, hemangioma, rheumatoid arthritis It is achieved by the anti-angiogenic food containing the above-mentioned disease preventive or therapeutic agent, which is gusset, psoriasis, or osteoarthritis, and the angiogenesis inhibitor.
発明の効果  The invention's effect
[0011] 本発明の血管新生阻害剤，シグナル伝達阻害剤は、増殖糖尿病網膜症等の疾病に おける血管新生を有意に抑制することから、いわゆる実験'研究用試薬として使用可 能である他、過剰な血管新生を伴う疾病の予防又は治療剤，あるいは健康食品等の 有効成分として有用である。 [0011] The angiogenesis inhibitor and signal transduction inhibitor of the present invention significantly suppress angiogenesis in diseases such as proliferative diabetic retinopathy, and thus can be used as a so-called experimental research reagent. Preventive or therapeutic agents for diseases with excessive angiogenesis, health foods, etc. Useful as an active ingredient.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0012] 以下、本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
[0013] 本発明の血管新生阻害剤やシグナル伝達阻害剤においては、有効成分としてビル ベリー抽出物を有効成分として用いる。  [0013] In the angiogenesis inhibitor and the signal transduction inhibitor of the present invention, a bilberry extract is used as an active ingredient as an active ingredient.
[0014] ピノレベリ1 ~~ (bilberry, whortleberry, huckleberry, Vaccinium myrtillus)とは、口 ~~ブッ シュ 'ブルーベリーと言われるブルーベリーの一種であり、つつじ科コケモモ属に属 する。ビルべリーは、アメリカやカナダ，北欧等に広く自生している。 [0014] Pinoreberi 1 ~~ (bilberry, whortleberry, huckleberry, Vaccinium myrtillus) is a kind of blueberry called mouth ~~ bush 'blueberry, and belongs to the azalea family bilberry genus. Bilberry is widely grown in the United States, Canada, Scandinavia, etc.
[0015] ビルべリー抽出物とは、主にビルべリーの果実からの抽出物が好適に用いられ、例え ば実を潰して、下記の抽出溶媒に浸漬することで、製造することができる。 [0015] As the bilberry extract, an extract mainly from bilberry fruits is preferably used. For example, the bilberry extract can be produced by crushing fruits and immersing them in the following extraction solvent. .
[0016] 抽出には、例えばアルコール等が用いられ、具体的にはエタノール，含水エタノール [0016] For the extraction, for example, alcohol or the like is used. Specifically, ethanol or hydrous ethanol is used.
(95%含水エタノール等)等が挙げられる。  (95% water-containing ethanol, etc.).
[0017] 抽出は、例えば、ビルべリー 100kgを、エタノール又は 95%含水エタノール 500〜2[0017] Extraction is performed by, for example, 100 kg of Bilberry, ethanol or 95% hydrous ethanol 500-2.
000リットルに、 5〜20時間，好ましくは 10〜17時間浸漬し、濾過することによって製 造することができる。 It can be produced by immersing in 000 liters for 5 to 20 hours, preferably 10 to 17 hours and filtering.
[0018] 本発明で用いられるビルべリー抽出物を、血管新生阻害剤やシグナル伝達阻害剤， 血管新生を伴う疾病に対する予防又は治療剤あるいは食品として使用する際には、 抽出液そのままで用いることもできるが、濃縮物，乾燥物として用いることもできる。乾 燥方法としては、スプレードライ (噴霧乾燥)，凍結乾燥，熱風乾燥等が挙げられる。  [0018] When the Bilberry extract used in the present invention is used as an angiogenesis inhibitor, a signal transduction inhibitor, a preventive or therapeutic agent for angiogenesis-related diseases, or a food, the extract is used as it is. However, it can also be used as a concentrate or a dry product. Examples of the drying method include spray drying (spray drying), freeze drying, and hot air drying.
[0019] 本発明の血管新生阻害剤やシグナル伝達阻害剤，血管新生を伴う疾病に対する予 防又は治療剤，あるいは食品には、その阻害効果や予防又は治療効果を阻害しな い範囲で、他の成分を含有させることができ、例えば薬学的に許容される担体として [0019] The angiogenesis inhibitor, the signal transduction inhibitor, the prophylactic or therapeutic agent for diseases associated with angiogenesis, or foods according to the present invention may be used as long as the inhibitory effect, the preventive or therapeutic effect is not inhibited. For example, as a pharmaceutically acceptable carrier
、賦形剤，滑沢剤，結合剤，崩壊剤，安定剤，矯味矯臭剤，希釈剤，界面活性剤， 乳化剤，可溶化剤，吸収促進剤，保湿剤，吸着剤，充填剤，増量剤，付湿剤，防腐 剤等の添加剤を用いて周知の方法で製剤化することができる。 , Excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption promoters, humectants, adsorbents, fillers, extenders It can be formulated by well-known methods using additives such as moisturizers and preservatives.
[0020] ここに、賦形剤としては、有機系賦形剤及び無機系賦形剤等が挙げられる。  [0020] Examples of excipients include organic excipients and inorganic excipients.
[0021] 本発明の血管新生阻害剤やシグナル伝達阻害剤，血管新生を伴う疾病に対する予 防又は治療剤，あるいは食品は、液状，シロップ状，粉末，顆粒，硬カプセル，軟カ プセル，錠剤，散剤，丸剤，トローチ，パップ剤，貼付剤， DDS製剤等の様々な形態 で用いることができる。 [0021] The angiogenesis inhibitor, the signal transduction inhibitor, the prophylactic or therapeutic agent for diseases associated with angiogenesis, or the food of the present invention is a liquid, syrup, powder, granule, hard capsule, soft capsule. It can be used in various forms such as psel, tablets, powders, pills, troches, poultices, patches, and DDS preparations.
[0022] 本発明の血管新生阻害剤やシグナル伝達阻害剤，血管新生を伴う疾病に対する予 防又は治療剤，あるいは食品の投与形態としては、静注等の静脈投与，筋肉内投与 ,経皮投与，経鼻投与，皮内投与，皮下投与，腹腔内投与，直腸内投与，粘膜投与 ,吸入，経口投与等が挙げられ、特に限定されない。  [0022] The angiogenesis inhibitor or signal transduction inhibitor of the present invention, a prophylactic or therapeutic agent for a disease associated with angiogenesis, or a food administration form includes intravenous administration such as intravenous injection, intramuscular administration, and transdermal administration. , Nasal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intrarectal administration, mucosal administration, inhalation, oral administration, etc., and are not particularly limited.
[0023] 眼科疾患の場合には、例えば眼局所投与用製剤を用いた投与は、血管新生阻害作 用を有する有効成分が、患部に迅速かつ確実に到達するため、好ましい。  [0023] In the case of ophthalmic diseases, for example, administration using a preparation for topical ocular administration is preferable because an active ingredient having an angiogenesis inhibitory action reaches the affected area quickly and reliably.
[0024] 当該眼局所投与用製剤は、前述のビルべリー抽出物を有効成分とし、汎用されてい る技術を用いて硝子体内投与用注射剤、点眼剤又は眼軟膏などの眼局所投与用製 剤に調製することにより製造することができる。  [0024] The preparation for topical ophthalmic administration comprises the aforementioned Bilberry extract as an active ingredient, and is a product for topical ophthalmic administration such as an injection for intravitreal administration, an eye drop or an eye ointment using a widely used technique. It can be manufactured by preparing an agent.
[0025] 例えば、硝子体内投与用注射剤は、常法によって前記ビルべリー抽出物を注射用 蒸留水に溶解させて製造することができ、適宜、マン-トール、塩ィ匕ナトリウム、ダルコ ース、ソルビット、グリセロール、キシリトーノレ、フルクトース、マルトース、マンノース等 の等張化剤、アルブミン等の安定化剤、ベンジルアルコール、パラヒドロキシ安息香 酸メチル等の保存剤等を製剤中に添加することができる。又、クェン酸等の酸、ジィ ソプロノ V—ルァミン等の塩基を pH調整剤として製剤中に添加することもできる。  [0025] For example, an injection for intravitreal administration can be produced by dissolving the Bilberry extract in distilled water for injection by a conventional method, and is appropriately prepared with mannitol, sodium chloride salt, Darcoel. Tonicity agents such as sucrose, sorbit, glycerol, xylitol, fructose, maltose and mannose, stabilizers such as albumin, preservatives such as benzyl alcohol and methyl parahydroxybenzoate can be added to the preparation. In addition, acids such as citrate and bases such as disoprono V-lamine can be added to the preparation as pH adjusters.
[0026] 硝子体内投与用注射剤は、用時溶解用の凍結乾燥製剤とすることもできる。凍結乾 燥製剤は、前記ビルべリー抽出物を常法により凍結乾燥することによって製造するこ とができ、適宜、上記、等張化剤、安定化剤、保存剤、 pH調整剤等を製剤中に添カロ することができる。 [0026] The injection for intravitreal administration may be a lyophilized preparation for dissolution at the time of use. The lyophilized preparation can be produced by lyophilizing the Bilberry extract by a conventional method. The above-mentioned tonicity agent, stabilizer, preservative, pH adjuster and the like are appropriately formulated. It can be added to the inside.
[0027] 点眼剤は、常法によって前記ビルべリー抽出物を蒸留水に溶解させて製造すること ができ、適宜、マン-トール、塩化ナトリウム、グルコース、ソルビット、グリセロール、キ シリトール、フルクトース、マルトース、マンノース、グリセリン等の等張化剤、ェデト酸 ナトリウム、アルブミン等の安定化剤、ベンジルアルコール、パラヒドロキシ安息香酸メ チル等の保存剤、ポリオキシエチレンモノォレート、ステアリン酸ポリオキシル 40等の 界面活性化剤等を製剤中に添加することができる。又、クェン酸等の酸、ジイソプロ ノ V—ルァミン等の塩基を pH調整剤として製剤中に添加することもできる。更に、より 優れた効果を得るために、点眼剤に通常用いられる上記の添加剤に加え、ヒアルロ ン酸又はその塩、及び/又はポリカーボフィル等のポリマーを適宜添加することもでき る。 [0027] Eye drops can be produced by dissolving the Bilberry extract in distilled water by a conventional method, and mannitol, sodium chloride, glucose, sorbit, glycerol, xylitol, fructose, maltose are appropriately used. , Isotonic agents such as mannose and glycerin, stabilizers such as sodium edetate and albumin, preservatives such as benzyl alcohol and methyl parahydroxybenzoate, polyoxyethylene monooleate, polyoxyl 40 stearate, etc. An activator or the like can be added to the formulation. In addition, acids such as citrate and bases such as diisoprono V-lamine can be added to the preparation as pH adjusters. And more In order to obtain an excellent effect, hyaluronic acid or a salt thereof, and / or a polymer such as polycarbophil can be appropriately added in addition to the above-mentioned additives usually used for eye drops.
[0028] また、本発明の血管新生阻害剤，シグナル伝達阻害剤，又は血管新生を伴う疾病に 対する予防又は治療剤は、従来知られている眼科疾患，腫瘍又は癌疾患，あるいは 慢性炎症の予防又は治療剤との合剤としても良い。  [0028] Further, the angiogenesis inhibitor, signal transduction inhibitor, or preventive or therapeutic agent for a disease associated with angiogenesis according to the present invention is a conventionally known ophthalmic disease, tumor or cancer disease, or prevention of chronic inflammation. Alternatively, a combination with a therapeutic agent may be used.
[0029] 本発明の血管新生阻害剤，シグナル伝達阻害剤，又は血管新生を伴う疾病に対す る予防又は治療剤は、血管新生を有意に抑制することから、血管新生を伴う眼科疾 患，腫瘍又は癌疾患，あるいは慢性炎症の予防又は治療剤として有効に用いられる  [0029] Since the angiogenesis inhibitor, signal transduction inhibitor, or preventive or therapeutic agent for diseases associated with angiogenesis of the present invention significantly suppresses angiogenesis, ophthalmic diseases and tumors associated with angiogenesis. It can be used effectively as a preventive or therapeutic agent for cancer diseases or chronic inflammation.
[0030] 本発明の予防又は治療剤の使用対象となる、血管新生を伴う眼科疾患，腫瘍又は 癌疾患，あるいは慢性炎症としては、加齢性黄斑変性症，糖尿病性網膜症，新生血 管緑内障，増殖性糖尿病網膜症，未熟児網膜症，滲出型加齢黄斑変性症，血管新 生緑内障，網膜静脈閉塞症，網膜動脈閉塞症，翼状片，ルべォ一シス，角膜新生 血管症，固型腫瘍，骨髄腫，血管腫，慢性関節性リウマチ，乾癬，又は変形性関節 症等が挙げられる。中でも、糖尿病性網膜症，新生血管緑内障，増殖性糖尿病網膜 症等に特に有効である。 [0030] The ophthalmic diseases, tumors or cancer diseases associated with angiogenesis, or chronic inflammations to be used for the preventive or therapeutic agent of the present invention include age-related macular degeneration, diabetic retinopathy, neovascular glaucoma. , Proliferative diabetic retinopathy, retinopathy of prematurity, wet age-related macular degeneration, neovascular glaucoma, retinal vein occlusion, retinal artery occlusion, pterygium, rubeosis, corneal neovascularization, solid Type tumor, myeloma, hemangioma, rheumatoid arthritis, psoriasis, or osteoarthritis. Among them, it is particularly effective for diabetic retinopathy, neovascular glaucoma, proliferative diabetic retinopathy and the like.
[0031] 本発明の血管新生阻害剤，シグナル伝達阻害剤，及び本発明の予防又は治療剤は 、食品成分として安全性が確認されて 、るビルべリー抽出成分を用いて 、るため好ま しい。また、眼局所に投与した場合、患部にて効果を発現した後、速やかに代謝され ることから、安全性が高ぐ糖尿病網膜症、特に増殖糖尿病網膜症等の眼科疾患の 予防又は治療に有効に用いることができる。  [0031] The angiogenesis inhibitor, the signal transduction inhibitor of the present invention, and the prophylactic or therapeutic agent of the present invention are preferable because the safety is confirmed as a food ingredient and the Bilberry extract ingredient is used. . In addition, when administered locally to the eye, it produces an effect at the affected area and is rapidly metabolized, so it is effective in preventing or treating diabetic retinopathy with high safety, especially proliferative diabetic retinopathy and other ophthalmic diseases. Can be used.
[0032] 本発明の血管新生阻害剤，シグナル伝達阻害剤，又は血管新生を伴う疾病に対す る予防又は治療剤の投与量は、患者の病態、年齢、体重、剤形等によって適宜選択 することができ、限定されるものではないが、例えば有効性成分の前記ビルべリー抽 出物を、 1回当たり 0.01〜： LOg,好ましくは 0.3〜3gを、一日 1〜数回投与する。  [0032] The dose of the angiogenesis inhibitor, the signal transduction inhibitor, or the prophylactic or therapeutic agent for a disease associated with angiogenesis according to the present invention should be appropriately selected depending on the patient's condition, age, weight, dosage form, and the like. For example, but not limited to, the Bilberry extract of the active ingredient is administered 0.01 to LOg, preferably 0.3 to 3 g per dose, once to several times a day.
また、眼科疾患の予防又は治療等における、眼局所投与用製剤を用いた投与の場 合には、有効性成分の前記ビルべリー抽出物を 0. 001〜5重量％含有する眼局所 投与用製剤を 1日に 1回又は数回に分けて適量投与すればよい。例えば、点眼剤の 場合、有効性成分の前記ビルべリー抽出物を 0. 001〜1重量％含有する当該製剤 を 1日 1回又は数回、 1滴〜数滴点眼すればよい。また、硝子体内投与用注射剤の場 合、有効性成分の前記ビルべリー抽出物を 0. 01〜1重量％含有する当該注射剤を 1日 1回、 0. 5ml〜： Lml投与すればよい。 In addition, in the case of administration using a preparation for topical ophthalmic administration in the prevention or treatment of ophthalmic diseases, the topical ocular containing 0.001 to 5% by weight of the active ingredient Bilberry extract is used. The dosage formulation should be administered once or several times a day. For example, in the case of eye drops, one to several drops of the preparation containing 0.001 to 1% by weight of the active ingredient Bilberry extract may be instilled once or several times a day. In addition, in the case of an injection for intravitreal administration, if the injection containing 0.01 to 1% by weight of the active ingredient Bilberry extract is administered once a day, 0.5 ml to: Lml. Good.
[0033] 本発明の血管新生阻害用の食品は、上記血管新生阻害剤，シグナル伝達阻害剤等 を、食品として用いられる成分とともに配合し、常法により製造することができる。その 形態としては、液状，粉末状，顆粒状，ペースト状，ゼリー状等の各種のものが挙げら れる。 [0033] The food for inhibiting angiogenesis of the present invention can be produced by a conventional method by blending the above-mentioned angiogenesis inhibitor, signal transduction inhibitor and the like together with ingredients used as food. Examples of the form include various forms such as liquid, powder, granule, paste, and jelly.
[0034] 以下に試験例を挙げて本発明の効果について説明する。  [0034] The effects of the present invention will be described below with reference to test examples.
[0035] 試験例 1 [0035] Test Example 1
血管新生抑制作用（血管新生キットを用いた in vitro試験）：  Antiangiogenic activity (in vitro test using angiogenesis kit):
本発明の糖尿病網膜症治療剤の血管新生抑制作用について、血管内皮細胞の管 腔形成を指標とする血管新生キットを用いて検討した。  The angiogenesis inhibitory action of the therapeutic agent for diabetic retinopathy of the present invention was examined using an angiogenesis kit using vascular endothelial cell lumen formation as an index.
[0036] (1)試験成分： [0036] (1) Test components:
ビルべリーのエタノール抽出物（粉末状）  Bilberry ethanol extract (powder)
ビルべリー 100kgに対し、エタノール又は 95%含水エタノール 1000リットルに浸漬 し、 15時間時間後、濾過し、更にスプレードライで乾燥することによって、粉末状のビ ルベリー抽出物を製造した。  100 kg of Bilberry was immersed in 1000 liters of ethanol or 95% water-containing ethanol, filtered for 15 hours, filtered and dried by spray drying to produce a powdery bilberry extract.
[0037] (2)試験材料及び試験方法：  [0037] (2) Test material and test method:
[0038] (試験材料）  [0038] (Test material)
ヒトさい帯静脈血管の血管内皮細胞と線維芽細胞の共培養系である血管新生キット（ クラボウ）を用いて検討した。 VEGF-A,血管新生専用培地— 2, CD31抗体は、本キ ットに付属しているものを使用した。  It was examined using an angiogenesis kit (Kurabo) which is a co-culture system of human umbilical vein vascular endothelial cells and fibroblasts. VEGF-A, angiogenesis medium-2, CD31 antibody was the one that came with this kit.
[0039] (試験方法） [0039] (Test method)
本試験は、当該血管新生キットの付属説明書に従って実施した。培養系として、正常 群 (N) , VEGF投与群 (V) , VEGF及び各種濃度の試験成分投与群の 3群を用意し た。試験成分は、 DMSO (dimethyl sulfoxideジメチルスルホキシド =溶剤 ·外用消炎 鎮痛剤に使用される試薬)で lOOmM溶液に調製後、 0. 1〜： LOO /z Mの濃度に希釈 して本試験に供した。 This test was performed according to the instructions attached to the angiogenesis kit. As a culture system, three groups were prepared: a normal group (N), a VEGF administration group (V), VEGF, and test component administration groups of various concentrations. Test components are DMSO (dimethyl sulfoxide = solvent) Reagents used for analgesics) were prepared in lOOmM solution, diluted to a concentration of 0.1-: LOO / zM, and used for this test.
[0040] 本試験の評価は、 1群あたり 4ゥエルを使用し、培地交換は、培養 1日（キット入荷日） 、 4日、 7日及び 9日目に行い、培養 11日目に細胞をエタノール固定後、 CD31抗体 により血管内皮細胞を染色した後、顕微鏡下で各ゥエルの上下左右及び中央の 5点 をデジタルカメラで撮影し、血管新生定量ソフトウェア Ver.2 (クラボウ）を用いて、〈1〉 管腔面積 (area)、〈2〉管腔長 (length)、〈3〉管腔分岐点数 (joint)及び〈4〉枝数 (pa th)の 4項目を解析することにより行った。  [0040] In this test, 4 wells per group were used, and the medium was changed on the 1st day of culture (date of kit arrival), 4th, 7th and 9th days. After fixation with ethanol, vascular endothelial cells were stained with CD31 antibody, and the top, bottom, left, right, and center of each well were photographed with a digital camera under a microscope. Using angiogenesis quantification software Ver.2 (Kurabo), The analysis was performed by analyzing four items: 1> lumen area (area), <2> lumen length (length), <3> lumen branch point (joint), and <4> branch number (path).
[0041] (統計処理）  [0041] (Statistical processing)
VEGFの血管新生促進効果を確認するため、正常群と VEGF投与群とを Student ' s t testで解析した。また、試験成分の血管新生抑制効果を確認するため、 VEGF投 与群と、 VEGFと試験成分を投与した群とを、 Dunnett' s multiple comparison testで解析した。両検定とも有意水準は 5%とした。  In order to confirm the pro-angiogenic effect of VEGF, the normal group and the VEGF administration group were analyzed by Student's test. In addition, in order to confirm the angiogenesis inhibitory effect of the test component, the VEGF administration group and the group administered with VEGF and the test component were analyzed by Dunnett's multiple comparison test. The significance level for both tests was 5%.
[0042] (3) 試験結果： [0042] (3) Test results:
試験結果は、上記〈1〉〜く 4〉の 4項目それぞれについて、撮影した上記 5点の平均値 を各ゥエル値とし、平均値士標準誤差を図 1〜4に示した（Mean士 S.E.M (n=3-6). ** p< 0.01 vs VEGF)。また、血管新生抑制作用を示す管腔形成図を図 5〜8に示した  The test results for each of the four items <1> to <4> above are the average values of the above five points taken as the respective well values, and the average error standard error is shown in Figs. 1-4 (Mean SEM ( n = 3-6). ** p <0.01 vs VEGF). In addition, the lumen formation diagram showing the angiogenesis inhibitory action is shown in FIGS.
[0043] 図 1〜4及び 5〜8に示すとおり、ビルべリーのエタノール抽出物は、 VEGFによる血管 新生促進効果を濃度依存的に、有意に抑制した。また、培養期間中の顕微鏡下に おける観察において、ビルべリーのエタノール抽出物の影響による細胞の形態変化 が認められず、ビルべリーのエタノール抽出物の細胞毒性は確認されなカゝつた。 [0043] As shown in FIGS. 1 to 4 and 5 to 8, Bilberry's ethanol extract significantly suppressed the angiogenesis-promoting effect of VEGF in a concentration-dependent manner. In addition, in the observation under the microscope during the culture period, no cell shape change was observed due to the effect of Bilberry ethanol extract, and the cytotoxicity of Bilberry ethanol extract was not confirmed.
[0044] 試験例 2  [0044] Test Example 2
血管新生抑制作用（マウス高酸素負荷網膜血管新生モデルによる in vivo試験）： 本発明の血管新生阻害剤の血管新生抑制作用について、マウス高酸素負荷網膜血 管新生モデルを用いて検討した。  Angiogenesis inhibitory action (in vivo test using mouse hyperoxia-loaded retinal neovascularization model): The angiogenesis inhibitory action of the angiogenesis inhibitor of the present invention was examined using a mouse hyperoxygen-loaded retinal neovascularization model.
[0045] (1)試験成分： [0045] (1) Test components:
ビルべリーのエタノール抽出物（粉末状） 試験例 1と同様にして製造した、粉末状のビルべリー抽出物を使用した。 Bilberry ethanol extract (powder) A powdery Bilberry extract produced in the same manner as in Test Example 1 was used.
[0046] (2)試験材料及び試験方法： [0046] (2) Test material and test method:
[0047] (試験材料） [0047] (Test material)
試験動物としては、妊娠 18日目のマウス C57/black6 (日本エスエルシー株式会社）を 購入し、その新生児を使用した。  As a test animal, mouse C57 / black6 (Japan SLC, Inc.) on the 18th day of pregnancy was purchased and the newborn was used.
[0048] (試験方法） [0048] (Test method)
(2)— 1 高酸素負荷条件：  (2) — 1 High oxygen load conditions:
本モデルはスミス等の方法 (スミス リー（Smith LE)等， Invest Ophthalmol Vis Sci. 19 This model is based on Smith's method (Smith LE, etc., Invest Ophthalmol Vis Sci. 19
94;35(1):101- 111.)に準じて行った。すなわち、生後 7日目（postnatal day 7:P7)のマウ スを 75 ± 1%の高酸素条件下、動物用チャンバ一で P12まで飼育した。 94; 35 (1): 101-111.). That is, mice on the 7th day of birth (postnatal day 7: P7) were raised to P12 in the animal chamber 1 under high oxygen conditions of 75 ± 1%.
酸素濃度の制御は、 PROOX model l lO (Biospherix)を使用した。  For control of the oxygen concentration, PROOX model 110 (Biospherix) was used.
高酸素負荷後、マウスを動物用チャンバ一力 取り出し、 P17まで正常条件下で飼育 した。  After the high oxygen load, the mice were taken out of the animal chamber and kept under normal conditions until P17.
[0049] (2) - 2 硝子体内投与  [0049] (2)-2 Intravitreal administration
高酸素負荷後 (P12)、新生児マウスを 2.5%イソフルレン下で麻酔し、左眼に Saline (溶 媒:生理食塩水）を、右眼に 0.3 mg/mLビルべリー抽出物を各々 1 μ L投与した。投与 針はハミルトン 'シリンジに 32Gの針を付けたものを使用した。溶媒又はビルべリー抽 出物を硝子体内に投与後、投与による細菌感染や炎症を抑えるため、眼表面にクラ ビット点眼液 (登録商標）（参天製薬株式会社)を 1 μ L滴下した。  After high oxygen load (P12), neonatal mice were anesthetized under 2.5% isoflurane, Saline (solvent: physiological saline) in the left eye and 0.3 μl / mL Bilberry extract in the right eye, 1 μL each. Administered. The administration needle used was a Hamilton 'syringe with a 32G needle. After administration of the solvent or Bilberry extract into the vitreous, 1 μL of Cravit eye drop (registered trademark) (Santen Pharmaceutical Co., Ltd.) was dropped on the ocular surface in order to suppress bacterial infection and inflammation caused by the administration.
[0050] 〈硝子体内投与の群構成〉  [0050] <Group composition of intravitreal administration>
[0051] [表 1]  [0051] [Table 1]
Figure imgf000009_0001
Figure imgf000009_0001
[0052] 硝子体の容積を約 10 Lとして、ビルべリー抽出物の硝子体内推定濃度を算出した [0053] (2) - 3 FITC- dextranによる網膜血管の染色 [0052] The vitreous extract estimated concentration in the vitreous was calculated with a vitreous volume of about 10 L. [0053] (2)-3 Retinal vessel staining with FITC-dextran
マウスをネンブターノレで麻酔後、左心室から 2 X 106 FITC- dextran (20 mg/mL, Sigm a)を 1 mL全身灌流した。全身灌流後、眼球を摘出し、 4%パラホルムアルデヒド中で 4 〜24時間固定した。固定した眼球は角膜 '水晶体を除去し網膜を採取してスライドグ ラス上でフラットマウントにした。フラットマウント状の網膜は VECTASHIELD(Vector) にて封入しカバーガラスをのせ、縁を透明なマ-ユキユアで覆った。  After anesthetizing the mouse with Nembutanol, 1 mL of 2 X 106 FITC-dextran (20 mg / mL, Sigma) was perfused systemically from the left ventricle. After systemic perfusion, the eyeballs were removed and fixed in 4% paraformaldehyde for 4-24 hours. The fixed eyeball was removed from the cornea's lens, and the retina was collected and flat-mounted on a slide glass. The flat-mount retina was encapsulated with VECTASHIELD (Vector), covered with a cover glass, and the edges were covered with a transparent matrix.
[0054] (2) -4 FITC- dextran染色された網膜血管の撮影  [0054] (2) -4 Imaging of retinal blood vessels stained with FITC-dextran
フラットマウントにした網膜を、ハロゲンランプ（U-UHL, OLYMPUS)を用いて顕微鏡（ BX50, OLYMPUS)下で観察し、各蛍光像を高感度冷却 CCDカメラ（DP30BW, OLY MPUS)で撮影した。  The flat-mounted retina was observed with a halogen lamp (U-UHL, OLYMPUS) under a microscope (BX50, OLYMPUS), and each fluorescent image was taken with a high-sensitivity cooled CCD camera (DP30BW, OLYMPUS).
[0055] (2) - 5 新生血管房領域の算出  [0055] (2)-5 Calculation of neovascular chamber region
CCDカメラで取得した画像は、画像解析ソフト（Metamorph, Molecular Devices)を用 いて新生血管房の面積を測定した。新生血管房の面積が小さい程、血管新生が抑 制されていることを示す。  The image acquired by the CCD camera was measured for the area of the new blood vessel using image analysis software (Metamorph, Molecular Devices). The smaller the area of the neovascular vessel, the more the angiogenesis is suppressed.
[0056] 結果は平均値士標準誤差で示した (mean士 SEM.)。また、 FITC-dextranの全身灌 流の際、灌流不良などの理由により網膜血管が染色されな力つた標本についてはデ 一タカも除外した。  [0056] The results are expressed as mean standard error (mean SEM.). In addition, when the whole body was perfused with FITC-dextran, the specimen was also excluded from the specimens that did not stain the retinal blood vessels due to poor perfusion.
[0057] (2) -6 統計処理  [0057] (2) -6 Statistical processing
溶媒投与群とビルべリー抽出物投与群の 2群間について Paired t testで解析し、有意 水準は 5%とした（*p < 0.05 vs. saline (paired t- test )。  The paired t test was used to analyze the two groups of the solvent administration group and the bilberry extract administration group, and the significance level was 5% (* p <0.05 vs. saline (paired t-test)).
[0058] (3) 試験結果：  [0058] (3) Test results:
マウス高酸素負荷網膜血管新生モデルを用いてビルべリー抽出物の血管新生抑制 効果について評価した。高酸素負荷後、溶媒 (Saline)およびビルべリー抽出物を硝 子体内投与した。  The anti-angiogenic effect of Bilberry extract was evaluated using a mouse hyperoxic retinal neovascularization model. After high oxygen load, vehicle (Saline) and Bilberry extract were administered intravitreally.
[0059] 高酸素負荷においた後、溶媒投与したマウス，及びビルべリー抽出物を投与したマ ウスの、網膜の血管造影図を各々図 9, 10として示す。  [0059] Retinal angiograms of mice administered with a solvent after administration of a high oxygen load and mice administered with Bilberry extract are shown in FIGS. 9 and 10, respectively.
[0060] 前網膜新生血管 (新生血管房)領域は溶媒投与群で 0.98 ±0.24 mm2 (n = 9)であつ たのに対し、ビルべリー抽出物投与群では 0.51 ±0.07 mm2 (n = 9)であった。 つまり、ビルべリー抽出物投与群は溶媒投与群に比して有意 (p < 0.05)に網膜血管 新生を抑制した (図 11)。 [0060] The area of the anterior retinal neovascularization (neovascular tuft) was 0.98 ± 0.24 mm 2 (n = 9) in the solvent-administered group, whereas 0.51 ± 0.07 mm 2 (n = 9). In other words, the bilberry extract administration group significantly suppressed retinal neovascularization (p <0.05) compared to the solvent administration group (Fig. 11).
なお、その他、被験動物に何ら異常は認められな力つた。  In addition, no abnormalities were observed in the test animals.
[0061] 従って、上記 in vitro及び in vivo試験の結果から、本発明の血管新生阻害剤は、 血管新生を有意に抑制し、眼科疾患，腫瘍又は癌疾患，あるいは慢性炎症等の血 管新生を伴う疾病の予防又は治療に用いることができることは明らかである。  [0061] Therefore, based on the results of the in vitro and in vivo tests, the angiogenesis inhibitor of the present invention significantly suppresses angiogenesis and suppresses angiogenesis such as ophthalmic diseases, tumors or cancer diseases, or chronic inflammation. It is clear that it can be used for the prevention or treatment of associated diseases.
[0062] 試験例 3  [0062] Test Example 3
VEGF-A (Vascular endothelial growth factor;血管内皮増殖因子）が誘導する HUVE C (Human umbilical vein endothelial cell;ヒト臍帯静脈内皮細胞）増殖に対する、ビル ベリー抽出物の作用試験：  Test of the effect of Bilberry extract on HUVE C (human umbilical vein endothelial cell) proliferation induced by VEGF-A (Vascular endothelial growth factor):
[0063] (1)試験成分： [0063] (1) Test components:
ビルべリーのエタノール抽出物（粉末状）  Bilberry ethanol extract (powder)
試験例 1と同様にして製造した、粉末状のビルべリー抽出物を使用した。  A powdery Bilberry extract produced in the same manner as in Test Example 1 was used.
[0064] (2)試験方法： [0064] (2) Test method:
[0065] 標記確認試験を、 Matsubaraらの増殖試験方法（Proliferation assay)を参照にして行 つた（Matsubara, K et al. J. Agric. Food Chem. 2005, 53, 6272-6275)。  [0065] The title confirmation test was conducted with reference to Matsubara et al. (Proliferation assay) (Matsubara, K et al. J. Agric. Food Chem. 2005, 53, 6272-6275).
HUVECを 1ゥエルあたり 2000 cellずつ 96ウェルマルチウエルプレートへ播種し、培養 1 2時間後、 2%FBS (ゥシ胎児血清）を含有した HuMedia- EB2 (倉敷紡績株式会社)へ 培地交換し、 6時間培養した。その後、図面に記載した濃度の、 VEGF-A (終濃度 10 ng/ml)及び Z又は各種濃度のビルべリー抽出物（終濃度それぞれ 0.3, 1, 3, 10, 30 μ g/ml)をそれぞれ添加した。培養 48時間後、 2%FBS含有 HuMedia- EB2培地へ交 換し、再度、 VEGF-A及び Z又はビルべリー抽出物をそれぞれ添加しさらに 48時間 口 ^しプ 。  Seed HUVEC in a 96-well multi-well plate at 2000 cells per well in a 96-well multi-well plate, and after 12 hours of culture, change the medium to HuMedia-EB2 (Kurashikibo Co., Ltd.) containing 2% FBS (Ushi Fetal Serum). Incubate for hours. After that, add VEGF-A (final concentration 10 ng / ml) and Z or various concentrations of Bilberry extract (final concentrations 0.3, 1, 3, 10, 30 μg / ml respectively) as shown in the drawing. Each was added. After 48 hours of culture, the medium was replaced with HuMedia-EB2 medium containing 2% FBS, and VEGF-A and Z or Bilberry extract were added again, and the mixture was further digested for 48 hours.
細胞増殖測定キットに使用する CCK-8溶液 (株式会社同仁科学研究所製)を各ゥ ルへ 10 /z lずつ添加し、 3時間、 37°Cにおいて反応させ、 492 nmの吸光度（参照波長 660 nm)を測定した。  CCK-8 solution (manufactured by Dojindo Laboratories Co., Ltd.) used for the cell proliferation measurement kit was added to each cell at 10 / zl, reacted at 37 ° C for 3 hours, and absorbance at 492 nm (reference wavelength 660 nm).
[0066] (3) 試験結果： [0066] (3) Test results:
ビルべリー抽出物添加により、濃度依存的かつ有意に VEGF-A誘導によって HUVE Cの増殖を抑制することが分力つた（図 12)。 By adding Bilberry extract, concentration-dependent and significantly HUVE by VEGF-A induction Suppression of C proliferation was a major factor (Fig. 12).
エラーバーは、標準誤差を示す。 **はコントロールに対して有意水準 0.01未満、 #は VEGF単独に対して有意水準 0.05未満、 は VEGF単独に対して有意水準 0.01未満 をそれぞれ示す。例数は 5〜8である。  Error bars indicate standard error. ** indicates a significance level of less than 0.01 for control, # indicates a significance level of less than 0.05 for VEGF alone, and indicates a significance level of less than 0.01 for VEGF alone. The number of examples is 5-8.
尚、ビルべリー抽出物単独 (VEGF-A無添加）による HUVECの減少は観察されなか つたことから、ビルべリー抽出物による細胞毒性はないと考えられる。  Since no reduction in HUVEC was observed with the bilberry extract alone (without VEGF-A), it is considered that there is no cytotoxicity with the bilberry extract.
[0067] 試験例 4 [0067] Test Example 4
VEGF-Aが誘導する HUVEC遊走に対する、ビルべリー抽出物の作用試験  Effect of Bilberry extract on HUVEC migration induced by VEGF-A
(本法は Kimらの方法を参照にして行った。 Kin, K.S. et al.， J. Biol. Chem. 2003, 27 8, 11449- 11456.)遊走試験（Migration assay)  (This method was performed with reference to the method of Kim et al. Kin, K.S. et al., J. Biol. Chem. 2003, 27 8, 11449-11456.) Migration assay
[0068] (1)試験成分： [0068] (1) Test components:
ビルべリーのエタノール抽出物（粉末状）  Bilberry ethanol extract (powder)
試験例 1と同様にして製造した、粉末状のビルべリー抽出物を使用した。  A powdery Bilberry extract produced in the same manner as in Test Example 1 was used.
[0069] (2)試験方法： [0069] (2) Test method:
[0070] 標記試験には、セルカルチャーインサート (株式会社べタトンディッキンソン）を用い た。セルカルチャーインサートの上部チャンバ一へ 0.1%BSA (ゥシ血清アルブミン）含 有の HuMedia- EB2にて懸濁した HUVECを 1ゥエル当たり 50000 cellずつ播種した。上 部チャンバ一へビルべリー抽出物、下部チャンバ一へ、図面に記載した濃度のビル ベリー抽出物及び Z又は VEGF-Aをそれぞれ添加し、培養した（終濃度：ビルべリー 抽出物 3, 10,30 μ g/ml,VEGF-A 10ng/ml)。  [0070] Cell culture insert (Betaton Dickinson Co., Ltd.) was used for the title test. HUVEC suspended in HuMedia-EB2 containing 0.1% BSA (usi serum albumin) was seeded in the upper chamber of the cell culture insert at a rate of 50000 cells per well. The bilberry extract was added to the upper chamber and the bilberry extract and Z or VEGF-A at the concentrations shown in the drawings were added to the lower chamber and cultured (final concentration: bilberry extract 3, 10,30 μg / ml, VEGF-A 10 ng / ml).
培養 4時間後、遊走していない HUVECを綿棒にて拭い取り、遊走している細胞を固 定した。固定後、セルカルチャーインサートの膜を切り離し、へマトキシリン染色を行 い、遊走細胞を染色した。染色した細胞をデジタルカメラで撮影し、染色した細胞の 数を数え、測定した。  After 4 hours of culture, non-migrated HUVECs were wiped off with a cotton swab to fix the migrating cells. After fixation, the cell culture insert membrane was cut off, stained with hematoxylin, and migrated cells were stained. The stained cells were photographed with a digital camera, and the number of stained cells was counted and measured.
[0071] (3) 試験結果： [0071] (3) Test results:
ビルべリー抽出物添加により、濃度依存的かつ有意に VEGF-A誘導によって HUVE Cの遊走を抑制することが分力つた（図 13)。  By adding Bilberry extract, concentration-dependent and significantly suppressed the migration of HUVE C by VEGF-A induction (Fig. 13).
エラーバーは、標準誤差を示す。 **はコントロールに対して有意水準 0.01未満、 は VEGF単独に対して有意水準 0.01未満をそれぞれ示す。例数は 5または 6である。 尚、ビルべリー抽出物単独 (VEGF-A無添加）による HUVECの減少は観察されなか つたことから、ビルべリー抽出物による細胞毒性はないと考えられる。 Error bars indicate standard error. ** indicates a significance level of less than 0.01 relative to the control. The significance level is less than 0.01 for VEGF alone. The number of examples is 5 or 6. Since no reduction in HUVEC was observed with the bilberry extract alone (without VEGF-A), it is considered that there is no cytotoxicity with the bilberry extract.
[0072] 試験例 3, 4の結果から、ビルべリー抽出物による VEGF-A誘導の管腔形成の抑制の メカニズムは、 HUVECの増殖及び遊走を抑制することにあることが判明した。  [0072] From the results of Test Examples 3 and 4, it was found that the mechanism of inhibition of VEGF-A-induced lumen formation by the Bilberry extract was to suppress the proliferation and migration of HUVEC.
[0073] 試験例 5  [0073] Test Example 5
VEGF- Aが誘導する ERKl, ERK2 (extracellular signal-regulated kinase 1 or 2) , PL C y (phospholipase C γ ) ,又は Akt (serine/ threonine protein kinase family protein k inase B)のリン酸ィ匕に対する、ビルべリー抽出物の作用試験  For ERKl, ERK2 (extracellular signal-regulated kinase 1 or 2), PL C y (phospholipase C γ), or Akt (serine / threonine protein kinase family protein k inase B) induced by VEGF-A, Action test of Bilberry extract
[0074] (1)試験成分： [0074] (1) Test components:
ビルべリーのエタノール抽出物（粉末状）  Bilberry ethanol extract (powder)
試験例 1と同様にして製造した、粉末状のビルべリー抽出物を使用した。  A powdery Bilberry extract produced in the same manner as in Test Example 1 was used.
[0075] (2)試験方法： roxyethyl)- 1 -piperazinyl] ethanesulfonic acid (HEPES)^g" のタノレべッコ 変づーグ ル培地 (D- MEM)へ培地交換を行い、培養した。 [0075] (2) Test method: roxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) ^ g "was changed to Tanolebecko's modified medium (D-MEM) and cultured.
培養 1時間後（Aktは 18時間後）、 10 ng/ml VEGF- A及び Z又は 30 μ g/mlビルべリー 抽出物を含有した 2%FBS (Aktは 0.5%FBS)及び 25 mM HEPES含有 D- MENで培地 交換を行い、反応させた。  1 hour after culture (18 hours after Akt), 2% FBS containing 10 ng / ml VEGF-A and Z or 30 μg / ml Bilberry extract (Akt is 0.5% FBS) and 25 mM HEPES The medium was exchanged with D-MEN and reacted.
反応後、 2回洗浄を行い、細胞溶解用 RIPA緩衝液 (シグマアルドリッチ株式会社製） を添加し、細胞を破壊し、— 80°Cにて保存した。  After the reaction, washing was performed twice, RIPA buffer for cell lysis (manufactured by Sigma-Aldrich) was added, the cells were destroyed, and stored at −80 ° C.
タンパク質は 7.5%SDS-ポリアクリルアミアミドゲル電気泳動を行 、、ウェスタンブロッ ティング法を用いてバンドの輝度を解析した。  The protein was subjected to 7.5% SDS-polyacrylamidamide gel electrophoresis, and the luminance of the band was analyzed using Western blotting.
一次抗体は抗リン酸化 ERK1/2抗体，抗トータル ERK1/2抗体，抗リン酸化 PLC γ抗 体，抗トータル PLC γ抗体，抗リン酸化 Akt抗体，抗トータル Akt抗体（セルシグナリン グノィォテクノロジ一社製)そして抗 j8 -ァクチン抗体 (シグマアルドリッチ株式会社） を用いた。  Primary antibodies are anti-phosphorylated ERK1 / 2 antibody, anti-total ERK1 / 2 antibody, anti-phosphorylated PLC γ antibody, anti-total PLC γ antibody, anti-phosphorylated Akt antibody, anti-total Akt antibody (Cersignalin Technology) And anti-j8-actin antibody (Sigma Aldrich Co.).
二次抗体としては、抗 ERK抗体，抗 PLC抗体，抗 Akt抗体に対しては、ャギ抗ゥサギ HRP (西洋わさびペルォキシダーゼ)抗体を、抗 |8ァクチン抗体に対しては、ャギ抗 マウス HRP抗体を使用した。 Secondary antibodies include anti-ERK antibody, anti-PLC antibody, and anti-Akt antibody. HRP (horseradish peroxidase) antibody was used, and for anti- | 8-actin antibody, goat anti-mouse HRP antibody was used.
[0076] (3) 試験結果： [0076] (3) Test results:
ビルべリー抽出物は、 VEGF- Α誘導による HUVECの増殖において、 PLC yより下流 であり、 ERK1又は ERK2より上流のシグナル経路を抑制することが明らかになった（ 図 14, 15)。  Bilberry extract was found to suppress signal pathways downstream of PLCy and upstream of ERK1 or ERK2 in HUVEC proliferation induced by VEGF-VEGF (Figs. 14 and 15).
また、ビルべリー抽出物は、 VEGF-A誘導による HUVECの遊走において、 Aktより上 流のシグナル経路を抑制することが明ら力となった（図 16)。  In addition, the Bilberry extract was clearly able to suppress the signal pathway upstream of Akt during VEGF-A-induced HUVEC migration (Fig. 16).
図 14, 15, 16において、エラーバーは、標準誤差を示す。 *はコントロールに対して 有意水準 0.05未満、 #は VEGF単独に対して有意水準 0.05未満、 は VEGF単独に 対して有意水準 0.01未満をそれぞれ示す。例数は 4〜7である。  In FIGS. 14, 15 and 16, error bars indicate standard errors. * Indicates a significance level of less than 0.05 for control, # indicates a significance level of less than 0.05 for VEGF alone, and indicates a significance level of less than 0.01 for VEGF alone. The number of examples is 4-7.
[0077] 以下に実施例を挙げて本発明をさらに具体的に説明する力 これらの実施例により 本発明は限定されるものではな 、。 [0077] The ability to explain the present invention more specifically with reference to the following examples. The present invention is not limited by these examples.
実施例  Example
[0078] 実施例 1, 2 (硝子体内投与用注射剤）  Examples 1 and 2 (Injection for intravitreal administration)
試験例 1と同様の方法で、粉末状ビルべリー抽出物を得る。この粉末状ビルべリー抽 出物（1重量部）を注射用蒸留水（10重量部又は 100重量部）に溶解し、 1mlずつ分 注してそれぞれ、実施例 1, 2の注射剤とする。  A powdered Bilberry extract is obtained in the same manner as in Test Example 1. Dissolve this powdered Bilberry extract (1 part by weight) in distilled water for injection (10 parts by weight or 100 parts by weight) and dispense 1 ml each to make the injections of Examples 1 and 2, respectively. .
[0079] 実施例 3, 4 (点眼剤）  [0079] Examples 3 and 4 (eye drops)
試験例 1と同様の方法で、粉末状ビルべリー抽出物を得る。この粉末状ビルべリー抽 出物（1重量部）を蒸留水（10重量部又は 100重量部）に溶解し、 10mlずつ分注して それぞれ、実施例 3, 4の点眼剤とする。  A powdered Bilberry extract is obtained in the same manner as in Test Example 1. Dissolve this powdered bilberry extract (1 part by weight) in distilled water (10 parts by weight or 100 parts by weight) and dispense 10 ml each to make eye drops of Examples 3 and 4, respectively.
産業上の利用可能性  Industrial applicability
[0080] 本発明の血管新生阻害剤，シグナル伝達阻害剤は、血管新生を有意に抑制するこ とから、いわゆる実験 ·研究用試薬として使用可能である他、血管新生を伴う、眼科 疾患，腫瘍又は癌疾患，あるいは慢性炎症の予防又は治療剤，あるいは健康食品 等の有効成分として有用である。 [0080] Since the angiogenesis inhibitor and signal transduction inhibitor of the present invention significantly suppress angiogenesis, they can be used as so-called experimental and research reagents, as well as ophthalmic diseases and tumors associated with angiogenesis. It is also useful as an active ingredient for preventing or treating cancer diseases or chronic inflammation, or for health foods.
図面の簡単な説明 [図 1]本発明の治療剤の、 in vitroでの血管新生（〈1〉管腔面積 (area) )抑制作用を 示す図である。尚、図中の Vとは VEGFを表す。 Brief Description of Drawings FIG. 1 is a graph showing an in vitro angiogenesis (<1> lumen area) inhibitory effect of the therapeutic agent of the present invention. In the figure, V represents VEGF.
[図 2]本発明の治療剤の、 in vitroでの血管新生（〈2〉管腔長 (length) )抑制作用を示 す図である。尚、図中の Vとは VEGFを表す。  FIG. 2 is a graph showing an in vitro angiogenesis (<2> lumen length (length)) inhibitory effect of the therapeutic agent of the present invention. In the figure, V represents VEGF.
[図 3]本発明の治療剤の、 in vitroでの血管新生（〈3〉管腔分枝点数 (joint) )抑制作 用を示す図である。尚、図中の Vとは VEGFを表す。  FIG. 3 is a view showing an in vitro angiogenesis (<3> lumen branch point (joint)) inhibitory action of the therapeutic agent of the present invention. In the figure, V represents VEGF.
[図 4]本発明の治療剤の、 in vitroでの血管新生（〈4〉枝数 (path) )抑制作用を示す 図である。尚、図中の Vとは VEGFを表す。  FIG. 4 is a graph showing the in vitro angiogenesis (<4> branch number (path)) inhibitory action of the therapeutic agent of the present invention. In the figure, V represents VEGF.
[図 5]VEGFの、 in vitroでの血管新生作用を示す管腔形成図である。  FIG. 5 is a luminal formation diagram showing the angiogenic action of VEGF in vitro.
[図 6]本発明の治療剤の、 in vitroでの血管新生抑制作用を示す管腔形成図である。 FIG. 6 is a luminal formation diagram showing in vitro angiogenesis inhibitory action of the therapeutic agent of the present invention.
[図 7]本発明の治療剤の、 in vitroでの血管新生抑制作用を示す管腔形成図である。 FIG. 7 is a luminal formation diagram showing in vitro angiogenesis inhibitory action of the therapeutic agent of the present invention.
[図 8]本発明の治療剤の、 in vitroでの血管新生抑制作用を示す管腔形成図である。 FIG. 8 is a luminal formation diagram showing the in vitro angiogenesis inhibitory action of the therapeutic agent of the present invention.
[図 9]高酸素負荷においた後、溶媒投与したマウスの、網膜の血管造影図である。 FIG. 9 is an angiogram of the retina of a mouse administered with a solvent after being subjected to a high oxygen load.
[図 10]高酸素負荷においた後、ビルべリー抽出物を投与したマウスの、網膜の血管 造影図である。 FIG. 10 is an angiogram of the retina of a mouse administered with Bilberry extract after being exposed to high oxygen load.
[図 11]本発明の治療剤の、 in vivoでの血管新生抑制作用を示す図である。  FIG. 11 is a diagram showing an in vivo angiogenesis inhibitory effect of the therapeutic agent of the present invention.
[図 12]本発明の治療剤の、血管新生抑制作用が、 HUVECの増殖を抑制することに 起因することを示す図である。  FIG. 12 is a graph showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is caused by inhibiting the proliferation of HUVEC.
[図 13]本発明の治療剤の、血管新生抑制作用が、 HUVECの遊走を抑制することに 起因することを示す図である。  FIG. 13 is a graph showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is due to inhibition of HUVEC migration.
[図 14]本発明の治療剤の、血管新生抑制作用が、 ERK1又は ERK2より上流のシグ ナル経路を抑制することよる HUVECの増殖抑制に起因することを示す図である。  FIG. 14 is a graph showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is caused by suppression of HUVEC proliferation by suppressing a signal pathway upstream of ERK1 or ERK2.
[図 15]本発明の治療剤の、血管新生抑制作用が、 PLC γより下流のシグナル経路を 抑制することよる HUVECの増殖抑制に起因することを示す図である。 FIG. 15 is a graph showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is due to inhibition of HUVEC proliferation by inhibiting a signal pathway downstream from PLC γ.
[図 16]本発明の治療剤の、血管新生抑制作用が、 VEGFレセプターより下流であり、 Aktより上流のシグナル経路を抑制することによる HUVECの遊走抑制に起因すること を示す図である。 FIG. 16 is a diagram showing that the angiogenesis inhibitory action of the therapeutic agent of the present invention is due to inhibition of HUVEC migration by suppressing a signal pathway downstream from the VEGF receptor and upstream from Akt.

Claims

請求の範囲 The scope of the claims
[1] ビルべリー抽出物を含むことを特徴とする、血管新生阻害剤。  [1] An angiogenesis inhibitor comprising a Bilberry extract.
[2] ビルべリー抽出物を含むことを特徴とする、 PLC y力も ERK1又は ERK2へのシグナ ル伝達の阻害剤。  [2] Inhibitor of signal transmission to ERK1 or ERK2 with PLC y force, characterized by containing Bilberry extract.
[3] ビルべリー抽出物を含むことを特徴とする、 VEGFレセプターから Aktへのシグナル 伝達の阻害剤。  [3] An inhibitor of signal transduction from VEGF receptor to Akt, characterized by comprising Bilberry extract.
[4] ビルべリー抽出物力 ビルべリーのエタノール抽出物又は含水エタノール抽出物で あることを特徴とする、請求項 1に記載の血管新生阻害剤。  [4] The angiogenesis inhibitor according to claim 1, which is an extract of bilberry or an aqueous ethanol extract of bilberry.
[5] 請求項 1乃至 4のいずれかに記載の血管新生阻害剤又はシグナル伝達阻害剤を有 効成分として含むことを特徴とする、血管新生を伴う疾病に対する予防又は治療剤。 [5] A preventive or therapeutic agent for a disease associated with angiogenesis, comprising the angiogenesis inhibitor or the signal transduction inhibitor according to any one of claims 1 to 4 as an active ingredient.
[6] 血管新生を伴う疾病が、眼科疾患，腫瘍又は癌疾患，あるいは慢性炎症である、請 求項 5記載の疾病予防又は治療剤。 [6] The disease preventive or therapeutic agent according to claim 5, wherein the disease accompanied by angiogenesis is ophthalmic disease, tumor or cancer disease, or chronic inflammation.
[7] 血管新生を伴う疾病が、加齢性黄斑変性症，糖尿病性網膜症，新生血管緑内障， 増殖性糖尿病網膜症，未熟児網膜症，滲出型加齢黄斑変性症，血管新生緑内障， 網膜静脈閉塞症，網膜動脈閉塞症，翼状片，ルべォ一シス，角膜新生血管症，固 型腫瘍，骨髄腫，血管腫，慢性関節性リウマチ，乾癬，又は変形性関節症である、請 求項 5記載の疾病予防又は治療剤。 [7] Diseases associated with angiogenesis are age-related macular degeneration, diabetic retinopathy, neovascular glaucoma, proliferative diabetic retinopathy, retinopathy of prematurity, wet age-related macular degeneration, neovascular glaucoma, retina Venous occlusion, retinal artery occlusion, pterygium, rubeosis, corneal neovascular disease, solid tumor, myeloma, hemangioma, rheumatoid arthritis, psoriasis, or osteoarthritis Item 5. A disease preventive or therapeutic agent according to Item 5.
[8] 請求項 1乃至 4のいずれかに記載の血管新生阻害剤又はシグナル伝達阻害剤を含 むことを特徴とする、血管新生阻害用の食品。 [8] A food for inhibiting angiogenesis, comprising the angiogenesis inhibitor or the signal transduction inhibitor according to any one of [1] to [4].
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