WO2011122321A1 - Agent d'inhibition de l'activité physiologique de la protéine se liant à l'héparine - Google Patents

Agent d'inhibition de l'activité physiologique de la protéine se liant à l'héparine Download PDF

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WO2011122321A1
WO2011122321A1 PCT/JP2011/055985 JP2011055985W WO2011122321A1 WO 2011122321 A1 WO2011122321 A1 WO 2011122321A1 JP 2011055985 W JP2011055985 W JP 2011055985W WO 2011122321 A1 WO2011122321 A1 WO 2011122321A1
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heparin
vegf
factor
binding
growth factor
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Japanese (ja)
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康二 西口
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国立大学法人名古屋大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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

Definitions

  • the present invention relates to a novel use of sulfated sugar chains such as heparin and heparan sulfate. Specifically, the present invention relates to a drug that inhibits the function of a specific protein using a sulfated sugar chain, and a preventive / therapeutic method using the drug.
  • This application claims priority based on Japanese Patent Application No. 2010-073967 filed on Mar. 29, 2010, the entire contents of which are incorporated by reference.
  • retinal blood vessels ⁇ Chronic elevation of blood glucose and rapid exposure to high-concentration oxygen cause existing retinal blood vessels to become occluded. From the vascular occlusion site, the retinal blood vessels are erroneously regenerated toward the vitreous, resulting in a series of changes that lead to a reduction in visual acuity. Examples of eye diseases that develop by such a mechanism include diabetic retinopathy and retinopathy of prematurity. These diseases are a concern that the number of patients is increasing all over the world due to an increase in the lifesaving rate of premature babies due to changes in lifestyle and dietary habits or the development of neonatal medicine. However, it is not known why retinal blood vessels tend to be directed toward the vitreous body in ischemic retinopathy, whereas developing retinal blood vessels are confined within the retina.
  • Heparan sulfate (HS) proteoglycan is composed of a sugar chain having a specific sequence called a core protein and one or more glycosaminoglycans (GAG) (Non-patent Document 1). These proteoglycans are ubiquitously present on the surface of all cells as a membrane-bound type, or on the extracellular matrix as a secreted type, and have important physiological activities. HS is strongly negatively charged due to many sulfate group modifications, and affects the interaction between various heparin-binding proteins and their receptors through ionic bonds (Non-patent Document 1).
  • Non-patent Document 2 tissue-specific expression profiles
  • membrane-bound HS proteoglycans also promote the binding of heparin-binding proteins and their receptors that also occur on the membrane surface
  • Non-Patent Documents 5 and 6 membrane-bound HS proteoglycans
  • soluble HS is ambiguous (Non-Patent Documents 5 and 6). It may vary depending on the environment and exhibit contradictory properties (Non-Patent Documents 6 and 7).
  • the role of soluble HS that adheres to the cell surface and can be changed to a membrane-bound type further complicates the role (Non-patent Document 3).
  • VEGF-A vascular endothelial growth factor
  • Non-patent Document 11 It was reported that the soluble HS in anterior aqueous humor suppresses the binding of angiogenesis-inducing factors (VEGF-A and basic fibroblast growth factor (bFGF)) and receptors (Non-patent Document 11). However, the physiological importance of soluble HS remains unclear.
  • Heparin a type of glycosaminoglycan, is a polymer in which ⁇ -D-glucuronic acid or ⁇ -L-iduronic acid and D-glucosamine are polymerized via 1,4-linkages, and is more sulfated than HS. It is characterized by a particularly high degree. However, there is no significant difference in chemical properties between heparin and HS sugar chains. In general, heparin is widely used as an anticoagulant. However, since many sulfate groups contained in the molecule are negatively charged, they interact with various physiologically active substances. In particular, it is known to act as a signal enhancement factor by promoting the binding of its receptor to many growth factors.
  • heparin has the function of enhancing the action of basic fibroblast growth factor (bFGF), which plays an important role in angiogenesis.
  • bFGF basic fibroblast growth factor
  • VEGF121 a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J. Biol. Chem. 270: 11322-11326. Funatsu, H., Yamashita, H., Noma, H., Mimura, T., Nakamura, S., Sakata, K., And Hori, S. 2005.
  • Aqueous humor levels of cytokines are related to vitreous levels and progression of diabetic retinopathy in diabetic patients. Graefes Arch. Clin. Exp. Ophthalmol. 243: 3-8. Aiello, LP, Avery, RL, Arrigg, PG, Keyt, BA, Jampel, HD, Shah, ST, Pasquale, LR, Thieme, H., Iwamoto, MA, Park, JE et al 1994. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 331: 1480-1487.
  • soluble HS shows a specific distribution in the intraocular fluid and exists at a high concentration, and as a result, functions as an endogenous inhibitor for VEGF.
  • This finding indicates that the physiological activity of VEGF can be inhibited by locally adjusting the concentration of HS or heparin, in other words, HS or the like is effective as an inhibitor of VEGF.
  • retinopathy especially proliferative retinopathy in young diabetic patients is one of the optimal targets for treatment with HS and the like.
  • vascular growth factors other than VEGF glycosaminoglycans such as heparin can bind to and inactivate vascular growth factors other than VEGF (eg bFGF), an additive or synergistic effect can be expected when used in combination with an anti-VEGF agent.
  • both heparin and HS have a large number of sulfate groups and are strongly negatively charged sugar chains. Therefore, sulfated sugar chains with similar characteristics, such as chondroitin sulfate, dermatan sulfate, ketalan sulfate, or their degradation products. Can also be said to function as an inhibitor of VEGF.
  • the inhibitory effect of VEGF by heparin and the like is based on the binding of both molecules, the binding occurs through the heparin binding domain present in VEGF, and a high concentration of soluble sulfated sugar chains are formed on the cell surface of heparan sulfate.
  • sulfated sugar chains such as heparin can be expected on various biomolecules having heparin-binding domains. That is, not only VEGF but also sulfated sugar chains such as heparin are used as a means for inhibiting the physiological activity of various biomolecules having heparin binding domains (growth factors, inflammatory factors, lipid metabolism-related factors, cell adhesion factors, etc.). It can be said that it is effective.
  • the present invention described below is mainly based on the above results or knowledge.
  • a physiologically active inhibitor for heparin-binding protein comprising a sulfated sugar chain.
  • the sulfated glycosaminoglycan is one or more compounds selected from the group consisting of heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, ketalan sulfate, and any degradation products or modifications thereof.
  • Growth factors include vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor family protein (FGF), midkine, hepatocyte growth factor (HGF) and beta-type mutant growth factor (TGF- ⁇ ) is a factor selected from the group consisting of tumor necrosis factor ⁇ (TNF- ⁇ ), stromal cell-derived factor 1 (SDF-1), platelets and T cell-derived eosinophils It is a factor selected from the group consisting of chemokine family proteins including chemotactic substances (RANTES) and monocyte chemotaxis activator (MCP-1), and lipid metabolism-related factors include Annexin V, Apolipo A factor selected from the group consisting of protein E (APOE), and the cell adhesion factor is a factor selected from the group consisting of L-selectin and P-selectin [4] The bioactivity inhibitor described in 1.
  • VEGF vascular endothelial growth factor
  • EGF epidermal growth factor
  • [9] Proliferative diabetic retinopathy, retinopathy of prematurity, wet age-related macular degeneration, myopic macular degeneration, idiopathic choroidal neovascular disease, ocular ischemic syndrome, retinal vein occlusion, iris neovascular disease, corneal blood vessels
  • the bioactivity inhibitor according to [9] which is administered to a patient with a vascular occlusion disease or a person with a high risk of suffering from a vascular occlusion disease.
  • the bioactivity inhibitor according to [11] which is a kit comprising a first component containing a sulfated sugar chain and a second component containing an anti-VEGF agent.
  • a therapeutically effective amount of a sulfated sugar chain is administered to a patient suffering from or likely to suffer from a disease caused by an abnormal increase in the expression level of heparin-binding protein and / or an abnormal increase in action.
  • HS heparan sulfate glycosaminoglycan
  • GAG heparan sulfate glycosaminoglycan
  • concentration is high in intraocular fluid.
  • A Profile of the core protein of HS proteoglycan in intraocular fluid. As a result of Western blotting using intraocular fluid collected from 15-day-old mice as a sample, perlecan, collagen XVIII (collagen XVIII), syndecan-3 (syndecan-3), syndecan-1 (syndecan-1) and syndecan-2 Bands corresponding to (syndecan-2) were observed.
  • B Time course of HS concentration in intraocular fluid.
  • D GS-stained image of a retinal extension specimen of an oxygen challenge model (OIR; a mouse that has induced pathological retinal neovascularization) Prior to exposure to oxygen (7 days of age), retinal blood vessels stretch toward the periphery (stars). After exposure to 80% oxygen (12 days of age), retinal blood vessels regress and disappear (arrows).
  • OIR oxygen challenge model
  • CG PBS (C), HIII (D), HIII and heparin (HIII + Hep; E), high concentration HS (HS-hi; F) or heparin (Hep; G) are administered to the eyes of 12-day-old mice did.
  • a GS-stained image (representative example) of the retina collected at 17 days of age is shown (top).
  • the lower row shows the neovascular area (red in the original image) and avascular area (blue in the original image).
  • FIG. 3 shows that VEGF-A bound with soluble heparin is inactivated while acquiring resistance to proteolysis.
  • A VEGF-A concentration in the intraocular fluid of OIR mice.
  • Human VEGF121 isoform (5 ng per eye) with heparin (20 ⁇ g per eye) was injected into one eye of a 12 day old mouse. The other eye was injected with the same amount of human VEGF121 isoform only.
  • VEGF165 (F) and VEGF121 (G) were subjected to degradation by plasmin (lanes 1 and 5; 400 ng, lanes 2 and 6; 80 ng, lanes 3 and 7; 16 ng, lanes 4 and 8; 3.2 ng). Evaluation was based on VEGF165 and VEGF121 (arrowheads) that had not undergone the degradation treatment. All data are expressed as mean ⁇ standard error (S.E.M.). The figure which shows that heparin and intraocular fluid suppress the proliferation of a HUVEC cell.
  • A Effect of heparin on the proliferation ability of HUVEC.
  • Heparin was added to cells cultured in EG2 medium supplemented with VEGF-A at various concentrations (each data is n 4). Soluble heparin suppressed the growth ability of HUVEC in a concentration-dependent manner. The experiment was repeated.
  • FIG. 1 The figure which shows that soluble HS and heparin glycosaminoglycan inhibits the binding of superficial heparin or VEGFR2 and VEGF165.
  • A Binding of VEGF-A to superficial heparin. The horizontal axis is the amount of superficial heparin.
  • B Effect of soluble heparin on binding of VEGF-A and superficial heparin. VEGF-A was added along with soluble heparin to a fixed amount of superficial heparin (heparin covalently bound to the bottom of the well). The horizontal axis is the amount of soluble heparin added.
  • E-J Effect of OF, soluble HS or soluble heparin on binding of VEGF165 or VEGF121 to superficial VEGFR2 (E-G) or VEGFR1 (H-J).
  • E-G superficial VEGFR2
  • VEGFR1 VEGFR1
  • HS-lo, HS-hi, OF (E) and heparin (G) inhibited the binding of VEGF165 and VEGFR2.
  • the inhibitory effect of HS / heparin glycosaminoglycan or OF on the binding of VEGF165 or VEGF121 to VEGFR1 was slight (H-J).
  • C) Scatter plot of HS concentration and age of PDR patients and results of regression analysis. There is no correlation (R 2 0.084).
  • production of CNV (choroidal neovascularization) is suppressed by heparin intraocular administration.
  • A, B Eyes treated with high concentrations of heparin (Hep; A) and PBS.
  • Relative values based on treatment with PBS are shown. Treated with low concentrations of heparin (100 ⁇ g / ml; C), high concentrations of heparin (10000 ⁇ g / ml; E) and concentrations in between (1000 ⁇ g / ml; D).
  • A-C Distribution of FITC-heparin 3 hours after laser irradiation (green in the original image) and GS staining (red in the original image).
  • FITC-heparin (arrowheads) accumulated in the photoreceptor layer (arrow A) and GS positive retinal blood vessels (arrows B and C) around the laser scar.
  • FIG. 1 The figure which shows the effect of heparin with respect to monocyte / macrophage migration.
  • A, B GS stained image 24 hours after laser irradiation and intraocular administration. GS positive cells around the laser scar are reduced in heparinized eyes compared to eyes treated with PBS (B) (A).
  • C, D Comparison of the number of GS positive cells inside and outside the laser mark. In heparinized eyes, the number of GS positive cells outside the laser mark is decreased.
  • A, B Measurement of VEGF and CCL2 secreted into the culture medium of human RPE.
  • the 1st aspect of this invention provides the bioactivity inhibitor with respect to a heparin binding protein.
  • the active ingredient of the physiologically active inhibitor of the present invention is a sulfated sugar chain.
  • a “sulfated sugar chain” is a sugar chain containing a sulfate group.
  • the composition, molecular weight and the like of the sugar chain are not particularly limited as long as they show the binding property to heparin-binding protein.
  • sulfated glycosaminoglycan hereinafter also referred to as “GAG”) is employed as the sulfated sugar chain.
  • GAG sulfated glycosaminoglycan
  • “Sulfated glycosaminoglycan” is a molecule constituting the sugar chain part of proteoglycan, and is usually linear.
  • GAG heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and ketalan sulfate are known. These GAGs all have a repeating structure of disaccharide units composed of uronic acid and amino sugar. When these GAGs are employed, the molecular weight is, for example, several hundred to several million. The reason why the range of molecular weight is large is that the minimum unit with physiological activity is desirable when emphasizing tissue permeability, whereas the larger medicinal effect is predicted when retention is important. Not only an aggregate having a uniform molecular weight but also an aggregate in which molecules having different molecular weights coexist can be used. Two or more types of GAGs may be used in combination.
  • Degradation products or modified products of GAG can also be used.
  • the “degradation product” here refers to a molecule having a relatively small molecular weight as a result of the decomposition as compared with the original GAG.
  • the method for obtaining the degradation product is not particularly limited. Examples of the decomposition method include enzymatic decomposition, chemical decomposition, thermal decomposition, and optical decomposition.
  • the molecular weight of the degradation product is, for example, several hundred to several million.
  • the “modified form” refers to a molecule in which some modification is applied to GAG.
  • the action and effect of the physiologically active inhibitor of the present invention depends on the binding property to the target (heparin-binding protein) possessed by the sulfated sugar chain as the active ingredient.
  • the sulfated sugar chain contained therein is required to have a binding ability to a heparin-binding protein. Therefore, degradation or modification as described above will be allowed as long as the property is maintained. Whether or not a degradation product or modified product of GAG binds to a heparin-binding protein can be confirmed by a standard binding experiment using a heparin-binding protein (for example, VEGF or bFGF).
  • the target of the physiologically active inhibitor of the present invention is a heparin binding protein.
  • heparin-binding protein refers to a protein that exhibits binding to heparin and has physiological activity.
  • heparin-binding proteins are vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor family protein (FGF), midkine, hepatocyte growth factor (HGF), beta mutant growth Various growth factors (growth factors) such as factor (TGF- ⁇ ), tumor necrosis factor ⁇ (TNF- ⁇ ), stromal cell-derived factor 1 (SDF-1), eosinophil chemotactic substances derived from platelets and T cells (RANTES), various inflammatory factors such as chemokine family proteins including monocyte chemotactic factor (MCP-1), lipid metabolism-related factors such as annexin V (Annexin V), apolipoprotein E (APOE), L selectin ( L-selectin), cell adhesion factors such as
  • VEGF is a growth factor produced by glial cells, macrophages and tumor cells, and plays an important role in angiogenesis, inflammation, tumor growth and edema. Therefore, when VEGF is targeted, the physiologically active inhibitor of the present invention can function as an anti-angiogenic agent, anti-inflammatory agent, anti-tumor agent, anti-edema agent and the like.
  • VEGF forms a family.
  • the VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-D, VEGF-E, placental growth factor (PlGF) -1 and PlGF-2.
  • VEGF-A has isoforms such as VEGF121, VEGF165, VEGF189, and VEGF206.
  • VEGF165 is a major VEGF isoform produced in the eye. Therefore, when the present invention is applied to the prevention / treatment of eye diseases, all isoforms including VEGF165 other than VEGF121 having no heparin binding domain can be targeted.
  • EGF plays an important role mainly in the growth, proliferation and differentiation of epithelial cells. Therefore, when EGF is a target, the physiologically active inhibitor of the present invention can function as an inhibitor of growth, proliferation or differentiation of epithelial cells.
  • FGF is a polypeptide having high affinity with heparin.
  • the FGF family includes aFGF (FGF1), bFGF (FGF2), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF11, FGF19, and the like.
  • FGF signal plays an important role in development, differentiation and metabolic regulation.
  • an abnormality in FGF signal contributes to canceration of cells and causes bone and cartilage diseases such as skull fusion, achondroplasia, and hypochondral dysplasia. Therefore, when FGF is targeted, the physiologically active inhibitor of the present invention can function as a metabolic regulator or a preventive / therapeutic agent for these diseases.
  • Midkine is a heparin-binding secreted protein (Tomomura, M., Kadomatsu, K., Nakamoto, M., Muramatsu, H., Kondoh, H., Imagawa, K. and Muramatsu, T. (1990) A retinoic, acid, responsive gene, MK, produced, a secreted, protein, with heparin, binding, activity, Biochem, Biophys, Res., Commun., 171, 603-609.).
  • the physiologically active inhibitor of the present invention can function as an apoptosis promoting agent, anti-inflammatory agent, anti-angiogenic agent and the like.
  • the physiologically active inhibitor of the present invention targets an angiogenesis-inducing factor and functions as an anti-angiogenic agent.
  • angiogenesis-inducing factor are VEGF (particularly VEGF-A) and FGF (particularly bFGF).
  • VEGF-A is targeted or one of the targets.
  • Anti-angiogenic agents are provided.
  • an anti-angiogenic agent targeting bFGF or one of the targets is provided.
  • the present invention when provided as an anti-angiogenic agent includes, for example, proliferative diabetic retinopathy, retinopathy of prematurity, wet age-related macular degeneration, myopic macular degeneration (neovascular macular degeneration), idiopathic choroidal neovascularization Used for prevention or treatment of cerebral dysfunction, ocular ischemic syndrome, retinal vein occlusion, iris neovascularization or corneal neovascularization.
  • proliferative diabetic retinopathy as an anti-tumor agent
  • macular edema as an anti-edema agent
  • one of the application fields of the present invention is the ophthalmological field, and ocular diseases associated with angiogenesis are suitable for prevention and treatment.
  • anti-VEGF agents are used to treat diseases associated with neovascularization and macular edema.
  • existing anti-VEGF agents may increase the risk of vascular occlusion diseases (cerebral infarction, myocardial infarction, etc.). It has been pointed out that it is difficult to use for patients with vascular occlusion disease or those who are at high risk of vascular occlusion disease (elderly, diabetics, etc.).
  • heparin which can be an active ingredient of the present invention has a blood anticoagulant action.
  • the physiologically active inhibitor of the present invention targets a heparin-binding protein, and is characterized by being capable of targeting two or more types simultaneously as long as it is a heparin-binding protein.
  • angiogenesis inducing factors such as bFGF
  • a high anti-angiogenic effect can be exhibited through an action on a plurality of angiogenesis inducing factors.
  • the anti-angiogenic agent (or anti-inflammatory agent, antitumor agent, anti-edema agent) of the present invention can be used in combination with an existing anti-VEGF agent to exert an additive or synergistic effect. That is, in one embodiment of the present invention, the bioactive agent of the present invention that functions as an anti-angiogenic agent is used in combination with an anti-VEGF agent.
  • anti-VEGF agents are pegaptanib (trade name: McGen (registered trademark)), ranibizumab (trade name: Lucentis (registered trademark)) and bevacizumab (trade name: Avastin (registered trademark)).
  • the anti-angiogenic agent of the present invention will be provided as a combination agent in which a sulfated sugar chain and an anti-VEGF agent are mixed.
  • the anti-angiogenic agent of the present invention in the form of a kit comprising a drug containing a sulfated sugar chain (first component) and a drug containing an anti-VEGF agent (second component).
  • first component and the second component will be administered at least once within the treatment period.
  • the administration schedule for each element can be set individually. Both elements may be administered simultaneously. “Simultaneous” here does not require strict simultaneity.
  • both elements are administered under the condition that there is no time difference, for example, both elements are mixed and then administered to the subject, both elements are immediately administered after one is administered.
  • both elements are immediately administered after one is administered.
  • the case where the administration is performed under conditions without a substantial time difference is also included in the concept of “simultaneous” herein.
  • the mode in which the anti-VEGF agent is used in combination is not limited to the above, for example, a new anti-vascular preparation containing a sulfated sugar chain as an active ingredient, so that the anti-VEGF agent is also administered within the treatment period. Also good.
  • Preparation of the physiologically active inhibitor of the present invention can be performed according to a conventional method.
  • other pharmaceutically acceptable ingredients for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like.
  • excipient lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used.
  • disintegrant starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer.
  • emulsifier gum arabic, sodium alginate, tragacanth and the like can be used.
  • suspending agent glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
  • soothing agent benzyl alcohol, chlorobutanol, sorbitol and the like can be used.
  • stabilizer propylene glycol, ascorbic acid or the like can be used.
  • preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
  • preservatives benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
  • the dosage form for formulation is not particularly limited. Examples of dosage forms are injections, eye drops, nasal tablets, powders, fine granules, granules, capsules, syrups, external preparations, inhalants and suppositories.
  • the physiologically active inhibitor of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (or preventive effect) (that is, a therapeutically effective amount).
  • the amount of the active ingredient in the physiologically active inhibitor of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within the range of about 0.01 wt% to about 95 wt% so as to achieve a desired dose.
  • the physiologically active inhibitor of the present invention is administered parenterally (instillation, intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) or oral depending on the dosage form. Applied to subjects by administration. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intraocular injection or the like is performed simultaneously with instillation or after a predetermined time has elapsed).
  • the physiologically active inhibitor of the present invention is particularly suitable for local administration due to its properties, but does not exclude application by systemic administration.
  • a drug delivery system (DDS) may be used to administer the active ingredient in a target tissue-specific manner.
  • the “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. , Sheep, dogs, cats, chickens, quails, etc.).
  • the present invention applies to humans.
  • a typical subject to which the present invention is applied is a patient suffering from or at risk of suffering from a disease caused by an abnormal increase in the expression level of heparin-binding protein and / or abnormal enhancement of action. In other words, patients or potential patients with diseases that develop due to or due to increased function of heparin-binding protein are targeted.
  • heparin-binding proteins are vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), midkine, hepatocyte growth factor (HGF), beta type as described above.
  • VEGF vascular endothelial growth factor
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • TGF- ⁇ mutant growth factor
  • TGF- ⁇ tumor necrosis factor ⁇
  • SDF-1 stromal cell-derived factor 1
  • RANTES eosinophil chemotactic substances derived from platelets and T cells
  • chemokine family proteins including monocyte chemotactic factor (MCP-1), lipid metabolism-related factors such as annexin V (Annexin V), apolipoprotein E (APOE), L selectin (L- selectin), cell adhesion factors such as P-selectin, and glycoproteins such as lactoferrin and transferrin.
  • subjects include patients with abnormal angiogenesis and edema in the eye due to abnormal expression of angiogenesis-inducing factors such as VEGF (eg proliferative diabetic retinopathy, retinopathy of prematurity, exudation type addition) Age-related macular degeneration, myopic macular degeneration (neovascular macular disease), idiopathic choroidal neovascular disease, proliferative vitreoretinopathy, uveitis, ocular ischemic syndrome, retinal vein occlusion, iris neovascular disease or corneal blood vessel New patients) or potential patients (eg, patients with nonproliferative diabetes, preproliferative diabetic retinopathy).
  • VEGF eg proliferative diabetic retinopathy, retinopathy of prematurity, exudation type addition
  • Age-related macular degeneration myopic macular degeneration (neovascular macular disease), idiopathic choroidal neovascular
  • the dose of the physiologically active inhibitor of the present invention is set so as to obtain the expected therapeutic effect.
  • a therapeutically effective dose the patient's symptoms, age, sex, weight, etc. are generally considered. A person skilled in the art can set an appropriate dose in consideration of these matters.
  • the administration schedule for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's symptoms and the duration of effect of the active ingredient can be taken into consideration.
  • the present application is intended to treat a patient suffering from or at risk of suffering from a disease caused by an abnormal increase in the expression level of heparin-binding protein and / or an abnormal increase in action.
  • a method for preventing or treating the disease characterized by administering an effective amount of a sulfated sugar chain.
  • a sulfated sugar chain is administered to a tissue or organ in which an abnormal increase in the expression level of heparin-binding protein and / or an abnormally enhanced action is observed (that is, a local area in the vicinity of the affected area).
  • a specific example of the tissue or organ is the eye (cornea, anterior chamber, vitreous, retina, etc.).
  • VEGF165 isoform as VEGF-A.
  • Recombinant VEGF165 and VEGF121 were purchased from PeprpTech.
  • Bovine kidney-derived heparan sulfate (HS) glycosaminoglycan was purchased from Seikagaku Corporation, and porcine intestine-derived non-fractionated heparin, heparinase III, heparinase I and chondroitinase ABC were purchased from Sigma-Aldrich.
  • Intraocular fluid was collected from the eyes of pigs (8 months old) within 3 hours after slaughter. After filter sterilization, the concentration of HS was measured (63.2 ⁇ g / ml) and stored at ⁇ 80 ° C. The porcine intraocular fluid thus prepared was used for cell experiments and binding experiments.
  • the collected intraocular fluid, urine and anterior aqueous humor were centrifuged (13000 g, 5 minutes), and the supernatant was used for analysis.
  • the level of VEGF-A in mouse and human body fluids was measured with an ELISA kit (R & D® Systems).
  • An ELISA kit manufactured by Seikagaku Corporation was used to measure the HS concentration in the body fluid.
  • Western blot using intraocular fluid or anterior aqueous humor the samples were treated with heparinase III (1 U / ml) and chondroitinase ABC (2 U / ml).
  • the sample was mixed with an equal amount of laemmli buffer (Biorad Laboratories), boiled, and subjected to SDS electrophoresis (reducing conditions) using a 4-20% acrylamide concentration gradient gel (Biorad Laboratories).
  • SDS electrophoresis reducing conditions
  • a 4-20% acrylamide concentration gradient gel Biorad Laboratories.
  • i-blot Invitrogen
  • the protein was transferred from the gel after the electrophoresis to a polyvinylidene fluoride membrane.
  • HS core protein was detected using an antibody (3G10, 500-fold diluted, Seikagaku Corporation) that recognizes an unsaturated structure produced by heparinase III treatment (Reference Document 12).
  • Heparinase III (0.005 U per eye) was injected into one eye of a 12-day-old mouse and PBS was injected into the other eye. After collecting intraocular fluid at the 14th day after birth, the HS concentration was measured by ELISA (in vivo assay). Intraocular fluid (1.5 ⁇ l) collected from 14-day-old mice was mixed with heparinase III (0.0025 U) or an equal volume of PBS and incubated at 37 ° C. overnight. The amount of HS glycosaminoglycan remaining was measured by ELISA (in vivo assay).
  • VEGF-A degradation assay Various amounts of mouse plasmin (3.2, 16, 80, 400 ng) and VEGF165 (20 ng) or VEGF121 (20 ng) in the presence or absence of heparin (100 ⁇ g)
  • the reaction buffer 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM dithiololeitol
  • the prepared sample was incubated at 37 ° C. overnight and then subjected to Western blotting.
  • HUVEC proliferation assay EG2 medium (containing 10 ng / ml epidermal growth factor, 1 ⁇ g / ml hydrocortisone, 3 ng / ml basic fibroblast growth factor (bFGF), 1% inactivated fetal bovine serum) Then, PBS or porcine intraocular fluid was used as a medium, and 3 to 5 passage HUVEC cells (Kurashikibo Co., Ltd.) were seeded in a 96-well plate (5,000 cells / 100 ⁇ l / well) and incubated at 37 ° C. Two hours later, heparin, VEGF-A (Kurashikibo Co., Ltd.) or PBS was added to the medium. After incubation for a total of 48 hours, cell proliferation reagent WST-1 (Promega) was added. After 4 hours, the amount of formazan dye (reflecting the amount of viable cells) was detected with a plate reader.
  • bFGF basic fibroblast growth factor
  • mice immediately after birth has no blood vessels. Retinal blood vessels develop radially from the optic nerve toward the periphery over the course of about 2 weeks after birth, and subsequently form a vascular network in the deep part of the retina.
  • FOG. 1A HS proteoglycan
  • FIG. 13 Multiple core proteins were present in mouse intraocular fluid (Ref. 13). From the molecular weight, these core proteins were presumed to be perlecan, collagen XVIII, syndecan 1, syndecan 2, and syndecan 3 (reference documents 14 and 15).
  • the concentration of HS sugar chains in the intraocular fluid of 7-60 day-old mice was measured using the ELISA method (FIG. 1B).
  • the concentration of HS sugar chains was found to be very high. For example, comparing the samples of 17 and 60 days after birth, the former was 15.8 times the concentration of the latter.
  • the sugar chain concentration in the intraocular fluid was found to be extremely high compared to plasma and urine (FIG. 1C). For example, on the 17th day after birth, the sugar chain concentration in the intraocular fluid was indeed 178 times that of plasma and 236 times that of urine.
  • heparinase III was administered to 12-day-old mice immediately after the end of oxygen exposure, and retinal blood vessels were evaluated at 17 days of age.
  • pathological retinal neovascularization increased 2.9-fold (FIGS. 2D, H).
  • heparinase III and heparin a sugar chain that is not degraded by heparinase III
  • the action of the enzyme was completely neutralized (FIGS. 2E and I).
  • Heparin has no core protein, but there is no significant difference in the scientific properties of HS and sugar chains (Reference 24).
  • VEGF165 bound to heparin is less susceptible to protease degradation>
  • Mouse VEGF-A isoforms VEGF164 (VEGF165 in human) and VEGF120 (VEGF121 in human) are expressed during the development of retinal blood vessels (Reference 26).
  • VEGF164 and VEGF165 are isoforms that play the most important role and have a heparin-binding domain (References 22 and 27). This can also be seen from the fact that the presence of VEGF164 is a necessary and sufficient condition for the formation of a normal mouse retinal vascular network (Reference 27).
  • VEGF164 and VEGF165 bind to cell surface HS via a heparin-binding domain, and also readily act on VEGFR on the cell surface. However, there are also soluble molecules in this isoform. Since VEGF120 and VEGF121 do not have a heparin binding domain, they exist only as soluble molecules.
  • human VEGF-A VEGF165 or VEGF121 was administered alone to one eye of the mouse, and the same amount of VEGF-A isoform was given to the other eye. was administered with heparin.
  • Plasmin is a serine proteolytic enzyme present in the eye (reference document 28) and is known to degrade VEGF-A (reference documents 26 and 29).
  • plasmin is a serine proteolytic enzyme present in the eye (reference document 28) and is known to degrade VEGF-A (reference documents 26 and 29).
  • Heparin inhibited VEGF165 degradation by plasmin (FIG. 3F), but did not affect VEGF121 degradation (FIG. 3G). These results indicate that the degradation of VEGF165 is suppressed by the binding of VEGF165 and heparin.
  • HUVEC were cultured by adding a certain amount of VEGF-A and various concentrations of heparin to a medium for vascular endothelium (EG2). As a result, the proliferation ability of HUVEC was suppressed depending on the heparin concentration (FIG. 4A).
  • PBS, EG2, or porcine intraocular fluid was used as a medium, and HUVEC were cultured in the presence and absence of VEGF-A (FIG. 4B). In PBS medium, the proliferation ability of HUVEC was hardly detected. On the other hand, the ability of HUVEC to grow was detected in porcine intraocular fluid medium, but it was inferior to EG2 medium.
  • HUVEC cultured in EG2 responded to VEGF-A stimulation. Furthermore, various concentrations of VEGF-A were added to the intraocular fluid or EG2 in the medium, and HUVEC were cultured. Then, in EG2 medium, the proliferation ability of HUVEC increased depending on the VEGF-A concentration (FIG. 4B). On the other hand, HUVEC did not respond to VEGF-A stimulation in the intraocular fluid medium.
  • the amount of VEGF-A bound to the well depends on the amount of surface heparin (FIG. 5A).
  • the amount of VEGF-A bound to the wells showed a negative correlation with the concentration of soluble heparin (FIG. 5B).
  • Soluble HS and porcine intraocular fluid similarly inhibited binding of VEGF-A and surface heparin (FIG. 5C).
  • various concentrations of VEGF-A were added based on intraocular fluid and PBS, and binding curves of surface heparin and VEGF-A were prepared and compared.
  • the intraocular fluid suppressed the binding of VEGF-A and surface heparin, and the intraocular fluid binding curve reverted to a PBS-shifted curve shifted to the right (FIG. 5D). This indicates that the intraocular fluid competitively inhibits the binding of VEGF-A and surface heparin.
  • VEGF-A receptors include VEGFR1 and VEGFR2.
  • VEGR2 plays a central role in the proliferation and migration of vascular endothelial cells (Reference 30).
  • VEGFR1 is thought to indirectly control the action of VEGF-A on VEGFR2 (Reference 30).
  • VEGFR1 and VEGFR2 are both heparin-binding proteins (References 31 and 32).
  • both soluble HS and intraocular fluid inhibited the binding of VEGF165 and VEGFR2 (FIG. 5E), but did not affect the binding of VEGF121 and VEGFR2 (FIG. 5F).
  • Soluble heparin also inhibited the binding of VEGF165 and VEGFR2 in a concentration-dependent manner, but had little effect on the binding of VEGF121 and VEGFR2 (FIG. 5G).
  • the binding of VEGF-A and VEGFR1 was hardly affected by soluble heparin, HS, or intraocular fluid (FIG. 5H-J).
  • ⁇ HS concentration in anterior aqueous humor of patients with proliferative diabetic retinopathy is decreased>
  • overproduction of VEGF-A is greatly involved in the presence of pathological new blood vessels (Refs. 10, 33).
  • the retina reference 34
  • liver references 35, 36
  • kidney references 37, 38
  • human references 39-41
  • skeletal muscles reference 42
  • Heparin has little effect on the binding of VEGFR1 and VEGF-A. As a result, there is a possibility that retinal blood vessels are protected from the occlusion mechanism by relatively increasing VEGFR1 signaling in the retina (Reference Document 46). On the other hand, manipulation of intraocular HS / heparin concentrations did not affect intraretinal neovascularization during development and retinal vascular regeneration. From these results, it is presumed that the anti-angiogenic action of soluble HS / heparin is specific to the extraretinal proliferation of blood vessels and is consistent with the intraocular distribution of intraocular fluid.
  • the concentration of HS sugar chains measured in human anterior aqueous humor was lower than the concentration of intraocular fluid (anterior aqueous humor + vitreous humor) of mice and pigs.
  • intraocular fluid anterior aqueous humor + vitreous humor
  • HS concentration of the sample that we measured for the mixture of vitreous and vitreous humor was much higher than that of the anterior aqueous humor.
  • a low level of soluble HS in the eye is at least an intraocular environment where extraretinal neovascularization is likely to occur. It is predicted.
  • the method is as follows. That is, after irradiating C57BL mice with radiation, GFP positive bone marrow cells were transplanted. Laser was irradiated to the fundus of mice transplanted with bone marrow or mice not transplanted with bone marrow, and heparin or phosphate buffered saline (PBS) was injected into the eye.
  • PBS heparin or phosphate buffered saline
  • CNV choroidal neovascularization
  • heparin The size of choroidal neovascularization (CNV), the distribution of heparin, and the number of inflammatory cells such as leukocytes invading into and out of CNV were quantified.
  • ARPE19 retinal pigment epithelial cells
  • heparin on the binding of VEGF, CCL2, and TNF- ⁇ to each receptor was examined.
  • FITC-labeled heparin was administered intraocularly and its localization was examined 3 hours later.
  • FITC-heparin was localized in blood vessels on the surface of the retina (FIGS. 9A-C).
  • FITC-heparin accumulated more strongly in the photoreceptor layer around the laser scar.
  • Heparin reduces VEGF and CCL2 secretion by retinal pigment epithelial cells>
  • the concentration of vascular growth factor VEGF in the culture supernatant increased (FIG. 11A).
  • the concentration of VEGF decreased. Similar results were obtained in the presence of the inflammatory cytokine TNF- ⁇ .
  • Soluble heparin inhibits binding of CCL2 and CCR2> Soluble heparin and porcine vitreous humor inhibited the binding of CCL2 and heparin coated on the surface and the binding of CCL2 and CCR2 in a concentration-dependent manner (FIG. 12D-E). On the other hand, soluble heparin had no obvious effect on the binding of TNF- ⁇ to the surface and the binding of TNF- ⁇ to TNFR1 and TNFR2 (FIGS. 12F and G).
  • Heparin suppressed the accumulation of inflammatory cells around CNV. From experiments using bone marrow transplanted mice, it was inferred that the inflammatory cells in which heparin suppresses accumulation are not derived from peripheral blood but are mainly inflammatory cells in the intraocular region. Combined with the results of in vitro experiments, heparin locally inhibits inflammation around the laser by inhibiting the action of CCL2, a leukocyte migration factor around CNV, or by suppressing the secretion of CCL2 by retinal pigment epithelial cells. It was thought to be suppressed.
  • heparin exhibits an anti-angiogenic action by inhibiting the action of VEGF, which is a vascular growth factor, and reducing VEGF secretion by retinal pigment epithelial cells.
  • VEGF vascular growth factor
  • VEGF-binding proteins such as VEGF and CCL2
  • vitreous bodies with low concentrations of HS showed increased binding of VEGF and parin to the surface. This suggests that when VEGF is produced in excess in the eye, VEGF readily binds retinal surface HS via the heparin-binding domain in young people with low concentrations of HS. A similar mechanism predicts that VEGF is likely to bind to VEGFR2 expressed in retinal blood vessels in young people.
  • VEGF produced excessively under retinal ischemia due to diabetic retinopathy can more efficiently act on the retinal blood vessels and cause intravitreal invasion of the retinal blood vessels There is sex.
  • the results of this study provide a scientific basis for the phenomenon that diabetic retinopathy tends to become more severe in young diabetics.
  • VEGF121 a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J. Biol. Chem. 270: 11322-11326. 9. Funatsu, H., Yamashita, H., Noma, H., Mimura, T., Nakamura, S., Sakata, K., And Hori, S. 2005.
  • Aqueous humor levels of cytokines are related to vitreous levels and progression of diabetic retinopathy in diabetic patients.Graefes Arch. Clin. Exp. Ophthalmol. 243: 3-8. 10. Aiello, LP, Avery, RL, Arrigg, PG, Keyt, BA, Jampel, HD, Shah, ST, Pasquale, LR, Thieme, H., Iwamoto, MA, Park, JE et al 1994. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 331: 1480-1487. 11.
  • Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.
  • Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization.
  • the fourth immunoglobulin-like loop in the extracellular domain of FLT-1, a VEGF receptor, includes a major heparin-binding site.
  • Heparan sulfate proteoglycan synthesis and its expression are decreased in the retina of diabetic rats.
  • the agent of the present invention can inhibit the physiological activity of heparin-binding protein.
  • the present invention is used as, for example, an angiogenesis inhibitor.
  • Specific examples of application include proliferative diabetic retinopathy, retinopathy of prematurity, wet age-related macular degeneration, myopic macular degeneration (neovascular macular degeneration), idiopathic choroidal neovascular disease Prophylactic / vitreous proliferative vitreoretinopathy, uveitis, ocular ischemic syndrome, retinal vein occlusion, iris neovascularization or corneal neovascularization is envisaged.
  • the drug of the present invention also has utility value as a medicine that replaces existing anti-vascular growth factor drugs (for example, anti-VEGF drugs).
  • anti-VEGF drugs for example, anti-VEGF drugs
  • it is expected to provide alternative medicine in situations where existing anti-vascular growth factor drugs are difficult to use, such as when it is not appropriate to use anti-VEGF drugs to increase the risk of vascular occlusion disease.
  • It is also envisaged to be used in combination with an anti-vascular growth factor drug in order to enhance the therapeutic effect.

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Abstract

Les objets de la présente invention sont d'élucider l'importance physiologique des glycosaminoglycanes tels que l'héparine et le sulfate d'héparane et de proposer de nouvelles applications de ces molécules. Cette invention concerne plus spécifiquement un agent d'inhibition de l'activité physiologique de la protéine se liant à l'héparine, qui contient un saccharide sulfaté à titre de principe actif. L'agent d'inhibition de l'activité physiologique de la protéine se liant à l'héparine selon l'invention est utilisé, par exemple, en tant qu'inhibiteur d'angiogenèse.
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WO2022062101A1 (fr) * 2020-09-22 2022-03-31 广东海洋大学 Utilisation de mucopolysaccharide d'héparine dérivé de vessie natatoire pour la préparation d'un inhibiteur d'angiogenèse

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WO2016194869A1 (fr) * 2015-05-29 2016-12-08 生化学工業株式会社 Composition comprenant un dérivé de glucosaminoglycane et un régulateur d'activité de récepteur de chimiokine
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WO2022062101A1 (fr) * 2020-09-22 2022-03-31 广东海洋大学 Utilisation de mucopolysaccharide d'héparine dérivé de vessie natatoire pour la préparation d'un inhibiteur d'angiogenèse

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