WO2020042449A1 - 葫芦素e在制备治疗血管新生不足相关疾病药物或生物医用材料中的用途和应用其的产品 - Google Patents

葫芦素e在制备治疗血管新生不足相关疾病药物或生物医用材料中的用途和应用其的产品 Download PDF

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WO2020042449A1
WO2020042449A1 PCT/CN2018/121847 CN2018121847W WO2020042449A1 WO 2020042449 A1 WO2020042449 A1 WO 2020042449A1 CN 2018121847 W CN2018121847 W CN 2018121847W WO 2020042449 A1 WO2020042449 A1 WO 2020042449A1
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insufficient
cucurbitacin
cucurbitin
angiogenesis
diseases related
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PCT/CN2018/121847
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French (fr)
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成文翔
王新峦
张鹏
孟祥波
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中国科学院深圳先进技术研究院
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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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • 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
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/22—Lipids, fatty acids, e.g. prostaglandins, oils, fats, waxes
    • A61L2300/222—Steroids, e.g. corticosteroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/422—Anti-atherosclerotic agents

Definitions

  • the present application relates to the field of medical technology, in particular to a use of cucurbitin E in the preparation of a medicine or a biomedical material for treating diseases related to insufficient angiogenesis, and a product using the same.
  • Angiogenesis refers to the process by which adults form new blood vessels in the form of germination on the basis of primordial vascular plexus or existing blood vessels. It has the function of maintaining the normal growth and balance of the human body. Generally, angiogenesis occurs in the embryo and later stages of embryonic development. Wound healing period. Angiogenesis plays an important role in the occurrence, development, outcome and prognosis of many diseases.
  • Bone tissue repair, slow fracture healing, trauma, coronary heart disease, myocardial ischemia, inadequate establishment of collateral circulation, occlusive vasculitis, etc. are all clinically frequent and common diseases, and are important factors affecting the health and quality of life of the people .
  • Insufficient angiogenesis is an important pathophysiological basis common to these diseases, so it is of great significance to carry out prevention and treatment of angiogenesis insufficiency diseases.
  • Studies have shown that angiogenesis is mainly related to the degradation of basement membrane, endothelial cell migration, proliferation, and tube formation. As angiogenesis plays an important role in disease, research on new angiogenesis drugs has become a research in promoting angiogenesis. hot spot.
  • Cucurbitin E can promote vascular endothelial cells to form tubes at a concentration that is not cytotoxic. Angiogenesis has a promoting effect.
  • Another object of the present application is to provide a medicament for treating diseases related to insufficient angiogenesis, which is mainly made of cucurbitin E and a pharmaceutically acceptable carrier.
  • a third object of the present application is to provide a biomedical material for treating diseases related to insufficient angiogenesis, including an effective amount of cucurbitacin E.
  • cucurbitin E in the preparation of a medicament or a biomedical material for the treatment of diseases associated with insufficient angiogenesis, said cucurbitin E being at a non-cytotoxic concentration.
  • the effective concentration of the cucurbitacin E is not higher than 50 nM, and preferably not higher than 30 nM.
  • the route of administration of cucurbitacin E includes topical, oral, parenteral, aerosolized inhalation or implanted biological materials.
  • the diseases related to insufficient angiogenesis include poor bone tissue repair, slow fracture healing, coronary heart disease, myocardial ischemia, insufficient establishment of collateral circulation compensation, and occlusive veins. Either tuberculitis or insufficient angiogenesis after organ transplantation.
  • the fracture includes a fracture caused by trauma or pathological osteoporosis
  • the coronary heart disease includes ischemic heart disease, coronary artery embolism, and trauma diseases.
  • a medicament for treating a disease related to insufficient angiogenesis is provided, the medicament is mainly made of cucurbitin E and a pharmaceutically acceptable carrier.
  • the dosage form of the drug includes tablets, powders, granules, capsules, suspensions, syrups, oral solutions, ointments, patches or injections.
  • a biomedical material for treating diseases related to insufficient angiogenesis including an effective amount of cucurbitacin E.
  • the biomedical material is a biomedical polymer material, preferably a biomedical polymer scaffold.
  • the cucurbitacin E exists in the form of itself, microspheres or nanoparticles.
  • the application provides the use of cucurbitin E at a non-cytotoxic concentration in the preparation of a medicament or a biomedical material for treating diseases related to insufficient angiogenesis.
  • cucurbitin E can greatly promote the formation of vascular endothelial cells in a safe tolerable concentration of endothelial cells and form a well-interwoven three-dimensional network structure.
  • bone repair experiments made with cucurbitacin E after bio-scaffolding also confirmed that cucurbitacin E can promote blood vessel formation and new bone formation in animal bone defects.
  • cucurbitin E promoted the expression of vascular endothelial cells hy.EA926-related genes showed that the expression of vascular endothelial growth factor receptor VEGFR was dose-dependent with cucurbitin E, and cucurbitin E could promote the up-regulation of VEGFR without cytotoxic concentration. , Suggesting that cucurbitacin E can promote angiogenesis.
  • the promotion effect of cucurbitin E on angiogenesis can be used to treat diseases related to insufficient angiogenesis, and it provides a new choice for the development of drugs or biomedical materials for the treatment of diseases related to insufficient angiogenesis.
  • Figure 1 is the molecular structure of cucurbitacin E
  • Example 2 is a relationship diagram between cucurbitacin E concentration and cell survival rate in Example 1 of the present application;
  • FIG. 3 is a photograph of cucurbitacin E promoting tube formation of vascular endothelial cells in Example 2 of the present application, where A is a photograph of a control group, B is a photograph of a 10nM cucurbitacin E group, and C is a photograph of a 30 nM cucurbitacin E group;
  • Example 4 is a photo of the cucurbitacin biocomposite stent prepared in Example 3 of the present application to promote blood vessel formation at a bone defect site, where A is a photograph of a control group and B is a photograph of a cucurbitacin E treatment group;
  • FIG. 5 is a photo of a cucurbitacin biocomposite scaffold prepared in Example 3 of the present application to promote new bone formation at a bone defect site, where A is a photograph of a control group and B is a photograph of a cucurbitacin E treatment group;
  • Figure 6 shows the effect of cucurbitacin E on the mRNA expression of VEGFR gene.
  • cucurbitin E in preparing a medicament or a biomedical material for treating diseases related to insufficient angiogenesis, in which the cucurbitin E is at a non-cytotoxic concentration.
  • cucurbitacin E promotes angiogenesis. This study found that cucurbitin E can greatly promote vascular endothelial cell tube formation at a safe tolerable concentration of vascular endothelial cells, that is, cucurbitacin E has the effect of promoting angiogenesis at a non-cytotoxic concentration.
  • Cucurbitacin E also known as cucurbitin E, is a tetracyclic triterpene compound, mainly distributed in Cucurbitaceae, and also found in higher plants such as Cruciferae and Scrophulariaceae and some large fungi. Its chemical formula is C 32 H 44 O 8 and its molecular structure is shown in Figure 1.
  • the source of cucurbitacin E is not limited, and can be obtained by using a commercially available existing product or using an existing method.
  • the non-cytotoxic concentration refers to a safe concentration that the endothelial cells can tolerate, that is, cucurbitin E does not show growth inhibition of the endothelial cells at this concentration.
  • the effective concentration of cucurbitin E is typically, but not limited to, not higher than 50 nM, and particularly not higher than 30 nM.
  • Angiogenesis also called angiogenesis, is the process by which new blood vessels are sprouted from existing blood vessels.
  • cucurbitacin E has a significant role in promoting angiogenesis in human umbilical vein endothelial cell proliferation experiments, microtubule formation experiments, and biological scaffold bone repair animal experiments, and can be used to prepare drugs or organisms for the treatment of diseases related to insufficient angiogenesis. Medical materials.
  • Inadequate angiogenesis refers to insufficient angiogenesis during the process of body repair or healing, which cannot meet the body's repair or healing needs. Inadequate angiogenesis can lead to poor local blood circulation and even tissue necrosis.
  • Insufficient angiogenesis-related diseases refer to diseases with insufficient angiogenesis as the pathological and physiological basis.
  • Typical but non-limiting examples include diseases such as poor bone repair, slow fracture healing, coronary heart disease, myocardial ischemia, inadequate establishment of collateral circulation or occlusive vasculitis; and also include, for example, dependence on angiogenesis Sexual diseases, such as insufficient angiogenesis after organ transplantation.
  • angiogenesis is helpful for the cure and outcome of certain diseases, such as fractures, osteonecrosis, occlusive vasculitis, and the establishment of collateral circulation in various parts of the body due to ischemia.
  • diseases such as fractures, osteonecrosis, occlusive vasculitis, and the establishment of collateral circulation in various parts of the body due to ischemia.
  • good angiogenesis is conducive to the microenvironment of organs and stem cells, and affects the outcome of disease after transplantation.
  • Bone tissue repair, slow fracture healing, trauma, coronary heart disease, myocardial ischemia, inadequate establishment of collateral circulation, occlusive vasculitis, etc. are all clinically frequent and common diseases, and are important factors affecting the health and quality of life of the people
  • Insufficient angiogenesis is an important pathological and physiological basis common to these diseases, so it is of great significance to carry out the prevention and treatment of angiogenesis insufficiency diseases.
  • the bone tissue repair referred to here refers to the repair of the bone and related blood vessels at the defect site after the bone defect caused by the disease, trauma or medicine.
  • the fracture healing described here includes fractures caused by trauma or pathological osteoporosis.
  • Coronary heart disease described here includes ischemic heart disease, coronary vascular embolism and injury diseases.
  • the myocardial ischemia mentioned here refers to the decrease of blood perfusion after the imbalance of the myocardial blood supply and demand caused by the heart due to some reasons, and the myocardial energy metabolism is imbalanced.
  • the insufficient establishment of the collateral circulation compensation described here is that after the blood flow of the main blood vessels in a certain part of the body is blocked, bypasses cannot be formed, and blood bypasses these bypasses in a roundabout manner, thereby failing to effectively establish a collateral circulation to make local Tissue suffers from insufficient blood supply.
  • the occlusive vasculitis mentioned here refers to a chronic peripheral vascular stenosis or occlusion disease. Among them, the arterioles and their accompanying veins and superficial veins have stenosis or occlusion, and the affected blood vessels present non-purulent inflammation of the entire wall of the blood vessel. There is thrombosis in the lumen, and the lumen presents progressive stenosis or even complete occlusion, causing limb ischemia, leading to ulcers and gangrene.
  • Insufficient angiogenesis after organ transplantation refers to the inability of new blood vessels to satisfy better growth or healing effects including organ transplantation and stem cell transplantation.
  • the pharmaceutical form made of cucurbitin E is not limited, and may be in the form of a pharmaceutical composition or a pharmaceutical preparation.
  • biomedical materials made of cucurbit E There are no restrictions on the types and materials of biomedical materials made of cucurbit E.
  • the biomedical materials mainly use cucurbitacin E as the active substance, and make biocomposite materials with other inert materials such as polymer materials, ceramics or metals, alloys, etc. .
  • the treatment target of drugs or biomedical materials for the treatment of diseases related to insufficient angiogenesis includes, but is not limited to, humans and other animals, such as dogs, rabbits, cats, pigs, ostriches, cattle, horses, mice, monkeys Waiting for mammals.
  • cucurbitacin E in the preparation of angiogenesis-promoting drugs.
  • cucurbitacin E can greatly promote the formation of vascular endothelial cells in a safe concentration that can be tolerated by endothelial cells, forming a well-woven three-dimensional network structure.
  • Bone repair experiments using cucurbitacin E to make biological scaffolds also confirmed that cucurbitacin E can promote blood vessel formation and new bone formation in animal bone defects.
  • Further cucurbitin E promoted vascular endothelial cell hy.EA926-related gene expression test showed that the expression of vascular endothelial growth factor VEGFR was dose-dependent with cucurbitin E.
  • cucurbitacin E can be administered by topical, oral, parenteral, aerosolized inhalation, and implanted biological materials.
  • Topical application mainly refers to smearing, topical application, etc.
  • Parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or input.
  • a medicament for treating diseases related to insufficient angiogenesis is provided.
  • the medicament is mainly made of cucurbitacin E and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field.
  • a curatively effective amount of cucurbitacin E and any pharmaceutically acceptable adjuvant can be made into a pharmaceutical preparation, and other angiogenic drugs that have no antagonistic effect with cucurbitacin E can also be added.
  • the preparation can be any pharmaceutical form, including but not limited to tablets, powders, granules, capsules, suspensions, syrups, oral solutions, ointments, patches or injections.
  • the appropriate pharmaceutical carrier is selected, including but not limited to fillers, adhesives, disintegrants, lubricants, solubilizers, suspending agents, wetting agents, pigments, flavors, solvents, surfactants, One or more of flavoring agents, buffers, isotonicity regulators, antioxidants, bacteriostatic agents and analgesics.
  • a biomedical material for treating diseases related to insufficient angiogenesis including an effective amount of cucurbitacin E.
  • Biomedical materials include, but are not limited to, biomedical polymer materials.
  • the biomedical material contains an effective amount of cucurbitacin E.
  • the typical biomedical polymer material is a stent of a biopolymer material.
  • cucurbitacin E When preparing a biopolymer scaffold, cucurbitacin E can be prepared by mixing it with stent materials in the prior art, or cucurbitacin E can be prepared into microspheres or nanoparticles before being combined with the stent in the prior art. The materials are mixed to prepare a biological composite material. That is, in the biocomposite material, the cucurbitacin E, which is a pharmaceutically active substance, may exist directly in its own form or in the form of microspheres or nanoparticles.
  • a stent of a biopolymer material containing cucurbitin E is prepared by 3D printing technology in the present application.
  • the weight percentage of cucurbitacin E, the active ingredient in the stent of the biopolymer material is 0.001-0.1%, for example, 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.05%, 0.06%, 0.08% Or 0.1%.
  • Drugs and / or biomedical materials made from cucurbitacin E can be used to treat diseases related to insufficient angiogenesis, especially for bone tissue repair, slow fracture healing, trauma, coronary heart disease, myocardial ischemia, and collateral circulation compensation Prevention and treatment of many angiogenesis insufficiency diseases such as insufficient establishment and occlusive vasculitis.
  • reagents used in the following examples were purchased directly from the market, for example: cucurbitacin E was purchased from French company EXTRASYNTHESE; CCK-8 reagent was purchased from Tongren Chemical; matrigel was purchased from Corning; PLGA (poly lactic-co-glycolic acid, Polylactic acid-glycolic acid copolymer) was purchased from Shandong Medical Device Research Institute. The molecular weight of PLGA is 100,000, and the molar ratio of lactic acid to glycolic acid is 70:30.
  • the above substances are not limited to those purchased from the above-mentioned manufacturers, and other raw materials or reagents used are also purchased from the market, and the manufacturers are not limited.
  • the rapid prototyping machine used in the following examples was developed by the Department of Mechanical Engineering of Tsinghua University, and its model is CLRF-2000-II.
  • the drug toxicity test uses the CCK-8 method.
  • the CCK-8 method is a method for determining the number of cell proliferation using colorimetry in living cells.
  • the principle is WST-8 [Chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfonic acid benzene)- 2H-tetrazolium monosodium salt], reduced by the dehydrogenase in the cell to highly water-soluble under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy) Sexual yellow formazan product, the amount of formazan produced is directly proportional to the number of living cells. Therefore, this feature can be used to directly analyze cell proliferation and toxicity.
  • vascular endothelial cell ea.hy926 cell suspension was seeded into a 96-well plate at 3000 cells / well, and after overnight adherence, cucurbitin E (1, 3, 10, 30, 100, 300 nM) containing different concentrations was added.
  • the culture medium was used for 72 hours, then 10 ⁇ L of CCK-8 reagent was added to each well, and incubated for 2 hours.
  • the light absorption value in each well was read at 490 nm on a microplate reader. According to the value, the median lethal concentration of the drug is 65.45 nM (as shown in Figure 2). Based on this result, it was determined that the safe concentration tolerated by endothelial cells was below 50 nM.
  • Matrigel is a soluble basement membrane extract obtained from EHS (Engelbreth-Holm-Swarm) mouse sarcoma. At room temperature, matrigel polymerizes into a biologically active matrix material, which functions similarly to the basement membrane of mammalian cells, and has an effect on cell adhesion and differentiation.
  • Vascular endothelial cells can be fused into microtubules on Matrigel and used This characteristic of vascular endothelial cells is used to study the effect of drugs on angiogenesis.
  • Matrigel was spread on a 24-well plate, and left to stand at 37 ° C in a 5% CO 2 incubator for 30 minutes until it solidified. Add 500 ⁇ L of the same volume of cells and different concentrations of cucurbitacin E to each well and mix. After incubating in a 5% CO 2 incubator at 37 ° C for 16-18 hours, observe the formation of microtubules under a microscope, calculate the number of microtubules and perform statistical analysis.
  • the microtubule formation photo is shown in Fig. 3, where A is a control group (without cucurbitacin E), B is a 10 nM cucurbitin E group, and C is a 30 nM cucurbitin E group.
  • A is a control group (without cucurbitacin E)
  • B is a 10 nM cucurbitin E group
  • C is a 30 nM cucurbitin E group.
  • Print parameters import the CLI file set in step 1 into the cark software, and set the printing process parameters as: printing speed 20mm / s, slurry filling speed 1.00mm / s, support layer thickness 120 ⁇ m, average pore size 500 ⁇ m .
  • 4Printing process After the temperature of the 3D printer is pre-cooled to -30 ° C, pour the slurry into the silo, and connect the silo to the printer with a hose. The printing process runs under the set parameters. The entire printing process is at -30. °C to ensure the activity of drug molecules in the slurry.
  • Freeze-drying After printing is completed, the prepared scaffold is placed in a freeze-dryer and freeze-dried under vacuum for 72 hours to remove organic solvents from the material.
  • Scan the animal specimen injected with contrast medium using micro-CT (SkyScan 1176) for small animals, place the tibia or spine specimen at the center of the machine tool, adjust the computer parameters, medium resolution (Resolution) (18um), filter (Al) 1mm , Voltage (Voltage) 60kV, Current (Current) 400 ⁇ A, Average Correction (Frame Averaging) 2 times.
  • Apply NRecon software to reconstruct the two-dimensional image of the distal femur specimen select the two-dimensional reconstruction area, preview the two mappings, and set the gray scale uniformly (Minimum or Maximum for CS Image Conversion): 0.00-0.10, select the entire specimen reconstruction area (Reconstruct ROI), reconstruction of angiogenesis.
  • Use CTAn software to select the location of the bone defect (Analysis ROI), set the background (Thresholding) to 40-255, and select a 3D model program for reconstruction.
  • the curcumin E biocomposite scaffold promotes blood vessel formation at the bone defect site as shown in Fig. 4, where A is a photograph of a control group (a curcumin E-free scaffold), and B is a curcumin E treatment group photograph. It can be seen from the figure that the use of cucurbitacin biocomposite scaffold can obviously promote blood vessel formation in rabbit bone defects.
  • CTAn software was used to select the bone defect location (Analysis ROI), set the background (Thresholding) to 63-255, and select 3D analysis to analyze the bone volume fraction (Percent bone volume) (BV / TV) of the new bone at the defect location.
  • BV / TV bone volume fraction
  • a density and gray standard curve can be established to obtain the bone density of the new bone at the defect location.
  • Selecting 3D modeling can generate a 3D reconstruction model of the new bone.
  • the cucurbit E biocomposite scaffold promotes new bone formation at the bone defect site, as shown in Figure 5, where A is a control group (a cucurbitin-free stent) photograph, and B is a cucurbitacin-E treated group photograph. It can be seen in the figure that the use of cucurbitacin biocomposite scaffold can significantly promote the formation of new bone in rabbit bone defects.
  • Hy.EA926 cells were seeded in a 6-well plate and incubated overnight. 30nM cucurbitacin E was added at 6, 12, 24, and 48 hours to extract RNA according to the instructions of the AxyGen RNA extraction kit. The total mRNA was reverse transcribed into cDNA. Design primers for VEGFR2.
  • the primer sequences are: upstream: 5′-CTCTCTCTGCCTACCTCACCTG-3 ′; downstream: 5′-CGGCTCTTTCGCTTACTGTTC-3 ′.
  • SYBR-Green Mix Kit (Takara) and Roche LC96 fluorescence quantitative PCR instrument were used to detect the change of VEGFR2 gene level.
  • the effect of cucurbitin E on the regulation of mRNA expression of VEGFR2 gene is shown in Figure 6.
  • the figure shows that the drug can promote the expression of vascular endothelial growth factor receptor VEGFR2 and is time-dependent.
  • Related gene expression promotes angiogenesis.
  • Cucurbitin E experimental results show that low-toxic cucurbitacin E has a significant angiogenic effect on human umbilical vein cells. Further in vivo experiments in animals have shown that it promotes angiogenesis at the site of injury and has a role in bone repair. This is because Low-dose cucurbitacin E can promote the up-regulation of VEGFR2 expression.

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Abstract

公开了一种葫芦素E在制备治疗血管新生不足相关疾病药物或生物医用材料中的用途，还公开了用于治疗血管新生不足相关疾病的药物或生物医用材料，能够用于骨组织修复、骨折愈合缓慢、创伤、冠心病、心肌缺血、侧支循环代偿建立不足、闭塞性脉管炎、器官移植后血管新生不足等诸多血管新生不足疾病防治。

Description

葫芦素E在制备治疗血管新生不足相关疾病药物或生物医用材料中的用途和应用其的产品
相关申请的交叉引用
本申请要求于2018年08月31日提交中国专利局的申请号为CN201811013796.9、名称为“葫芦素E在制备治疗血管新生不足相关疾病药物或生物医用材料中的用途和应用其的产品”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及医药技术领域，具体而言，涉及一种葫芦素E在制备治疗血管新生不足相关疾病药物或生物医用材料中的用途和应用其的产品。
背景技术
血管新生是指成人在原始血管丛或已存在血管的基础上以发芽方式形成新生血管的过程，具有维持人体正常的生长和平衡的功能，通常血管新生发生在胚胎和胚胎发育后期，生殖周期和伤口愈合期。血管新生在许多疾病的发生、发展及转归和预后中扮演着重要的角色。
骨组织修复、骨折愈合缓慢、创伤、冠心病、心肌缺血、侧支循环代偿建立不足、闭塞性脉管炎等均为临床多发病、常见病，是影响民众健康和生活质量的重要因素。血管新生不足是这些疾病共同的重要病理生理基础，因此开展血管新生不足疾病的防治有着重要的意义。研究显示，血管新生主要涉及基底膜的降解、内皮细胞迁移、增殖和管形成等过程，由于血管形成在疾病中扮演着重要角色，研究新的促血管形成的药物，成为促进血管新生中的研究热点。
有鉴于此，特提出本申请。
发明内容
本申请的目的之一在于提供一种葫芦素E在制备治疗血管新生不足相关疾病的药物或生物医用材料中的用途，葫芦素E在无细胞毒性的浓度下可促进血管内皮细胞成管，对血管新生有促进作用。
本申请的目的之二在于提供一种用于治疗血管新生不足相关疾病的药物，主要由葫芦素E和药学上可接受的载体制成。
本申请的目的之三在于提供一种用于治疗血管新生不足相关疾病的生物医用材料，包括有效量的葫芦素E。
为了实现本申请的上述目的，特采用以下技术方案：
第一方面，提供了一种葫芦素E在制备治疗血管新生不足相关疾病的药物或生物医用 材料中的用途，所述葫芦素E处于无细胞毒性浓度下。
优选地，在本申请提供的技术方案的基础上，所述葫芦素E的有效浓度不高于50nM，优选不高于30nM。
优选地，在本申请提供的技术方案的基础上，所述葫芦素E的给药途径包括外用、口服、肠胃外、雾化吸入或植入生物材料。
优选地，在本申请提供的技术方案的基础上，所述血管新生不足相关疾病包括骨组织修复不良、骨折愈合缓慢症、冠心病、心肌缺血、侧支循环代偿建立不足、闭塞性脉管炎或器官移植后血管新生不足中的任一疾病。
优选地，在本申请提供的技术方案的基础上，所述骨折包括创伤或病理性骨质疏松引起的骨折；
优选地，所述的冠心病包括缺血性心脏病、冠脉血管栓塞及损伤性疾病。
第二方面，提供了一种用于治疗血管新生不足相关疾病的药物，所述药物主要由葫芦素E和药学上可接受的载体制成。
优选地，在本申请提供的技术方案的基础上，所述药物的剂型包括片剂、散剂、颗粒剂、胶囊剂、混悬剂、糖浆剂、口服液、软膏剂、贴剂或注射剂。
第三方面，提供了一种用于治疗血管新生不足相关疾病的生物医用材料，包括有效量的葫芦素E。
优选地，在本申请提供的技术方案的基础上，所述生物医用材料为生物医用高分子材料，优选为生物医用高分子支架。
优选地，在本申请提供的技术方案的基础上，所述葫芦素E以自身形态、微球或纳米粒形式存在。
与已有技术相比，本申请具有如下有益效果：
本申请提供了葫芦素E无细胞毒性的浓度下在制备治疗血管新生不足相关疾病的药物或生物医用材料中的用途。实验表明，葫芦素E在内皮细胞可耐受的安全浓度下可大大促进血管内皮细胞成管，形成交织良好的三维网络结构。同时用葫芦素E制成生物支架后进行骨修复实验也证实葫芦素E能促进动物骨缺损处的血管形成以及新骨形成。进一步葫芦素E促进血管内皮细胞hy.EA926相关的基因表达试验表明血管内皮生长因子受体VEGFR的表达变化与葫芦素E有剂量依赖性，无细胞毒性浓度下葫芦素E可促进VEGFR的表达上调，提示基因水平葫芦素E可以促进血管新生。葫芦素E对血管新生的促进作用可用于治疗血管新生不足相关疾病，为开发用于治疗血管新生不足相关疾病的药物或生物医用材料提供了新的选择。
附图说明
图1为葫芦素E的分子结构；
图2为本申请实施例1葫芦素E浓度与细胞存活率之间的关系图；
图3为本申请实施例2葫芦素E促进血管内皮细胞成管照片，其中A为对照组照片，B为10nM葫芦素E组照片，C为30nM葫芦素E组照片；
图4为本申请实施例3制得的葫芦素E生物复合材料支架促进骨缺损部位血管形成照片，其中A为对照组照片，B为葫芦素E处理组照片；
图5为本申请实施例3制得的葫芦素E生物复合材料支架促进骨缺损部位新骨形成照片，其中A为对照组照片，B为葫芦素E处理组照片；
图6为葫芦素E对VEGFR基因mRNA表达的影响。
具体实施方式
下面将结合实施例对本申请的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本申请，而不应视为限制本申请的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
根据本申请的一个方面，提供了一种葫芦素E在制备治疗血管新生不足相关疾病的药物或生物医用材料中的用途，该用途中，葫芦素E处于无细胞毒性浓度下。
目前还没有现有技术中披露过葫芦素E对血管新生有促进作用。本研究发现葫芦素E在血管内皮细胞可耐受的安全浓度下可大大促进血管内皮细胞成管，即葫芦素E处于无细胞毒性浓度下具有促进血管新生的效果。
葫芦素E(Cucurbitacin E)也称葫芦苦素E，为一种四环三萜化合物，主要分布于葫芦科植物中，在十字花科、玄参科等高等植物及一些大型真菌中也有发现。其化学式为C 32H 44O 8，分子结构如图1所示。对葫芦素E的来源不作限定，可采用市售的现有产品或采用现有方法制备得到。
无细胞毒性浓度是指内皮细胞可耐受的安全浓度，即葫芦素E在该浓度下内皮细胞未出现生长抑制。
葫芦素E的有效浓度(无细胞毒性浓度)典型但非限制性的不高于50nM，特别可以不高于30nM。
血管新生也称为血管生成(angiogenesis)，是从已有血管发芽生成新血管的过程。
本研究表明，葫芦素E在人脐带静脉内皮细胞增殖实验、微管形成实验以及生物支架骨修复动物实验中有明显的促进血管新生的作用，可用于制备治疗血管新生不足相关疾病的药物或生物医用材料。
血管新生不足是指在机体修复或愈合过程中血管生成不足，不能满足机体修复或愈合 需要。血管新生不足会导致局部血液循环不良、甚至组织坏死。
血管新生不足相关疾病是指以血管新生不足为病理、生理基础的疾病。典型但非限制性的例如包括骨组织修复不良、骨折愈合缓慢症、冠心病、心肌缺血、侧支循环代偿建立不足或闭塞性脉管炎等疾病；还可包括例如对血管新生有依赖性的疾病，例如器官移植后血管新生不足等。
促进血管形成则有助于某些疾病的治愈和转归，例如骨折、骨坏死，闭塞性脉管炎以及机体各个部位由于局部缺血后侧支循环的建立。在器官移植包括脏器移植以及干细胞移植，良好的血管新生有利于脏器以及干细胞生存的微环境，影响移植后疾病的转归。
骨组织修复、骨折愈合缓慢、创伤、冠心病、心肌缺血、侧支循环代偿建立不足、闭塞性脉管炎等均为临床多发病、常见病，是影响民众健康和生活质量的重要因素，血管新生不足是这些疾病共同的重要病理和生理基础，因此开展血管新生不足疾病的防治和治疗有着重要的意义。
这里所述的骨组织修复是指疾病、创伤或药物导致骨缺损后的缺损部位的骨以及相关血管的修复。这里所述的骨折愈合包括创伤或病理性骨质疏松等引起的骨折。这里所述的冠心病包括缺血性心脏病、冠脉等血管栓塞及损伤性疾病。这里所述的心肌缺血指心脏由于某些原因导致的心肌血液供需失衡后的血液灌注减少，心肌能量代谢失调。这里所述的侧支循环代偿建立不足是是机体某一局部的主要血管的血流受阻后，不能形成旁路，使血液迂回地通过这些旁路，从而无法有效的建立侧支循环使局部组织遭受供血不足。这里所述的闭塞性脉管炎是指慢性周围血管狭窄或闭塞的疾病，其中、小动脉及其伴行的静脉和浅静脉发生狭窄或闭塞，受累血管呈现血管壁全层的非化脓性炎症，管腔内有血栓形成，管腔呈现进行性狭窄以至完全闭塞，引起肢体缺血，导致溃疡及其坏疽。
器官移植后血管新生不足是指包括脏器移植以及干细胞移植后新生血管不能满足更好的生长或愈合效果。
对葫芦素E制成的药物形式不作限定，可以是药物组合物的形式，也可以是药物制剂的形式。
对葫芦素E制成的生物医用材料的种类和材质不作限定，该生物医用材料主要以葫芦素E作为活性物质，与其他惰性材料如高分子材料、陶瓷或金属、合金等制成生物复合材料。
需要注意的是，治疗血管新生不足相关疾病的药物或生物医用材料的治疗对象包括但不限于人，也可以是其他动物，例如狗、兔、猫、猪、鸵鸟、牛、马、鼠，猴等哺乳类动物。
本申请公开了葫芦素E在制备促进血管新生药物中的应用，实验表明，葫芦素E在内 皮细胞可耐受的安全浓度下可大大促进血管内皮细胞成管，形成交织良好的三维网络结构。用葫芦素E制成生物支架后进行骨修复实验也证实葫芦素E能促进动物骨缺损处的血管形成以及新骨形成。进一步葫芦素E促进血管内皮细胞hy.EA926相关的基因表达试验表明血管内皮生长因子VEGFR的表达变化与葫芦素E有剂量依赖性，低剂量葫芦素E可促进VEGFR的表达上调，提示基因水平葫芦素E可以促进血管新生。葫芦素E对血管新生的促进作用可用于治疗血管新生不足相关疾病，为开发用于治疗血管新生不足相关疾病的药物或生物医用材料提供了新的选择。
在一种优选的实施方式中，为实施治疗和/或预防血管新生不足相关疾病的方法，葫芦素E可通过外用、口服、肠胃外、雾化吸入以及植入生物材料给药。
外用主要指涂抹、外敷等方式，肠胃外包括皮下、皮内、静脉内、肌肉内、关节内、动脉内、滑膜腔内、胸骨内、鞘内、病灶内及颅内注射或输入。
根据本申请的第二个方面，提供了一种用于治疗血管新生不足相关疾病的药物，药物主要由葫芦素E和药学上可接受的载体制成。
药学上可接受的载体是指药学领域常规的药物载体。
根据本申请可以将治疗有效量的葫芦素E和药学上允许的任意辅料制成药物制剂，也可以添加其他与葫芦素E没有拮抗作用的促血管新生药物。其制剂可以是药学上的任意一种剂型，包括但不限于片剂、散剂、颗粒剂、胶囊剂、混悬剂、糖浆剂、口服液、软膏剂、贴剂或注射剂等。
根据剂型的不同，选择合适的药物载体，包括但不限于填充剂、粘合剂、崩解剂、润滑剂、增溶剂、助悬剂、润湿剂、色素、香精、溶剂、表面活性剂、矫味剂、缓冲剂、等渗调节剂、抗氧剂、抑菌剂和止痛剂中的一种或几种。
制成不同的剂型有利于根据实际需要进行临床应用，提高药物活性成分葫芦素E的利用率。
根据本申请的第三个方面，提供了一种用于治疗血管新生不足相关疾病的生物医用材料，包括有效量的葫芦素E。
生物医用材料包括但不限于生物医用高分子材料。
所述的生物医用材料包含有效量的葫芦素E。
优选地，典型的生物医用高分子材料为生物高分子材料的支架。
制备生物高分子材料的支架时，可以是葫芦素E与现有技术中的支架材料混合制备而成，也可以先将葫芦素E制备成微球或纳米粒后再与现有技术中的支架材料混合制备成生物复合材料。即生物复合材料中，药物活性物质葫芦素E可以自身形态直接存在，也可以微球或纳米粒的形态存在。
优选地，本申请通过3D打印技术制备了含葫芦素E的生物高分子材料的支架。
优选地，生物高分子材料的支架中药物有效成分葫芦素E的重量百分比为0.001-0.1％，例如为0.001％、0.005％、0.01％、0.015％、0.02％、0.05％、0.06％、0.08％或0.1％。
由葫芦素E制成的药物和/或生物医用材料可应用于治疗血管新生不足相关疾病，特别是用于骨组织修复、骨折愈合缓慢、创伤、冠心病、心肌缺血、侧支循环代偿建立不足、闭塞性脉管炎等诸多血管新生不足疾病的防治。
为了有助于更清楚的理解本申请的内容，现结合具体的实施例和附图详细介绍。如未明确指出，以下实施例中涉及的实验操作方法为常用的医学操作方法，涉及的试剂、仪器为常规的市售试剂或者仪器。
以下实施例中所用各试剂均直接购于市场，例如：葫芦素E购自法国EXTRASYNTHESE公司；CCK-8试剂购自同仁化学；基质胶购自康宁公司；PLGA(poly lactic-co-glycolic acid，聚乳酸-羟基乙酸共聚物)购自山东省医疗器械研究所，PLGA的分子量为10万，其中乳酸与乙醇酸的摩尔比为70:30。上述各物质不限于购于以上所述厂家，所用其他原料或试剂也均购于市场，购买厂家不限。以下实施例所用快速成型机由清华大学机械工程系研制，型号为CLRF-2000-II。
实施例1 葫芦素E对血管内皮细胞毒性检测
1、原理：药物毒性实验采用CCK-8方法。CCK-8方法是一种在活细胞中运用比色法确定细胞增殖数量的测定方法。原理为WST-8[化学名：2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐]，在电子载体1-甲氧基-5-甲基吩嗪鎓硫酸二甲酯(1-Methoxy PMS)的作用下被细胞中的脱氢酶还原为具有高度水溶性的黄色甲瓒产物，生成的甲瓒物的数量与活细胞的数量成正比。因此可利用这一特性直接进行细胞增殖和毒性分析。
2、方法：将血管内皮细胞ea.hy926细胞悬液按照3000细胞/孔接种于96孔板中，过夜培养贴壁后，分别加入含有不同浓度葫芦素E(1,3,10,30,100,300nM)的培养基作用72小时，然后每孔加入10μL CCK-8试剂，孵育2小时，酶标仪上490nm处读出每个孔中的光吸收值。根据数值得出药物的半数致死浓度为65.45nM(如图2所示)。根据此结果确定50nM以下为内皮细胞可耐受的安全浓度。
实施例2 葫芦素E促进血管内皮细胞EA.hy926成管实验
1、原理：基质胶是从EHS(Engelbreth-Holm-Swarm)小鼠肉瘤中抽提得到的可溶性的基底膜抽提物。在室温下，基质胶聚合为一种具有生物活性的基质材料，作用与哺乳动物细胞基底膜类似，对于细胞的粘附和分化有效应作用，血管内皮细胞可在Matrigel上融合成微管，利用血管内皮细胞的这种特性来研究药物对血管形成的影响。
2、方法：将基质胶铺到24孔板上，于37℃，5％CO 2培养箱放置30分钟待其凝固。每孔加入相同体积的细胞和含不同浓度葫芦素E的培养基共500μL，混匀。于37℃，5％CO 2培养箱中培养16-18h后在显微镜下观察微管形成情况，计算微管形成数量并进行统计学分析。
微管形成情况照片如图3所示，其中A为对照组(未给予葫芦素E)，B为10nM葫芦素E组，C为30nM葫芦素E组。血管内皮细胞在基质胶中生长16-18h后，细胞由原来的多角形变成长梭形，在基质胶中延伸生长，单个细胞卷成管状并相互连接，形成三维网状结构。图中可以看出，与对照组相比，给予葫芦素E之后，内皮成管过程大大被促进，形成交织良好的三维网络结构，说明葫芦素E可以促进内皮细胞微管形成。
实施例3 葫芦素E生物复合材料支架的制备
(1)PLGA+TCP+葫芦素E溶剂的配制
称取5.0g PLGA和1.25g TCP置于100ml烧杯中,加入50ml 1,4-二氧六环，搅拌使其溶解。在打印之前，称取20mg葫芦素E置于1.5ml离心管中，加入500ul 1,4-二氧六环使其溶解，在加入到PLGA/TCP溶液中，备用。
(2)3D打印
①建立支架模型：设计支架的长宽高为20mm×20mm×20mm的立方体，生成STereo Lithography(STL)。将STL文件导入Aurora软件中进行分层切割，设计单层厚度0.12mm,生成command-line interface(CLI)文件。
②设置打印参数：将步骤1设置好的CLI文件，导入cark软件中，打印过程参数设定为：打印速度20mm/s，浆料填充速度1.00mm/s,支架层厚120μm，平均孔径大小500μm。
③测量溶浆的粘度：使用粘度计测量PLGA/TCP/CuE溶液的粘度，将其粘度控制在600cp-700cp范围内。
④打印过程：3D打印机的温度预冷至-30℃后，将浆料倒入料仓，使用软管将料仓与打印机连接，打印过程在设定的参数下运行，整个打印过程在-30℃条件下进行，以保证浆料中药物分子的活性。
冷冻干燥：打印结束后，将制备的支架置于冷冻干燥机中，在真空条件下冷冻干燥72小时，去除材料中的有机溶剂。
实施例4 葫芦素E生物复合材料支架促进兔骨缺损处血管形成
将注入造影剂的动物标本应用小动物用微CT(SkyScan 1176)扫描，置胫骨或脊柱标本于机床中心，调节电脑参数，分辨率(Resolution)中级(18um)，滤光器(Filter)Al 1mm，电压(Voltage)60kV，电流(Current)400μA，平均校正(Frame Averaging)2次。应用NRecon软件重建股骨远端标本二维图，选择二维重建区域，预览(Preview)二制图，统 一设置灰度范围(Minimum or Maximum for CS to Image Conversion)：0.00-0.10，选择全标本重建区域(Reconstruct ROI)，重建血管形成。用CTAn软件选取骨缺损位置(Analysis ROI)，设置背景(Thresholding)为40-255，选择3D模型程序进行重建。
葫芦素E生物复合材料支架促进骨缺损部位血管形成照片如图4所示，其中A为对照组(未含有葫芦素E的支架)照片，B为葫芦素E处理组照片。图中可以看出，使用葫芦素E生物复合材料支架能明显促进兔骨缺损处血管形成。
实施例5 葫芦素E生物复合材料支架促进兔骨缺损处新骨形成
应用小动物用微CT(SkyScan 1176)，置胫骨或脊柱标本于机床中心，调节电脑参数，分辨率(Resolution)中级(18um)，滤光器(Filter)Al 1mm，电压(Voltage)60kV，电流(Current)400μA，平均校正(Frame Averaging)2次。应用NRecon软件重建股骨远端标本二维图，选择二维重建区域，预览(Preview)二制图，统一设置灰度范围(Minimum or Maximum for CS to Image Conversion):0.00-0.10，选择全标本重建区域(Reconstruct ROI)，添加重建并开始运行。应用CTAn软件选取骨缺损位置(Analysis ROI)，设置背景(Thresholding)为63-255，选择3D分析进行分析可检测缺损位置新生骨的骨体积分数(Percent bone volume,BV/TV)。根据大鼠标样建立密度与灰度标准曲线，可获得缺损位置新生骨的骨密度。选择3D建模可以生成新生骨3D重建模型。
葫芦素E生物复合材料支架促进骨缺损部位新骨形成照片如图5所示，其中A为对照组(未含有葫芦素E的支架)照片，B为葫芦素E处理组照片。图中可以看出，使用葫芦素E生物复合材料支架能明显促进兔骨缺损处新骨形成。
实施例6 葫芦素E促进内皮细胞hy.EA926相关的基因表达试验
将hy.EA926细胞种在6孔板中，孵育过夜后，加30nM葫芦素E分别于时间点6,12,24,48小时按AxyGen的RNA提取试剂盒说明书提取RNA，逆转录总mRNA为cDNA，设计VEGFR2的引物。
引物序列为：上游：5′-CTCTCTCTGCCTACCTCACCTG-3′；下游：5′-CGGCTCTTTCGCTTACTGTTC-3′。使用SYBR-Green Mix kit(Takara)应用Roche LC96荧光定量PCR仪来检测VEGFR2基因水平的变化。葫芦素E对VEGFR2基因mRNA表达调控的影响如图6所示，图中显示药物可以促进血管内皮生长因子受体VEGFR2的表达，并具有时间依赖性，该结果提示在葫芦素E可调控血管新生相关的基因表达从而促进血管新生。
葫芦素E经实验结果表明，无毒性的低剂量葫芦素E对人脐静脉细胞有显著的成血管效果，进一步动物体内实验证明其促进损伤部位的血管新生以及具有骨修复的作用，这是由于低剂量葫芦素E可促进VEGFR2的表达上调。
尽管已用具体实施例来说明和描述了本申请，然而应意识到，在不背离本申请的精神和范围的情况下可以作出许多其它的更改和修改。因此，这意味着在所附权利要求中包括属于本申请范围内的所有这些变化和修改。

Claims (10)

  1. 葫芦素E在制备治疗血管新生不足相关疾病的药物或生物医用材料中的用途，所述葫芦素E处于无细胞毒性浓度下。
  2. 按照权利要求1所述的用途，其特征在于，所述葫芦素E的有效浓度不高于50nM，优选不高于30nM。
  3. 按照权利要求1所述的用途，其特征在于，所述葫芦素E的给药途径包括外用、口服、肠胃外、雾化吸入或植入生物材料。
  4. 按照权利要求1-3任一项所述的用途，其特征在于，所述血管新生不足相关疾病包括骨组织修复不良、骨折愈合缓慢症、冠心病、心肌缺血、侧支循环代偿建立不足、闭塞性脉管炎或器官移植后血管新生不足中的任一疾病。
  5. 按照权利要求4所述的用途，其特征在于，所述骨折包括创伤或病理性骨质疏松引起的骨折；
    优选地，所述的冠心病包括缺血性心脏病、冠脉血管栓塞及损伤性疾病。
  6. 一种用于治疗血管新生不足相关疾病的药物，其特征在于，所述药物主要由葫芦素E和药学上可接受的载体制成。
  7. 按照权利要求6所述的用于治疗血管新生不足相关疾病的药物，其特征在于，所述药物的剂型包括片剂、散剂、颗粒剂、胶囊剂、混悬剂、糖浆剂、口服液、软膏剂、贴剂或注射剂。
  8. 一种用于治疗血管新生不足相关疾病的生物医用材料，其特征在于，包括有效量的葫芦素E。
  9. 按照权利要求8所述的用于治疗血管新生不足相关疾病的生物医用材料，其特征在于，所述生物医用材料为生物医用高分子材料，优选为生物医用高分子支架。
  10. 按照权利要求8或9所述的用于治疗血管新生不足相关疾病的生物医用材料，其特征在于，所述葫芦素E以自身形态、微球或纳米粒形式存在。
PCT/CN2018/121847 2018-08-31 2018-12-18 葫芦素e在制备治疗血管新生不足相关疾病药物或生物医用材料中的用途和应用其的产品 WO2020042449A1 (zh)

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