WO2021068962A1 - 一种用于血管新生、淋巴管新生相关疾病的多肽及其用途 - Google Patents

一种用于血管新生、淋巴管新生相关疾病的多肽及其用途 Download PDF

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WO2021068962A1
WO2021068962A1 PCT/CN2020/120360 CN2020120360W WO2021068962A1 WO 2021068962 A1 WO2021068962 A1 WO 2021068962A1 CN 2020120360 W CN2020120360 W CN 2020120360W WO 2021068962 A1 WO2021068962 A1 WO 2021068962A1
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polypeptide
disease
cancer
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angiogenesis
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田中纯美
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors

Definitions

  • the present invention relates to a preventive, inhibitory and/or therapeutic agent containing Semaphorin protein (Semas) and its pharmaceutically acceptable components as active and effective ingredients, and its preparation for preventing, inhibiting and/or treating angiogenesis diseases, lymphatic vessels Use in medicine for new-born related diseases.
  • Semaphorin protein Semaphorin protein
  • it is used in the prevention and treatment of cancer-related diseases, scarring, cardiovascular diseases, organ aging diseases, and Alzheimer's disease, especially in the prevention and treatment of diseases related to angiogenesis in ophthalmology.
  • Human cells maintain their activities and functions by ingesting nutrients and oxygen supplied by blood vessels around the cells. The number of necessary cells is also strictly controlled by human primitive functions. However, cancer cells cannot be controlled and proliferate very actively. Compared with normal cells, cancer cells that carry out such activities require a lot of nutrients and oxygen, so they begin to make new blood vessels, that is, new blood vessels. The new blood vessels produced by cancer cells have become a source of support for their activities.
  • Angiogenesis is the use of existing vasculature to form new blood vessels, such as wound healing, cancer diseases, aging macular degeneration, tumor growth and diabetic retinopathy, and many other physiological processes, which play a vital role in pathological conditions. .
  • Cancers related to angiogenesis are about the following: breast cancer (especially metastatic breast cancer), colorectal cancer, colorectal cancer, progressive colorectal cancer, esophageal cancer, superficial esophageal cancer, early esophageal cancer, laryngeal cancer, Malignant tumor of the accessory nasal cavity, gastrointestinal stromal tumor (GIST), renal (renal cell) cancer, liver (primary adult) cancer (especially liver cancer that cannot be operated), lymphoma, lymphangiomatosis/Gorham disease , Huge lymphatic malformations, melanoma (malignant melanoma), lung cancer, non-small cell lung cancer (NSCLC), metastatic non-small cell lung cancer, ovarian cancer, epithelial ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, nasal cavity tumors , Gynecological malignant tumors, cervical cancer (especially early invasive cancer), endometrial cancer, bladder cancer (superficial bladder cancer, invasive cancer).
  • breast cancer especially meta
  • cancer cells metastasize to the lymph nodes, they may metastasize to the far end through the lymph vessels.
  • the difficulty of metastasis to the liver is that there are almost no symptoms at first, and it is very easy to become severe. Cancer that has metastasized is difficult to treat, so early prevention and early treatment are extremely important.
  • angiogenesis inhibitors used in the treatment of human cancer are several anti-angiogenesis agents approved by the U.S. Food and Drug Administration (FDA), which can delay tumor growth, but recent research results have shown innate in animal studies. The possibility of sexual abnormalities has been identified as the possibility of complications.
  • FDA U.S. Food and Drug Administration
  • angiogenesis inhibitor treatment includes bleeding, arterial thrombosis (causing stroke or heart attack), high blood pressure, and urine protein (3-5). Perforation of the digestive tract and gastrointestinal fistula are also considered side effects of certain angiogenesis inhibitors.
  • the anti-VEGF antibody Bevacizumab (trade name AVASTIN) can not be used in patients with squamous cell carcinoma and diagnosed tumors infiltrating large blood vessels due to the risk of bleeding, nor can it be used in patients with a history of high bleeding risk. For patients with brain metastases, they cannot be used because of the risk of bleeding from tumor lesions.
  • Angiogenesis diseases also include: keloid, rheumatoid arthritis (RA), psoriasis (psoriasis), arteriosclerosis, tuberous sclerosis, Alzheimer’s disease such as vascular Alzheimer’s, aging, acute Coronary artery disease induced by myocardial infarction, cytomegalovirus and chlamydia infection.
  • Vascular endothelial dysfunction is caused by lifestyle diseases (obesity, stress, smoking, lack of exercise, etc.) and diseases such as high blood pressure, dyslipidemia, diabetes, sleep apnea syndrome, chronic kidney disease, and aging, gender, menopause, genetics, etc. Caused by damage to vascular endothelial cells.
  • lifestyle diseases ovalchiec, diabetes, sleep apnea syndrome, chronic kidney disease, and aging, gender, menopause, genetics, etc.
  • the paralysis of vascular function promotes the occurrence of cardiovascular disease, obesity, etc. make the blood vessels narrow and hard, and the cholesterol and neutral fat in the blood increase and deposit, which intensifies the development of arteriosclerosis. It can cause angina pectoris and renal dysfunction, cerebral infarction and myocardial infarction after thrombosis, and cerebral hemorrhage caused by rupture of blood vessels.
  • the arteries begin to harden, causing severe cerebral infarction and myocardial infarction.
  • psoriasis psoriasis
  • RA psoriasis
  • sirolimus a drug that has various side effects, and there is no certain opinion on its effect.
  • Kidney tumors and lung LAM are usually involved in the prognosis of life when they are severe.
  • Ophthalmic angiogenesis diseases include: diabetic fundus vascular disease, omental hemangiomas, von Hippel-Lindau disease, soft hemangioma on the body, macular degeneration, myopic choroidal neovascularization, diabetic omentopathy, and immature infant retinopathy , Neovascular glaucoma, diabetic macular disease, omental vein occlusive disease, intraocular neovascularization with uveitis, Behcet disease, tuberculous uveitis, choroidal neovascularization with high myopia, malignant lymphoma, conjunctival malignant lymphoma , Choroidal tumors, cytomegalovirus diseases such as cytomegalovirus retinitis, intraocular malignancies such as retinoblastoma, choroidal malignant melanoma, malignant lymphomas that appear in the eyeball, ocular adnexal tumors such as optic nerve tumor
  • Laser treatment leads to a decrease in the amount of blood required by the retina and a blood shortage condition. If the new blood vessels blocking the angle of the chamber are not cured, the intraocular pressure will hardly drop. Only glaucoma surgery can be performed at this stage, but the effect of neovascular glaucoma surgery is not good.
  • Intraocular inflammation of tuberculous uveitis is treated with anti-tuberculosis treatment and oral corticosteroids to treat new blood vessels.
  • corticosteroids cause high intraocular pressure, glaucoma, cataracts, corneal fungus, etc., and affect the development of children.
  • cytomegalovirus retinitis is oral and intravenous infusion of drugs, but the existing drugs often cause bone marrow suppression and renal insufficiency, which makes the treatment difficult.
  • Steroids, interferon alpha, propranolol, chemotherapy (vincristine), etc. are used clinically for tumor disease/Gorham disease, but the therapeutic effect is limited. If there are chest lesions, such as breasts, the prognosis is not good. If the disease spreads to multiple organs, causing various symptoms, it often leaves permanent diseases such as chronic respiratory failure and motor dysfunction. Many cases require long-term treatment, and the cure rate is extremely low.
  • lymphatic malformations Cervical and facial lesions
  • the present invention provides a method for preventing, inhibiting and inhibiting angiogenesis and lymphangiogenesis related diseases, especially cancer, ophthalmic diseases, arteriosclerosis, cardiovascular diseases, aging diseases, scar formation, Alzheimer's disease and other diseases. /Or a therapeutic polypeptide or a fragment comprising the polypeptide as an active ingredient, and its use in the preparation of medicines for preventing, inhibiting and/or treating angiogenesis diseases and lymphangiogenesis-related diseases. In particular, it is used in the prevention and treatment of cancer-related diseases, arteriosclerosis, Alzheimer's disease, and scar formation, especially in the prevention and treatment of diseases related to angiogenesis in ophthalmology.
  • the invention also relates to a polypeptide for preventing, inhibiting and/or treating angiogenesis and lymphangiogenesis related diseases, especially cancer, ophthalmic diseases, arteriosclerosis, cardiovascular diseases, aging diseases, scar formation, Alzheimer's disease, etc. Methods of disease.
  • the present invention also relates to a polypeptide, which is used to prevent, inhibit and/or treat angiogenesis and lymphangiogenesis-related diseases, especially cancer, ophthalmic diseases, arteriosclerosis, cardiovascular diseases, aging diseases, scar formation, and Alzheimer's Diseases such as Haimer's disease.
  • a and/or B when used to link open ending words such as “includes”, in one embodiment, it may only refer to A (optionally including other than B In another embodiment, it can only refer to B (optionally including components other than A); in yet another embodiment, it refers to A and B (optionally including other components) and the like.
  • Angiogenesis mainly based on the original capillary blood and (or) venules, through the proliferation and migration of vascular endothelial cells, new ones are generated in the form of budding or non-sprouting (or intussusception) from pre-existing blood vessels Capillaries.
  • RA is a chronic inflammatory disease, which invades all joints.
  • the inflammatory synovium of RA has early capillaries. Small blood vessels such as posterior capillary venules are regenerated significantly, and they are the site of inflammatory cell infiltration. As the nutritional blood vessels of the synovial membrane, it is deeply involved in the disease state. At present, the treatment of RA also relies on angiogenesis inhibitors.
  • Arteriosclerosis is accompanied by the progression of hyperlipidemia, hypertension, and aging.
  • Vasa Vasorum can be observed to stretch new blood vessels near the atherosclerotic plaque.
  • PDGF, TNF- ⁇ , FGF and TGF- ⁇ factors are secreted to induce angiogenesis.
  • arteriosclerosis occurs, the blood vessels become narrow and hard, and the cholesterol and neutral fat in the blood increase and deposit inside the blood vessels, which intensifies the development of arteriosclerosis.
  • the narrowing and hardening of blood vessels can cause angina pectoris and renal dysfunction. After thrombosis, it can cause cerebral infarction and myocardial infarction, and rupture of blood vessels can cause cerebral hemorrhage.
  • Tuberous sclerosis is renal angiosarcoma
  • Lymphangioleiomyomatosis is a disease that causes systemic hamartoma, such as angiofibroma of the face and fundus. Renal angiosarcoma lipoma has many new blood vessels, which sometimes cause rupture when enlarged. As the number of tumor cells increases, malignant transformation may occur.
  • LAM disease When tuberous sclerosis diseases occur with LAM disease, most of them occur in women of childbearing age. Diseases other than LAM are likely to occur in many organs (brain, skin, heart, lung, kidney, etc.), accompanied by epilepsy, Skin lesions and other symptoms of tuberous sclerosis.
  • Sinus tumors are tumors caused by sinus tissues (mainly mucosal epithelium). Representative benign tumors of sinus tumors are papilloma and hemangioma. Among malignant tumors, squamous cell carcinoma is the most common. In addition, the nasal cavity also produces malignant tumors of various pathological tissue types, including olfactory neuroblastoma. For example, the clinical characteristics of papilloma include a high recurrence rate and the possibility of malignant transformation. The recurrence rate is about 30%, and most of them relapse within 2 years after surgery. Malignant transformation is considered to be about 10%, usually into squamous cell carcinoma.
  • cervical cancer The most common gynecological malignancies are cervical cancer, placental angiomas, and cervical cavernous hemangioma associated with pregnancy.
  • angiogenesis is deeply involved in the growth of metastatic lesions that have the greatest prognosis.
  • Dementia is closely related to blood vessels. Blood vessel obstruction leads to vascular dementia and Alzheimer's disease. The improvement of symptoms requires the restoration of cerebral blood flow.
  • AD Alzheimer's disease
  • a ⁇ beta amyloid
  • Immunotherapy in basic and clinical trials has shown that reducing amyloid deposits is useful for improving cognitive function.
  • antibody or vaccine immunotherapy against A ⁇ is considered promising, but side effects such as angiogenic edema and cerebral microhemorrhage are obvious.
  • the hyperpermeability of the blood-brain barrier (BBB) that exists between the brain and the blood is closely related to cerebral vascular endothelial cells. It is also found that the blood vessel density of AD patients is also increasing.
  • the blood vessels throughout the body not only transport oxygen and nutrients to every corner of the body, but also move inflammatory cells to this part when abnormalities such as tissue damage or inflammation occur to promote tissue repair.
  • the vascular endothelial cells covering the vascular cavity are activated by TNF ⁇ (an inflammatory cytokine) and help the inflammatory cells to migrate out of the blood vessel and play a role in normally causing inflammation.
  • TNF ⁇ an inflammatory cytokine
  • IL-6 levels in patients with acute myocardial infarction are elevated.
  • Plasma IL-6 levels rise within a few hours after the onset of infarction, and reach a peak after 24 to 48 hours ( Figure 2).
  • CRP in the blood increases.
  • the maximum IL-6 value during this process is significantly correlated with the maximum CRP value.
  • Cytomegalovirus directly acts on endothelial cells to enhance IL-6 production.
  • Blankenberg et al. suggest that patients with cytomegalovirus antibody positive and high blood IL-6 levels are at high risk of death from heart disease in the future, but even if cytomegalovirus antibodies are positive and blood IL-6 levels are not high, the patient is not at risk. of.
  • the representative lesion that impairs ocular transparency is angiogenesis.
  • the newly formed blood vessels are fragile, prone to repeated bleeding and exudation, forming a proliferative membrane, accompanied by the proliferation of other cells, so the visual function is obviously impaired.
  • Intraocular angiogenesis diseases typical examples include diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration, angiogenic glaucoma, etc., which are all refractory diseases that lead to blindness.
  • VEGF a chemical substance called VEGF
  • This "new blood vessel” is very easy to rupture and is the root cause of retinal hemorrhage and vitreous hemorrhage.
  • neovascular glaucoma The causes of neovascular glaucoma include diabetic retinopathy syndrome, central retinal vein occlusion and ocular ischemia syndrome.
  • blood vessels in the retina are damaged by diabetes, blood circulation is blocked, causing severe visual impairment.
  • New blood vessels may block the angle of the chamber, block the outlet of the aqueous humor, and cause a sudden rise.
  • the intraocular pressure of about 20mmHg becomes 50mmHg or higher.
  • the macula is located in the center of the retina and is the most important part of observing things. Due to diabetic diseases, capillary hemangioma, etc. often appear near the macula, blood components ooze, and symptoms caused by macular swelling are called diabetic macular disease.
  • neovascularization inhibitors Before the disease progresses seriously, there may be no symptoms, and it is important to use neovascularization inhibitors early.
  • the current clinical use of laser coagulation treatment for the purpose of preventing angiogenesis in the avascular area, photocoagulation of the coagulation scar of omental tissue degeneration will expand over time, so as to affect the macular vision and visual field.
  • Hypertension is one of the causes of arteriosclerosis, but even in the absence of hypertension, arteriosclerosis will develop. If the root of the retinal vein is blocked, it will become blocked in the central retinal vein and bleed throughout the retina. Lead to sudden loss of vision, visual field disorder, vision change, vitreous hemorrhage, and visual acuity decreased significantly. Combined with angiogenic glaucoma.
  • Intraocular neovascularization with uveitis Intraocular neovascularization with uveitis
  • Harada disease causes a large number of retinal and choroidal detachment, optic nerve head edema, and new blood vessels on the posterior part of the nipple. Treatment requires large-scale steroids to contract new blood vessels.
  • Behcet disease causes neovascularization in the retina and nipple. Treatment requires local administration of steroid drugs and immunosuppressive drugs.
  • myopic retinopathy The prevalence of myopic retinopathy is 1.7%, and the prevalence of women is about twice that of men. If the eyeball stretches back and the retina/choroid is damaged, pathological blood vessels (choroidal neovascularization) will develop, leading to bleeding and swelling.
  • Choroidal hemangioma caused by abnormal blood vessel proliferation, often occurs at the same time as Sturge-Weber syndrome, which is a frequent occurrence of systemic hemangioma.
  • Metastatic choroidal tumors are blood-line metastatic malignant tumors that occur in all organs of the body. Choroidal metastases of female breast cancer and male lung cancer are common.
  • the tumor occurs in a particularly important part of the macular area, or the retinal detachment around the tumor reaches the macular area, vision deterioration will occur. As the tumor grows, the visual field will be damaged and disappear.
  • Cytomegalovirus retinitis is in patients with weakened immune function, such as AIDS patients and patients receiving anti-cancer treatment, retinitis can occur as an opportunistic infection.
  • retinitis can occur as an opportunistic infection.
  • the incidence is high.
  • the eye part is the eyeball and eye appendages (eyelid, conjunctiva, eye socket, lacrimal gland, etc.), and tumors can occur in various parts. Poor prognosis even affects the survival rate of life.
  • Choroidal malignant melanoma is a malignant tumor that occurs in the uvea (choroid, ciliary body, iris) of the eyeball of an adult. If it progresses and the tumor is large, the eyeball needs to be removed.
  • lymphoma of the central nervous system a subtype of lymphoma of the brain.
  • systemic chemotherapy, radiotherapy and other medical treatments are carried out, but it is difficult to be completely cured at present.
  • preventive treatment methods have been reported, so far there is no reliable method for practical application.
  • Eyelid tumors salivaous gland cancer
  • the tumors that appear in the eyelids are common in eye tumors, and recurrences require surgery or radiotherapy.
  • Lacrimal cancer treatment requires surgery to completely remove the tumor. However, even if the tumor is completely removed, it is impossible to prevent subsequent recurrence.
  • Ocular sarcoma tumor compresses the deviation of the eyeball and is usually difficult to completely remove. It requires a combination of surgery, chemotherapy and radiotherapy.
  • Medulloma is a tumor produced by the sheath that includes the optic nerve. When the tumor is removed, it usually leads to blindness.
  • Lymphatic vessels are the passages through which lymphatic fluid passes. It collects the lymphatic fluid produced from various corners of the body, filters the bacteria and viruses contained in the lymphatic fluid, and finally returns them to the blood vessels. Many diseases related to lymphatic vessels are difficult diseases.
  • lymphatic vessels can not only serve as the circulatory system for excessive drainage of extracellular fluid, but also have a great influence on the immune system. It is important to clarify the pathological conditions such as lymph node metastasis and lymphedema of malignant tumors.
  • Lymphatic vessels play an important role in maintaining the stability of the microenvironment in the body, immune response, and tumor lymphatic metastasis. Lymphatic neoplasia is closely related to embryonic development, trauma repair, congenital lymphedema, inflammatory outcome and tumor metastasis. Under the action of chemokines and growth factors, lymphatic endothelial cells migrate, proliferate and form lumens to form new lymphatic vessels. In recent years, it has been discovered that lymphatic endothelial progenitor cells are involved in lymphangiogenesis. Lymphatic endothelium specifically expresses Prox-1, podopiann, VEGFR-3 and LYVE-1, etc. These factors and receptors regulate lymphangiogenesis.
  • VEGF-C/VEGFR-3 or VEGF-D/VEGFR-3 signaling pathway plays an important role in the process of lymphangiogenesis.
  • VEGF-C, VEGF-D and VEGFR-3 can be used as targets for gene therapy, which are expected to treat lymphangiogenesis disorders, as well as resistance to post-transplantation immune rejection and anti-tumor lymphatic metastasis.
  • Lymphangiomatosis/Gorham disease (designated as refractory disease 277 in Japan)
  • Lymphangiomatosis/Gorham disease is also known as diffuse lymphangiomatosis, Gorham-Stout syndrome, also known as massive osteolysis.
  • the lymphatic proliferation of esophageal squamous cell carcinoma can be based on lymphangiogenesis and predict metastasis.
  • esophageal cancer is highly malignant compared with colon cancer and gastric cancer and is one of the tumors with a poor prognosis.
  • lymphatic vessels In recent years, the growth and metastasis of malignant tumors involve the formation of blood vessels and lymphatic vessels. It is speculated that angiogenesis is related to tumor growth, and lymphangiogenesis in tumor tissues is related to lymph node metastasis. Chikuhou et al. measured the distribution and density of lymphatic vessels and showed the ratio of the number of lymphatic vessels in tumor areas and non-tumor areas. There is a significant increase in the number of lymphatic vessels in the lamina muscular of the tumor. The percentage of lymphatic vessels in the number of blood vessels has also been observed to increase significantly.
  • Lymphatic malformations consist of large and small lymphatic cysts with vascular disease. Intraorbital lymphangioma may cause intraorbital bleeding or infection, leading to blindness. If the lymphatic disease spreads to the skin and mucous membranes, local lymphangioma appears, and the symptoms are repeated lymphatic fistulas, bleeding, and infections. In the course of the disease, infection and bleeding may occur in any part of the disease, and repeated acute swelling and inflammation.
  • Semaphorin3A Semaphorin3A
  • Polypeptide (b) A polypeptide consisting of more than 70 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 and containing the region from 166 aa to 235 aa in SEQ ID No. 2.
  • Polypeptide (c) A polypeptide is a polypeptide in which a few (preferably 1 to a few) amino acid residues of the polypeptide of (a) or (b) are substituted, deleted and/or inserted.
  • the peptide is a polypeptide having 80% or higher, preferably 90% or higher, more preferably 95% or higher, and still more preferably 98% or higher homology with the original sequence.
  • the region composed of the polypeptide of (c) above can also produce the physiological activity of Semaphorin3A protein, similar to the above 70aa region.
  • Amino acid sequence "homology” means to align two amino acid sequences so that the amino acid residues of the two amino acid sequences to be compared match as much as possible, and divide the number of matching amino acid residues by the total number of amino acid residues Expressed as a percentage. When aligning, the gap is appropriately inserted into one or both of the two sequences to be compared as needed.
  • This sequence alignment can be performed using known programs, such as BLAST, FASTA, CLUSTAL and so on.
  • the total number of amino acid residues is the number of residues that count one gap as one amino acid residue.
  • the homology (%) is the total number of amino acid residues in the longer sequence, which is calculated by dividing by.
  • the 20 amino acids that constitute natural proteins are neutral amino acids with low polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro) and neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, Trp) are known to be combined together and the substitution between them is usually not Will change the nature of the peptide.
  • the active ingredient used as an active ingredient for the prevention, inhibition or treatment of corneal diseases in the conjunctiva can be obtained by substituting between these respective groups. Peptides. Alternatively, the possibility of maintaining the preventive, suppressive or therapeutic activity of corneal injury is increased.
  • polypeptide (c) is a polypeptide having 80% or higher sequence homology with a polypeptide composed of the full length of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 (ie, Semaphorin3A protein) , Or a polypeptide with 80% or higher sequence homology with the Sema3A protein fragment containing 70 amino acid regions.
  • the active ingredient polypeptide c is a polypeptide composed only of these amino acids, or a polypeptide obtained by adding amino acids or polypeptides to one or both ends of the polypeptide (c), and/or has corneal diseases or corneal damage to blood vessels, etc.
  • the preventive, inhibitory or therapeutic activity of the polypeptide is a polypeptide composed only of these amino acids, or a polypeptide obtained by adding amino acids or polypeptides to one or both ends of the polypeptide (c), and/or has corneal diseases or corneal damage to blood vessels, etc.
  • the preventive, inhibitory or therapeutic activity of the polypeptide is a polypeptide composed only of these amino acids, or a polypeptide obtained by adding amino acids or polypeptides to one or both ends of the polypeptide (c), and/or has corneal diseases or corneal damage to blood vessels, etc.
  • the preventive, inhibitory or therapeutic activity of the polypeptide is a polypeptide composed only of these amino acids, or a polypeptide obtained by
  • a polypeptide composed of the full length of SEQ ID NO: 2 or 4 ie Sema3A protein
  • has 80% or more sequence homology A sexual polypeptide, a polypeptide having 80% or more sequence homology with a Sema3A protein fragment having corneal disease or corneal disease or corneal damage or preventing, inhibiting, or treating activity, and 80% or more of a region comprising 70 amino acids Sequence homology.
  • a chimeric protein obtained by fusing a region having a secretion domain with a secreted semaphorin other than Semaphorin3A.
  • the active ingredient polypeptide c a polypeptide whose homology in the 70aa region is higher than the homology of the entire region composed of the polypeptide of (c) is particularly preferred.
  • the active ingredient polypeptide c is a polypeptide having 80% homology with the polypeptide having the amino acid sequence of SEQ ID NO: 2, it is preferably a polypeptide having more than 80% homology in the 70aa region, especially 70aa.
  • the homology of this region is 90% or more, more preferably 95% or more, further preferably 98% or more, and more preferably, polypeptides with the same sequence in the 70aa region are preferred.
  • the active ingredient polypeptide c is a chimeric protein in which a region having 80% sequence homology with the Sema domain of human Sema3A protein and a region other than the Sema domain of secreted Semaphorin other than Sema3A are fused .
  • the homology of the 70aa region in the Sema domain is greater than 80%, especially the homology in the 70aa region is 90% or higher, more preferably 95% or higher, and even more preferably 98%. % Or higher, more preferably 90% or higher.
  • polypeptides having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 are particularly 80% or more, preferably 90% or more, and more preferably 95% or more , More preferably, a polypeptide having 98% or higher homology, and wherein, as described above, a polypeptide having a homology higher than that of the entire polypeptide in the 70aa region is preferable.
  • the polypeptide contained as an active ingredient in the preventive, inhibitory or therapeutic agent of the present invention is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • sugar chains or polyethylene glycol (PEG) chains are added to the polypeptide, or at least one amino acid constituting the polypeptide.
  • PEG polyethylene glycol
  • the technique of using D-type amino acids as a part and adding an Fc domain is a well-known and used technique.
  • polypeptide used in the present invention may be a polypeptide that has undergone these known in vivo stabilization modifications, as long as it has preventive, inhibitory, or therapeutic activity against corneal diseases or corneal damage.
  • polypeptide in the specification and claims is used in a sense, which also includes those modified for in vivo stabilization.
  • sugar chains can bind to the N-terminus, C-terminus, or amino acids between them, but preferably bind to the N-terminus or C-terminus so as not to inhibit the activity of the polypeptide.
  • the number of sugar chains to be added is preferably one or two, and preferably one.
  • the sugar chain is preferably a monosaccharide to a tetrasaccharide, and more preferably a disaccharide or trisaccharide.
  • the sugar chain may be connected to the free amino or carboxyl group of the polypeptide directly or through a spacer structure such as a methylene chain having about 1-10 carbon atoms.
  • the PEG chain can be connected to the N-terminus, the C-terminus or the amino acid between them, and usually one or two PEG chains are connected to the free amino or carboxyl group on the polypeptide.
  • the molecular weight of the PEG chain is not particularly limited, but is usually about 3,000 to about 7,000, preferably about 5,000.
  • amino acids that constitute a polypeptide are in the D-form.
  • all the amino acids that constitute the polypeptide are D-form amino acids instead of only those that form the D-form. Because the activity of the polypeptide is not inhibited.
  • a method of adding an Fc domain to a polypeptide is also known, and can be produced, for example, using a commercially available Fc fusion protein expression vector such as pFUSEN-Fc (manufactured by InvivoGen).
  • Semaphorin 3A used as the active ingredient of the present invention can be easily prepared by a conventional method using, for example, a commercially available peptide synthesizer. Moreover, it can be easily prepared using known genetic engineering techniques.
  • the cDNA of the Semaphorin gene is prepared by RT-PCR from RNA extracted from the tissue expressing the Semaphorin3A gene, and the full length or a desired part of the cDNA is integrated into the expression vector, and it can be introduced to obtain the target polypeptide.
  • RNA extraction, RT-PCR, integration of cDNA into the vector, and introduction of the vector into host cells can be performed by known methods.
  • vectors and host cells to be used are also well known, and various vectors and host cells are commercially available.
  • the method for producing the chimeric protein is also well known in the art, and the chimeric protein can be produced, for example, by the method described in the article by Koppel et al. above.
  • the above-mentioned stabilization modification can be easily performed by the known methods described in the above-mentioned respective documents.
  • the present invention relates to a polypeptide for preventing, inhibiting and/or treating angiogenesis diseases and lymphangiogenesis diseases, wherein the polypeptide is selected from any one of the following polypeptides (a) to (c):
  • the polypeptide of (c) above is a polypeptide in which a small number (preferably 1 to a few) of the amino acid residues of the polypeptide of (a) or (b) are substituted, deleted and/or inserted.
  • the peptide is a polypeptide having 80% or higher, preferably 90% or higher, more preferably 95% or higher, and still more preferably 98% or higher homology with the original sequence.
  • the present invention also relates to a polypeptide (hereinafter referred to as "active ingredient polypeptide c" for convenience), which has a region containing the polypeptide of (c) above, and has a preventive effect on corneal diseases or corneal damage. , Inhibitory or therapeutic activity. Similar to the above-mentioned active ingredient polypeptides a and b, they can be used to prepare the preventive, inhibitory or therapeutic agent of the present invention.
  • the polypeptide (c) used as an active ingredient can be obtained by mutual substitution between the groups defined herein, As the preventive, inhibitory or therapeutic agent of the present invention.
  • the above-mentioned polypeptide (c) has 80% or higher sequence homology with a polypeptide composed of the full length of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 (ie, Semaphorin3A protein) Polypeptide, or a polypeptide having 80% or higher sequence homology with the Sema3A protein fragment containing a 70 amino acid region.
  • the active ingredient polypeptide c is a polypeptide composed only of these amino acids, or a polypeptide obtained by adding amino acids or polypeptides to one or both ends of the polypeptide of (c), and/or has corneal diseases or corneal diseases.
  • the active ingredient polypeptide c is similar to the above-mentioned active ingredient polypeptides a and b, and is not particularly limited.
  • a polypeptide composed of the full length of SEQ ID NO: 2 or 4 ie, Sema3A protein
  • has 80% Or higher sequence homology ie, Sema3A protein
  • the sequence has homology, and a chimeric protein obtained by fusing a region having a secretion domain with other regions of secreted semaphorin other than Semaphorin3A.
  • the homology in the 70aa region is higher than the homology in the entire region composed of the polypeptide of (c).
  • the active ingredient polypeptide c is a polypeptide having 80% homology with the polypeptide having the amino acid sequence of SEQ ID NO: 2, it is preferably a polypeptide having more than 80% homology in the 70aa region, especially 70aa.
  • the homology of this region is 90% or more, more preferably 95% or more, and still more preferably 98% or more,
  • polypeptides with the same sequence in the 70aa region are preferred.
  • the active ingredient polypeptide c is a chimeric protein in which a region having 80% sequence homology with the Sema domain of human Sema3A protein and a region other than the Sema domain of secreted Semaphorin other than Sema3A are fused .
  • the homology of the 70aa region in the Sema domain is greater than 80%, especially the homology in the 70aa region is 90% or higher, more preferably 95% or higher, and even more preferably 98%. % Or higher, most preferably 90% or higher.
  • Preferred are chimeric proteins in which the sequences in the 70aa region are the same.
  • the polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 particularly has 80% or more, preferably 90% or more, More preferably 95% or more, more preferably a polypeptide having 98% or more homology, and wherein, as described above, a polypeptide having a homology higher than that of the entire polypeptide in the 70 aa region is preferable.
  • the polypeptide contained as an active ingredient in the preventive, inhibitory or therapeutic agent of the present invention is a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • the polypeptide used in the present invention may be a polypeptide that has undergone known in vivo stabilization modifications, as long as it has the preventive, inhibitory, or therapeutic activity of the present invention.
  • it is a glycosylated polypeptide.
  • the sugar chain can bind to the N-terminus, C-terminus or amino acids between them, but preferably binds to the N-terminus or C-terminus so as not to inhibit the activity of the polypeptide. .
  • the number of sugar chains to be added is preferably one or two, and preferably one.
  • the sugar chain is preferably a monosaccharide to a tetrasaccharide, and more preferably a disaccharide or trisaccharide.
  • the sugar chain may be connected to the free amino or carboxyl group of the polypeptide directly or through a spacer structure such as a methylene chain having about 1-10 carbon atoms.
  • the polypeptide used in the present invention is also preferably a PEGylated polypeptide.
  • the PEG chain can be connected to the N-terminus, C-terminus or amino acids between them, and usually one or two PEG chains are connected to the polypeptide on the Free amino or carboxyl connection.
  • the molecular weight of the PEG chain is not particularly limited, but is usually about 3,000 to about 7,000, preferably about 5,000.
  • At least a part of the amino acids constituting the polypeptide used in the present invention are in the D form; it is preferable to add an Fc domain to the polypeptide, and it is more preferable to use, for example, a commercially available Fc fusion protein expression vector such as pFUSEN-Fc (manufactured by InvivoGen) To produce peptides.
  • a commercially available Fc fusion protein expression vector such as pFUSEN-Fc (manufactured by InvivoGen)
  • Semaphorin 3A used as the active ingredient of the present invention can be easily prepared by a conventional method using, for example, a commercially available peptide synthesizer. Moreover, it can be easily prepared using known genetic engineering techniques.
  • the cDNA of the Semaphorin gene is prepared by RT-PCR from RNA extracted from the tissue expressing the Semaphorin3A gene, and the full-length or desired part of the cDNA is integrated into the expression vector, and it can be introduced to obtain the target polypeptide.
  • RNA extraction, RT-PCR, integration of cDNA into the vector, and introduction of the vector into host cells can be performed by known methods.
  • Semaphorin 3A is the active ingredient in the preventive, inhibitory or therapeutic agent of the present invention, and is effective for preventing, inhibiting or treating vascular/lymphangiogenesis diseases.
  • the present invention also relates to a preventive, inhibitor or therapeutic agent, which comprises the above-mentioned polypeptide (preferably Semaphorin 3A) or pharmacologically acceptable excipients, stabilizers, preservatives, buffers suitable for each administration form Agent.
  • a preventive, inhibitor or therapeutic agent which comprises the above-mentioned polypeptide (preferably Semaphorin 3A) or pharmacologically acceptable excipients, stabilizers, preservatives, buffers suitable for each administration form Agent.
  • additives such as solubilizers, emulsifiers, diluents, tonicity agents, etc. can also be appropriately mixed to prepare formulations.
  • the formulation methods and additives that can be used as described above are well known in the field of pharmaceutical formulations, and any methods and additives can be used.
  • the present invention also relates to the use of the aforementioned polypeptides in the preparation of medicaments for the prevention, inhibition and/or treatment of angiogenesis diseases and lymphangiogenesis diseases.
  • it also relates to the preparation of the aforementioned polypeptides for the prevention, inhibition and/or treatment of cancer.
  • polypeptide is selected from any one of the following (a) to (c):
  • a polypeptide having 80% or higher sequence homology with the polypeptide of (a) or (b), preferably 90% or higher, more preferably 95% or higher, still more preferably 98% or higher Homologous polypeptides are especially preferred as chimeric proteins, in which the sequences in the 70aa region are the same.
  • polypeptide is a glycosylated polypeptide
  • the polypeptide is a PEGylated polypeptide; optionally, the molecular weight of the PEG chain is about 3000 to about 7000, preferably about 5000.
  • angiogenesis disease or lymphangiogenesis disease is cancer
  • the above-mentioned cancer is one or more diseases selected from the group consisting of breast cancer, (especially metastatic breast cancer), colorectal cancer, colorectal cancer, progressive colorectal cancer, esophageal cancer, Superficial esophageal cancer, early esophageal cancer, laryngeal cancer, paranasal malignant tumor, gastrointestinal stromal tumor (GIST), kidney (kidney cell) cancer, liver (primary adult) cancer (especially those that cannot be operated on Liver cancer), lymphoma, lymphangiomatosis/Gorham disease, huge lymphatic malformations, melanoma (malignant melanoma), lung cancer, non-small cell lung cancer (NSCLC), metastatic non-small cell lung cancer, ovarian cancer, epithelial Ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, nasal cavity tumors, gynecological malignancies, cervical cancer (especially early invasive cancer), endometrial cancer, bladder cancer (
  • angiogenesis disease or lymphangiogenesis disease is an ophthalmic angiogenesis disease or lymphangiogenesis disease.
  • the above-mentioned ophthalmic angiogenesis disease or lymphangiogenesis disease is selected from one or more of the following diseases: diabetic vascular proliferative ocular fundus disease, omental angioma, von Hippel-Lindau disease, body surface Soft hemangioma, macular degeneration, myopic choroidal neovascularization, diabetic omentum disease, immature infant retinopathy, neovascular glaucoma, diabetic macular disease, omental vein occlusive disease, intraocular neovascularization with uveitis, Behcet Disease, tuberculous uveitis, high myopia choroidal neovascularization, malignant lymphoma, conjunctival malignant lymphoma, choroidal tumor, cytomegalovirus disease such as cytomegalovirus retinitis, intraocular malignant tumor such as retinoblastoma , Choroidal malignant mela
  • diseases
  • the above-mentioned neovascular glaucoma includes angiogenesis of the fundus accompanying systemic diabetes and hypofunction of blood vessels and lymphatic vessels in secondary angiogenic glaucoma.
  • the above-mentioned angiogenesis disease or lymphangiogenesis disease is one or more diseases selected from the group consisting of keloid, rheumatoid arthritis (RA), psoriasis (psoriasis), arteriosclerosis , Tuberous sclerosis, Alzheimer's disease such as vascular Alzheimer's disease, aging, acute myocardial infarction, cytomegalovirus and chlamydia infection induced coronary artery disease.
  • diseases selected from the group consisting of keloid, rheumatoid arthritis (RA), psoriasis (psoriasis), arteriosclerosis , Tuberous sclerosis, Alzheimer's disease such as vascular Alzheimer's disease, aging, acute myocardial infarction, cytomegalovirus and chlamydia infection induced coronary artery disease.
  • the above-mentioned angiogenesis disease or lymphangiogenesis disease is a scar tissue formation related disease.
  • the above-mentioned scar tissue formation-related disease is one or more diseases selected from the following: diseases caused by scars, ulcers, wounds, necrosis caused by infarction, and tissue defects of various organs.
  • the above-mentioned angiogenesis disease or lymphangiogenesis disease is one or more diseases selected from the group consisting of: aging of aging organs, blood vessels and lymphatic function of aging organs in a state of deficient blood caused by decreased vascular function Diminish.
  • the above-mentioned organ aging diseases include: vascular dementia and Alzheimer's disease.
  • the cause of the vascular/lymphangiogenesis disease is not particularly limited, and it may be a localized disease or a systemic disease.
  • the preventive and/or therapeutic agent of the present invention may consist of Semaphorin 3A alone, or consist of pharmacologically acceptable excipients, stabilizers, preservatives, and buffers suitable for each dosage form.
  • additives such as solubilizers, emulsifiers, diluents, tonicity agents may be appropriately mixed to prepare a formulation. And the preparation method can use any method and additives.
  • the target of administration of the preventive, inhibitory or therapeutic agent of the present invention is a mammal, and examples include humans, dogs, cats, rabbits, hamsters and the like.
  • the use of the aforementioned polypeptide (preferably Semaphorin 3A) of the present invention from the same species as the patient to be prevented, inhibited, or treated is considered to be more effective in preventing, inhibiting, or treating it, and from the viewpoint of safety in clinical application Also expected. Therefore, for example, when the target of administration of the preventive, suppressive or therapeutic agent of the present invention is a human, it is particularly preferable to include the above-mentioned active ingredient polypeptide b as an active ingredient for preventive, suppressive or therapeutic agents.
  • the preventive, suppressive or therapeutic agent of the present invention for ophthalmic diseases can be used by administering the cornea to be prevented or the corneal pathology to be treated.
  • As the administration method topical administration (administration of eye drops on the cornea, administration of subconjunctival injection, etc.) and the like can be mentioned.
  • Dosage forms include contact lens types, eye ointments, injections, eye drops, etc.
  • the aforementioned polypeptide of the present invention preferably Semaphorin 3A
  • the aforementioned polypeptide of the present invention can be contained in a transparent film having a contact lens-like spherical surface, and the aforementioned polypeptide of the present invention (preferably Semaphorin 3A) released from the film can be administered In the cornea.
  • the dosage of the aforementioned polypeptide (preferably Semaphorin 3A) of the present invention can be appropriately selected according to symptoms, age, weight, administration method, etc., and is not particularly limited.
  • the unit dose of the aforementioned polypeptide (preferably Semaphorin 3A) of the present invention is about 6000 to 60000 U, preferably about 4000 to 40,000 U, and more preferably about 500 to 5000 U.
  • the dose of eye drops may be about 1 to 3000 ng, about 20 to 900 ng, about 50 to 900 ng, about 50 to 500 ng, about 80 to 500 ng.
  • the concentration of the eye drops is about 10 ng/ml to about 1000 ng/ml, such as about 10 ng/ml, about 100 ng/ml, about 300 ng/ml, about 1000 ng/ml.
  • the above dosage forms and dosages are preferably once or several times a day, or every few days for several days to several months, depending on the degree of improvement of symptoms.
  • the amount of the aforementioned polypeptide (preferably Semaphorin 3A) of the present invention is usually about 500 to 5000 U per day, preferably about 500 to 5000 U, and for the test animal, it can be administered once or several times separately.
  • the amount of subconjunctival injection may be about 0.001 to 10 ng/day, about 0.05 to 5 ng/day.
  • the concentration is about 10 ng/ml to 1000 ng/ml, for example, about 10 ng/ml, about 100 ng/ml, about 300 ng/ml, about 1000 ng/ml.
  • the administration may be carried out regularly, once or several times a day, or once or several days a day, for several days to several months.
  • the aforementioned polypeptide (preferably Semaphorin 3A) of the present invention can also be prepared into the following dosage forms:
  • the dosage form is eye drops for the treatment of dry eye, keratitis, conjunctivitis, conjunctival lymphoma, angiogenesis inhibition, corneal chemical burn, corneal epithelial healing, meibomian adenocarcinoma: ,
  • the active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the concentration of the active ingredient is about 100-300ng/ml.
  • the dosage form is eye drops, which are used to treat angiogenesis inhibition, corneal chemical burn, conjunctival lymphoma, corneal epithelial healing, and meibomian adenocarcinoma: among them,
  • the active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the active ingredient concentration is: about 100ng/ml
  • the dosage form is eye drops for the treatment of dry eye, keratitis, conjunctivitis, conjunctival lymphoma, angiogenesis inhibition, corneal chemical burn, corneal epithelial healing, and meibomian adenocarcinoma: among them,
  • the concentration of the active ingredient in the eye drops is about 300ng/ml
  • 5ml/bottle containing the aforementioned polypeptide (preferably Sema3A) 1500ng/bottle;
  • the dosage form is an eye ointment, which is used to treat refractory dry eye, keratitis, conjunctivitis, conjunctival lymphoma, corneal epithelial healing, angiogenesis inhibition, corneal chemical burn, meibomian adenocarcinoma:
  • the active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • the dosage form is a corneal soft contact film, which is used to inhibit inflammation and pathological vascular and lymphangiogenesis and normalize its function, repair corneal chemical burn scars, and maintain normal corneal function;
  • the corneal soft contact medicinal film is also used for the treatment of chronic conjunctivitis, refractory dry eye, electrooptic ophthalmia, hypertonitis, corneal chemical burn, corneal laser burn, Stevens Johnson disease inflammation, cannibalism Keratitis, meibomian adenocarcinoma and other refractory corneal, conjunctival, and strong membrane eyelid diseases:
  • the active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • Diameter 13.5mm-14.5mm; Thickness: 0.01mm-0.09mm.
  • the corneal soft contact medicinal film has excellent moisturizing, water-absorbent material and viscoelasticity, and helps the corneal soft contact medicinal film to play a quick-acting and long-acting effect.
  • the dosage form is an injection, which is used to inhibit inflammation and pathological blood vessels, lymphangiogenesis and normalize its function, and to repair corneal chemical burn scars:
  • the active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • the injection is used to reduce inflammatory cells, control symptoms, normalize tissue function, inhibit diseased blood vessel/lymphangiogenesis, and normalize blood vessel/lymphatic function.
  • the injection is also used for the following inflammation-related diseases:
  • the dosage form is an oral liquid, which is used to treat allergy and inflammation of the respiratory tract and restore the normal function of the mucous membrane:
  • the active ingredient is: Sema3A;
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • the aforementioned polypeptide (preferably Sema3A) is 0.05% to 0.15% per mouse.
  • the dosage form is a mouthwash, which is used to treat allergies and inflammations of the oral cavity and respiratory tract, and restore the normal function of the mucous membrane:
  • the active ingredients are: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • the aforementioned polypeptide (preferably Sema3A) is 0.05% to 0.15% per mouse.
  • the dosage form is a drug film adhesive sheet, which is used for the treatment of mucosal diseases, skin diseases, burns/traumas and other wounds:
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • the medicinal film adhesive sheet includes: 1 oral adhesive sheet, 2 gynecological mucosal adhesive sheet; 3 mechanical trauma, chemical trauma, ulcer and other adhesive sheets.
  • the oral adhesive tablet is prepared by the following method: using the mucosal adhesive carbomer (CP934), sodium alginate, and low-viscosity sodium carboxymethyl cellulose (SCMC), the Sema3A oral adhesive tablet is prepared by direct compression. .
  • CP934 mucosal adhesive carbomer
  • SCMC low-viscosity sodium carboxymethyl cellulose
  • the dosage form is a cream, which is used to treat mucosal disorders, skin disorders, burns/wounds and other wounds:
  • the active ingredients are: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • the dosage form is a suppository, which is used to treat gynecological diseases, hemorrhoids:
  • the active ingredients are: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • the dosage form is a dressing for wound care:
  • the active ingredients are: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • 1Hydrogel wound dressings hydrogel wound dressings for treating laser burns and burns, and medical colloidal dressings with antibacterial and healing functions;
  • the dosage form is a spray, wherein:
  • the active ingredients are: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the active ingredient concentration is: about 100 ⁇ 300ng/ml
  • the sprays can be divided into the following categories: nasal sprays; oral sprays; gynecological sprays: a biological preparation used to treat gynecological inflammation.
  • the dosage form is a coating agent, which is used for the treatment of mucosal diseases, skin diseases, burns/traumas and other wounds:
  • the active ingredients are: the aforementioned polypeptide (preferably Semaphorin 3A);
  • the concentration of the active ingredient is: 100-300ng/ml.
  • the prophylactic, inhibitor, or therapeutic agent of the present invention can be used alone or in combination with other prophylactic, inhibitor, or therapeutic agents.
  • Other preventive, inhibitory or therapeutic agents include antibiotics, anti-inflammatory agents, antiviral agents, cell growth promoters, wound healing agents, extracellular matrix components, vitamins and the like.
  • Semaphorin 3A/hyaluronic acid ointment such as Semaphorin 3A/hyaluronic acid ointment, Semaphorin 3A/neomedrol EE ointment, Semaphorin 3A/Oders Linderon ointment, Semaphorin 3A/Touou Ma ointment 0.1%.
  • Semaphorin 3A/hyaluronic acid ointment can be used as a preparation for hyaluronic acid ophthalmic surgery.
  • Neomedrol EE ointment is a combination drug of antibiotics and synthetic adrenal cortex hormones. It can be used in combination with Semaphorin 3A to expect high anti-inflammatory effects. The same is true for the Linderon A ointment mentioned above.
  • the components of adrenocortical hormone can be appropriately reduced from the components in neomedrol EE ointment and administered.
  • Semaphorin acts gently in the body.
  • the administration of Semaphorin can activate the division ability without causing significant morphological changes of corneal endothelial cells, the preventive, inhibitory or therapeutic agent of the present invention is unlikely to cause side effects.
  • the present invention provides a method for preventing, inhibiting or treating corneal diseases or corneal damage, including the step of administering Semaphorin to a mammal.
  • the present invention provides Semaphorin 3A for preventing, inhibiting or treating corneal diseases or corneal damage.
  • the present invention provides the use of Semaphorin 3A in the preparation of a medicine for preventing, inhibiting or treating corneal diseases or corneal damage.
  • Figure 1 is a schematic diagram of the biological fluorescein staining for animal models according to the present invention, wherein the upper two images are observation images of corneal injury, and the lower is a schematic diagram, where (a) is the animal biological fluorescein staining of the corneal alkali burn model The photo under indoor light; (b) is the photo under the blue free filter of the slit lamp.
  • Figure 2 is a photograph of the whole corneal epithelium after scratching and staining with fluorescein after an alkaline corneal burn model is made.
  • Figure 3 is a comparison photograph of clinical symptoms of epithelial deficiency.
  • Figure 4 is an enlarged view of the photo under the microscope of the PBS administration control group 14 days after treatment.
  • Figure 5 shows the evaluation of corneal epithelial damage. The symptoms of the 100ng/ml and 1000ng/ml treatment groups were significantly improved.
  • Figure 6 is an evaluation of corneal angiogenesis, and the results show that Sema3A inhibits the formation of new blood vessels in a concentration-dependent manner.
  • Fig. 7 is a photograph of CD31 antibody and LYVE-1 antibody immunostaining of corneal pavement 14 days after debridement.
  • Figure 8 shows that Sema3A effectively inhibited the pathological angiogenesis of the cornea after alkali burn.
  • FIG. 9 shows that Sema3A effectively inhibited pathological lymphangiogenesis after corneal alkali burn.
  • Figure 10 is an immunostaining photograph of inflammatory cell infiltration in corneal tissue. (Photo of ⁇ IIItubulin and F4/80 immunostaining of corneal tissue).
  • Figure 11 shows the evaluation of the number of F4/80 antibody positive cells (macrophages).
  • Figure 12 is a photograph of nerve fiber immunostaining in a corneal section.
  • Figure 13 is the evaluation of corneal ⁇ IIItubulin-positive nerve fiber density.
  • the purpose of this example is to verify the polypeptides of the present invention, especially the angiogenesis/lymphangiogenesis of Semaphorin 3A, and diseases related to abnormal vascular endothelial dysfunction, especially cancer, ophthalmological diseases, arteriosclerosis, cardio-cerebrorenal vascular diseases, Prevent, inhibit and/or treat diseases such as Alzheimer's disease, aging disease, scar formation, etc.
  • the corneal alkali injury model was made by dripping 2ml 0.15M NaOH, and the following inspections were performed. The drug is administered once every other day and lasts for 7 days from the day when the alkaline damage occurs.
  • Semaphorin 3A (2 ⁇ l) manufactured by LSBio, was administered subconjunctivally on the 0th, 3rd, 6th and 9th days after surgery.
  • the control group was given PBS solution under the conjunctiva.
  • the experimental animals were randomly divided into the following groups, each with 8 animals:
  • a slit lamp blue free filter (blue no filter) is used, which selectively transmits only the green fluorescent color emitted by fluorescein, so an observation image of corneal damage can be obtained.
  • Fluorescein staining test Use fluorescein staining to observe the condition of the cornea. If there is staining, it means that the condition of corneal erosion and corneal epithelium is unstable.
  • This test is used to examine corneal ulcers (corneal wounds) by using the properties of osmotic epithelial defects and weak adhesion areas between epithelial cells.
  • Corneal opacity Five grades with a score of 0-4 according to the degree of statistics.
  • Grading of intracorneal neovascularization The length and extent of the invasion into the cornea are classified into five grades from 0 to 4 according to the degree.
  • Corneal epithelial defect area Since epithelial defects are usually elliptical, they are evaluated by the ratio of the long axis of the defect area to the corneal diameter.
  • the drug group and the control group were compared with the unpaired Student's test. **P ⁇ 0.01, ***P ⁇ 0.001; ns: no significant difference.
  • the corneal opacity scoring system is: neovascularization (NV) and corneal epithelial defect area. Defining the scoring system to describe the degree of corneal opacity (0-4) and NV (0-4) in semi-quantitative terms. On days 2, 5, 6, 10, and 14 after the corneal injury model was made, the blind observer assigned a opacity score for each corneal opacity:
  • the blood vessel length is less than half of the corneal radius
  • the blood vessel length is less than half of the corneal radius in two or more quadrants
  • the test was used to statistically compare the clinical scores of the untreated damaged cornea (control) and the Sema3A-treated cornea at each time point.
  • Figure 1(a) is a photograph of a corneal alkali burn model taken under indoor light.
  • the white broken line in Figure 1(a) is the corneal area. After the corneal alkali burn, the corneal epithelium falls off.
  • the black solid line frame is the accumulation domain of epithelial tissue degeneration and shedding after burn.
  • Figure 1(b) is a photograph of fluorescein stained after alkaline corneal burn and taken under a slit lamp blue free filter.
  • the white broken line in the figure is the corneal area, and the arrow points to the corneal epithelial defect area where the white part of fluorescein is attached.
  • Figure 2 shows a photograph of fluorescent staining after scratching the whole corneal epithelium. As shown in Figure 2, the area of the white broken line is the corneal area. After debridement, the epithelial tissue on the corneal surface is completely peeled off, the whole cornea is colored with fluorescein, and the whole cornea becomes cloudy like ground glass.
  • Figure 3 shows a comparison of the clinical symptoms of corneal epithelial deficiency.
  • the corneal repair changes in the treatment group and the control group were observed, and the epithelial under-damage was stained with fluorescein, and the photos were taken under the microscope.
  • corneal alkali burn animal model after 2 weeks of observation the first 1, 3, 5, and 7 photos show the changes in the clinical symptoms of the corneal epithelium on the 3rd, 6th, 9th, and 14th day under indoor light;
  • the 8 rows of photos show the changes in the clinical symptoms of the corneal epithelium on the 3rd, 6th, 9th, and 14th day of the slit lamp blue free filter (under the blue no filter) after fluorescein staining.
  • Fig. 4 is an enlarged view of the photo under microscope of the control group administered with PBS in Fig. 3 after 14 days of treatment.
  • the area of the white broken line in Figure 4(a) is the area of corneal tissue, and the arrow indicates corneal neovascularization after alkali burn. Thick blood vessels affect the perspective of the cornea, and the visual acuity of the animal model drops sharply.
  • the area of the white broken line is the area of corneal tissue, and the white arrow indicates that the total corneal degeneration after alkali burn shows white spots and is opaque.
  • the range of the white solid line is the area of corneal degeneration scar proliferation, and the black arrow indicates the tissue degeneration, hyperplasia and hypertrophy, showing an opaque state. Animal models have high vision loss or blindness.
  • Figure 5 shows the evaluation of corneal epithelial damage.
  • the corneal repair changes, fluorescein staining, and clinical points were observed (Figure 5).
  • the degree of corneal epithelial defect is scored into five levels:
  • the concentration depends on the changes of clinical symptoms.
  • the symptoms of the 100ng/ml and 1000ng/ml treatment groups improved significantly.
  • the treatment group with a concentration of 100ng/ml had a significant effect on the 6th day (**P ⁇ 0.01); there was a significant difference on the 14th day (*P ⁇ 0.05).
  • the corneal burn tissue of the treatment is significantly repaired, the corneal epithelium is healed well, and the cornea in the treatment group at a concentration of 100ng/ml is not different from the normal cornea.
  • Figure 6 shows the evaluation of corneal angiogenesis. At 2, 5, 6, 10, and 14 days after alkaline trauma, the assessment is divided into five stages according to the grading standard of the length and degree of neovascularization.
  • Neovascularization The length and extent of its invasion of the cornea are scored on a scale of 0 to 4.
  • New blood vessel and lymphatic vessel scoring system methods are as follows:
  • the blood vessel/lymphatic vessel length is less than half of the corneal radius
  • Sema3A inhibited the formation of new blood vessels in a concentration-dependent manner.
  • 100ng/ml Sema3A showed inhibition of cardiovascular formation on the 4th day; there was a significant difference on the 10th day (**P ⁇ 0.01), which was significantly inhibited by the administration of this treatment The formation and growth of new blood vessels in the cornea.
  • Figure 7 shows that 14 days after debridement, CD31 antibody (vascular endothelial marker) and LYVE-1 antibody (lymphatic endothelial marker) were used to immunostain corneal tiles.
  • the blood vessels and lymphatic vessels of the treatment group and the control group with Sema3A concentrations of 10ng/ml, 100ng/ml, and 1000ng/ml were immunostained. Quantitative evaluation is performed using the percentage of the area of blood vessels and lymphatic areas.
  • the PBS-treated control group had a large area of vascular and lymphatic angiogenesis.
  • the Sema3A concentration of 100ng/ml treatment group significantly inhibited the vascular and lymphatic angiogenesis, and the central part of the cornea maintained normal transparency. . It shows that Sema3A can effectively promote epithelial formation.
  • Figure 10 is a photograph of ⁇ IIItubulin and F4/80 immunostaining of corneal tissue (day 14 after debridement). It demonstrates the infiltration of inflammatory cells in the corneal tissue.
  • Triangular white arrows indicate immunostaining positive inflammatory cells
  • Medium B Anti-F4/80 antibody is used for mouse macrophage detection.
  • the infiltration of F4/80 positive cells (macrophages) in the corneal tissue of the Sema3A treatment group at a concentration of 100ng/ml was significantly reduced compared with the control group. It shows that Sema3A inhibits the inflammatory response that occurs after alkaline trauma of the cornea.
  • Figure 11 shows the evaluation of the number of F4/80 antibody-positive cells (macrophages).
  • Figure 12 shows an immunostained photograph of nerve fibers in a corneal section.
  • the corneal sections were immunostained with anti- ⁇ III tubulin antibody and PGP9.5 (neural marker).
  • the white arrow indicates the part of the nerve fiber.
  • the corneal nerve fibers in the cornea and the corneal tissues of the control group and the treatment group were stained by immunofluorescence staining.
  • Figure 13 quantifies the effect of the corneal sensory nerve (trigeminal nerve). Use ⁇ IIItubulin-positive nerve fiber density evaluation.
  • the corneal nerve fiber area was quantified.
  • the corneal nerve fibers were quantified as the percentage of the positive threshold area for ⁇ -tubulin III staining in the representative confocal image shown in the figure.
  • corneal epithelial defects corneal neovascularization
  • corneal neovascularization corneal neovascularization
  • inflammatory cell infiltration was significantly inhibited, and it was compared with normal mice with ⁇ -III tubulin-positive nerve fibers. Compared with the cornea, there is no reduction.
  • Semaphorin3A effectively promotes epithelialization, inhibits pathological angiogenesis, and inhibits inflammation that occurs after alkaline trauma to the cornea.

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Abstract

一种用于血管新生/淋巴管新生，血管内皮机能异常相关疾病，尤其是癌症、眼科疾病、动脉硬化、心脑肾血管疾患、阿尔茨海默病、老化性疾病、瘢痕形成等疾病的预防、抑制和/或者治疗或促进现有治疗药物的代谢和速效性的辅助制剂的多肽或者包含该多肽作为活性有效成分组成的片段，以及其在制备用于预防、抑制和/或者治疗血管新生疾病、淋巴管新生相关疾病的药物中的用途。尤其是在癌症相关的疾病、动脉硬化、阿尔茨海默病、瘢痕形成、老化相关的预防和治疗中的用途，特别是在眼科血管新生相关的疾病的预防和治疗中的用途。

Description

一种用于血管新生、淋巴管新生相关疾病的多肽及其用途 技术领域
本发明涉及含有Semaphorin蛋白(Semas)、其药学上可接受的成分作为活性有效成分的预防、抑制和/或者治疗剂，以及其在制备用于预防、抑制和/或者治疗血管新生疾病、淋巴管新生相关疾病的药物中的用途。尤其是在癌症相关的疾病、瘢痕形成、心血管疾病，器官老化性疾病、阿尔茨海默病的预防和治疗中的用途，特别是在眼科血管新生相关的疾病的预防和治疗中的用途。
背景技术
人体细胞是靠摄取细胞周围血管供应的营养和氧气来维持其活动和功能。必要细胞的数量也严格受人类原始功能的控制，然而，癌细胞无法控制并且增殖非常活跃。由于与正常细胞相比，进行此类活动的癌细胞需要大量的营养和氧气，因此开始制造新的血管即新生血管。癌细胞产生的新血管已成为其活动的支持来源。
血管新生是利用已经存在的脉管系统形成新血管，如伤口愈合，癌症疾患，老化性黄斑变性，肿瘤生长和糖尿病视网膜病变等在内的许多生理过程，病理状况中起着至关重要的作用。
与血管新生相关的癌症约有以下：乳腺癌(特别是转移性乳腺癌)、大肠癌、结直肠癌、进行性结肠直肠癌、食道癌、浅表型食管癌、早期食道癌、喉头癌、副鼻腔恶性肿瘤、胃肠道间质瘤(GIST)、肾(肾细胞)癌、肝脏(成人原发性)癌症(特别是不能实施手术的肝癌)、淋巴瘤、淋巴管瘤病/Gorham病、巨大的淋巴管畸形、黑色素瘤(恶性黑色素瘤)、肺癌、非小细胞肺癌(NSCLC)、转移性非小细胞肺癌、卵巢癌、上皮性卵巢癌、胰腺癌、前列腺癌、胃癌、鼻腔肿瘤、妇科恶性肿瘤、子宫颈癌(特别是早期浸润癌)、子宫内膜癌、膀胱癌(浅表型膀胱癌、浸润性癌)。
最近的研究表明，包括乳腺癌，子宫内膜癌，结肠癌，膀胱癌，这些血管新生因子的表达被识别出来，其表达程度与预后之间有直接相关性。此外，乳腺癌肿瘤组织中的血管新生密度与转移相关，而食道癌，结肠癌等，血管新生则是影响癌症患者预后的重要因素。而乳腺癌转移至淋巴结的概率是最高的。所以抑制淋巴管增殖对抑制乳腺癌转移有很重要的作用。食道癌无论进展程度如何，都可能扩散到淋巴结。前列腺癌最常见的是转移到前列腺周围的淋巴结。
癌细胞如果转移到淋巴结，就可能通过淋巴管向远端转移。而转移到肝脏的困难点是最初几乎不会出现自觉症状，非常容易重症化。已经发生转移的癌症治疗很困难，因此早期预防、早期治疗极其重要。
目前，用于人类癌症治疗的血管新生抑制剂是美国食品和药物管理局(FDA)批准的几种抗血管新生活性药剂，可以延迟肿瘤生长，但最近的研究结果在动物研究中显示了先天性异常的可能性，已经明确有并发症的可能性。
血管生成抑制剂治疗副作用包括出血，动脉血栓形成(引起中风或心脏病发作)，高血压和尿蛋白(3-5)。消化道穿孔和胃肠瘘也被认为是某些血管生成抑制剂的副作用。
比如抗VEGF抗体的Bevacizumab(商品名AVASTIN)由于有出血风险，不能用于鳞状细胞癌 和被确诊肿瘤渗入大血管的患者，也不能用于既往病史有出血风险高的患者。而对于脑转移的患者，由于肿瘤病变有出血的风险，也无法使用。
另外，现有的抗VEGF制剂和化疗药联合疗法，进行维持治疗的仍存在高药价的财务问题，即使维持治疗，联合使用高价药阿瓦斯汀和培美曲塞治疗很多患者也无法承受。
血管新生疾病还包括：瘢痕瘤、类风湿性关节炎(RA)、银屑病(牛皮癣)、动脉硬化、结节性硬化症、阿尔茨海默病如血管性阿尔茨海默、老化、急性心筋梗塞、巨细胞病毒和衣原体感染诱发的冠状动脉病变。
由于生活方式疾病和超老龄化社会，动脉硬化相关疾病正在迅速增加。根据卫生、劳动和福利部的重要统计数据，脉硬化相关疾病(中风，心肌梗死)死因占死亡率的三分之一左右和恶性肿瘤死因相匹敌。然而，因为在大多数情况下直到发病都没有症状，等到发现时为时已晚，所以比如提早投入本药剂，预防疾病的发生极其重要。
血管内皮机能障碍初期是由生活习惯病(肥胖，压力，吸烟，缺乏运动等)和高血压、血脂异常、糖尿病、睡眠呼吸暂停综合症、慢性肾病等疾病，及衰老、性别、更年期、遗传等使血管内皮细胞受损引起的。血管机能瘫痪促使心血管疾病的发生，肥胖等使血管变窄发硬，血液中的胆固醇和中性脂肪增加并沉积，加剧动脉硬化发展。引起心绞痛和肾功能障碍，血栓形成后引起脑梗塞和心梗塞，血管破裂引起脑出血。老化进展时的动脉开始硬，引起严重的脑梗塞和心肌梗塞。
这些疾病中，例如银屑病(牛皮癣)，尽管持续治疗很长时间，但改善并不容易，目前没有基本的治疗方法；而目前RA的治疗也是依靠血管新生抑制剂；结节性硬化症的疾病与LAM病伴随发生时，目前以美国为中心，使用名为西罗莫司的药物，因为有各种副作用，对其效果没有一定的意见。肾肿瘤和肺LAM病情严重时通常涉及生命预后。
眼科血管新生疾患包括：糖尿病性血管增值性眼底病变、网膜血管肿、von Hippel-Lindau病、体表软血管瘤、黄斑变性、近视性脉络膜新生血管、糖尿病网膜症、未成熟儿视网膜症、新生血管性青光眼、糖尿病黄斑症、网膜静脉闭塞症、葡萄膜炎合并的眼内新生血管、Behcet病、结核性葡萄膜炎、高度近视的脉络膜新生血管、恶性淋巴瘤，结膜恶性淋巴瘤、脉络膜肿瘤、巨细胞病毒性疾患例如巨细胞病毒性视网膜炎、眼内的恶性肿瘤例如视网膜母细胞瘤、脉络膜恶性黑素瘤、眼球中出现的恶性淋巴瘤、眼附属器肿瘤例如视神经肿瘤。
外伤性、化学烧伤性角膜血管新生，目前没有药物可以治疗，唯一的办法是角膜移植；血管新生性青光眼随着视网膜新血管形成的进展而发展，并在整个眼球中扩散，纤维小梁束被新生血管覆盖，导致房水的流出阻力使眼压升高。降低眼压的方法有静脉滴注和内科治疗，但无论如何效果只是暂时的。激光治疗导致视网膜所需的血液量减少，血液不足的状况。如果不治愈阻塞房角的新生血管，眼压几乎不会下降。在这个阶段只能进行青光眼手术，但新生血管性青光眼的手术效果不佳。
结核性葡萄膜炎的眼内炎症通过抗结核治疗和口服皮质类固醇治疗新血管。皮质类固醇的副作用引起高眼压，青光眼，白内障，角膜真菌发病等，而且影响小儿发育。
病理性近视眼脉络膜新生血管的治疗，视网膜光凝固，疗效不佳，并发症频发。黄斑移位术，新生血管摘除术等手术治疗效也没有有效数据。目前临床的抗VEGF注射药物治疗，有可能伴随视力恶 化。而且副作用很难避免，眼内炎，中风，白内障、视网膜脱离、视网膜色素上皮裂孔等，国内外在临床试验中已有中风报道。高额治疗费也是目前的抗VEGF注射药物的缺点。
巨细胞病毒性视网膜炎的治疗是口服和静脉输注药物，但现有药物时常引起骨髓抑制和肾功能不全等原因使得治疗困难。
瘤病/Gorham病临床用类固醇，干扰素α，普萘洛尔，化疗(长春新碱)等，但治疗效果有限。如果有胸部病变，如乳房，预后不好。病变如果扩散到多个器官，引起各种症状，经常留下永久性疾病，如慢性呼吸衰竭和运动功能障碍。许多病例需要长期治疗，治愈率极低。
任何治疗都很难恢复巨大的淋巴管畸形(颈部面部病变)。它不仅对治疗有抵抗力，而且导致持续的功能障碍(呼吸障碍，进食/吞咽障碍，视力障碍，听力障碍等)，从出生那一刻起，就需要终生就医。
然而直到目前，仍无报告明确指出Sema3A在血管新生疾病、淋巴管新生疾病相关疾病，尤其是在癌症、瘢痕形成和眼科疾病中的作用及功能。
发明内容
本发明提供了一种用于血管新生、淋巴管新生相关疾病，尤其是癌症、眼科疾病、动脉硬化、心血管疾病，老化性疾病、瘢痕形成，阿尔茨海默病等疾病的预防、抑制和/或者治疗的多肽或者包含该多肽作为活性有效成分组成的片段，以及其在制备用于预防、抑制和/或者治疗血管新生疾病、淋巴管新生相关疾病的药物中的用途。尤其是在癌症相关的疾病、动脉硬化、阿尔茨海默病、瘢痕形成的预防和治疗中的用途，特别是在眼科血管新生相关的疾病的预防和治疗中的用途。
本发明还涉及一种多肽预防、抑制和/或者治疗血管新生、淋巴管新生相关疾病，尤其是癌症、眼科疾病、动脉硬化、心血管疾病，老化性疾病、瘢痕形成，阿尔茨海默病等疾病的方法。
本发明还涉及一种多肽，其用于预防、抑制和/或者治疗血管新生、淋巴管新生相关疾病，尤其是癌症、眼科疾病、动脉硬化、心血管疾病，老化性疾病、瘢痕形成，阿尔茨海默病等疾病。
在一个实施方案中，上述多肽选自以下(a)至(c)中的任何一种多肽：
(a)由序列编号4所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号4中166aa～235aa区域的多肽；
(b)由序列编号2所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号2中166aa～235aa区域的多肽；
(c)与(a)或(b)的多肽具有80％或更高序列同源性的多肽。
本发明技术方案的具体实施方式及其含义将在下文中进行详细说明。
定义
除非另有说明，本文使用的所有技术和科学术语具有与本发明所属领域的技术人员通常理解相同的含义，但如有冲突，则以本说明书中的定义为准。
如说明书和权利要求书中所用，单数形式“一”、“一个”和“该(所述)”包括复数形式，除非上下文 另有明确说明。
如无特殊说明，本说明书中的百分比(％)均为重量百分比(重量％)。
在说明书和权利要求书中使用的涉及组分量、工艺条件等的所有数值或表述在所有情形中均应理解被“约”修饰。术语“约”当指数量或数值范围时，意思是所指数量或者数值范围是试验变异性内(或统计学实验误差内)的近似值，因此该数量或者数值范围可以在所述数量或数值范围的例如±5之间变化。
涉及相同组分或性质的所有范围均包括端点，该端点可独立地组合。由于这些范围是连续的，因此它们包括在最小值与最大值之间的每一数值。还应理解的是，本申请引用的任何数值范围预期包括该范围内的所有子范围。
当本发明针对物理性质例如分子量或者针对化学性质范围时，应包括范围的所有组合和亚组合以及其内的具体实施方式。术语“包含”(以及相关术语例如“含有”或“含”或“具有”或“包括”)包括这样一些实施方式，该实施方式为例如，物质、组合物、方法或过程等的任何组合，其“由所描述的特征组成”或者“基本上由所描述的特征组成”。
本说明书和权利要求中使用的“和/或”，应当理解为相关联的组分“二者择一或二者”，即组分在一些情况中联合存在而在另一些情况中分开存在。多个用“和/或”列出的组分应当以同样的方式理解，即“一种或多种”相关联的组分。除了“和/或”从句具体确定的组分，其它组分可任选地存在，无论与那些具体确定的组分相关还是不相关。因此，作为非限制性实例，提及“A和/或B”，当用于连接开放式结尾的文字如“包括”，在一个实施方案中，可仅指A(任选地包括除B外的组分)；在另一实施方案，可仅指B(任选地包括除A外的组分)；在再一实施方案中，指A和B(任选的包括其它组分)等。
本说明书和权利要求书中所用的“或”，应当理解为和上述定义的“和/或”具有相同的含义。例如，当在列表中分隔项目时，“或”或“和/或”应当译为包括在内的，即，包括多个或列表组分中的至少一个，但也包括多于一个，和任选的其它未列出的项目。只有术语明确地指向对立面，如“仅一个”或“正好一个”，或在权利要求书中使用的“由…组成”应当指的是包括多个或列表组分中的正好一个。通常，本文所用的术语“或”仅仅当有排他性的先行词如“要么”“之一”“仅一个”或“正好一个”时才被认为指向排他性的选择(即，一个或另一个但不是两者)。
应当理解，除非明确地相反指示，否则在本文要求保护的包括多于一步或一个行为的任何方法中，该方法的步骤和行为的顺序不必限制于所叙及的方法的步骤和行为的顺序。
本发明使用的缩写具有在食品、生物学和化学领域的通常含义。
A.血管新生疾病
血管新生(angiogenesis)：主要在原有的毛细血与(或)微静脉基础上通过血管内皮细胞的增殖和迁移，从先前存在的血管处以芽生或非芽(或称套叠)的形式生成新的毛细血管。
具体疾病如下所示例：
类风湿性关节炎(RA)
RA是一种慢性炎症性疾病，多见侵入所有关节，RA的炎性滑膜有早期毛细血管，小血管如后毛细血管小静脉显着的新生，并且是炎症细胞浸润的部位，新生血管还作为滑膜的营养血管深入参与 疾病病态。目前，RA的治疗也是依靠血管新生抑制剂。
银屑病(牛皮癣)
Nickoloff等在银屑病患者的病变部，观察到与正常皮肤相比IL-8(血管生成因子)和TSP-1的产生减少，根据病变部位组织建立的培养表皮细胞方法，从大鼠角膜中确认了血管新生增强。
动脉硬化
动脉硬化伴随高脂血症，高血压，衰老等病程进展。血管(vasa Vasorum)在动脉粥样硬化斑块附近可以观察到新血管伸展。在形成动脉粥样硬化病变时，分泌PDGF，TNF-α，FGF和TGF-β因子，诱导血管新生。
血管内皮机能障碍出现后，动脉硬化发生，血管变窄发硬，血液中的胆固醇和中性脂肪增加并沉积在血管内部，加剧动脉硬化发展。
血管变窄发硬后引起心绞痛和肾功能障碍，血栓形成后引起脑梗塞和心梗塞，血管破裂引起脑出血。
结节性硬化症
结节性硬化症(tuberous sclerosis complex：TSC)是肾血管肌肉脂肪瘤；
淋巴管平滑肌瘤病(lymphangioleiomyomatosis：LAM)是引起全身性错构瘤的疾病，例如面部和眼底血管纤维瘤。肾脏血管肌肉脂肪瘤有许多血管新生，增大有时会导致破裂。随着肿瘤的细胞增加，可能会发生恶变。
结节性硬化症的疾病与LAM病伴随发生时，其中大多数会在育龄妇女中发病，LAM以外的疾病很可能发生在许多器官(脑，皮肤，心脏，肺，肾等)，伴随癫痫，皮肤病变和其他结节性硬化症症状。
癌症
鼻腔肿瘤
鼻窦肿瘤是由鼻窦组织(主要是粘膜上皮)引起的肿瘤。代表性的鼻窦肿瘤良性肿瘤是乳头瘤和血管瘤。在恶性肿瘤中，鳞状细胞癌是最常见的，除此之外，鼻腔还产生各种病理组织类型的恶性肿瘤，包括嗅神经母细胞瘤。例如乳头瘤的临床特征包括高复发率和恶性转化的可能性。复发率约为30％，大多数在术后2年内复发。恶性转化被认为是约10％，通常转化为鳞状细胞癌。
妇科恶性肿瘤
妇科恶性肿瘤最多见的是宫颈癌，胎盘血管肿，妊娠合并的宫颈海绵状血管瘤。
发生在脑脊髓海绵状血管瘤，血管新生深度参与对预后影响最大的转移性病变的生长。
阿尔茨海默病
血管性阿尔茨海默病
痴呆与血管密切相关，血管阻塞导致血管性痴呆和阿尔茨海默病，症状改善需要恢复脑血流量。
阿尔茨海默病(AD)是痴呆症之一。病因被认为是β淀粉样蛋白(Aβ)在大脑中的积累。基础和临床试验中的免疫疗法显示减少淀粉样蛋白沉积对认知功能的改善是有用的。用于从脑中去除Aβ，针对Aβ的抗体或疫苗免疫疗法被认为很有前景，但是诸如血管生成性水肿和脑微出血的副作用却很 明显。脑和血液之间存在的血-脑屏障的透过性亢进(BBB)，与脑血管内皮细胞密切相关，还发现AD患者的血管密度也在增加。
老化
遍布全身的血管不仅向身体的每个角落输送氧气和营养物质，而且还在发生组织损伤或炎症等异常时将炎症细胞移动到该部位，促进组织修复。在此过程中，覆盖血管腔的血管内皮细胞被TNFα(一种炎性细胞因子)激活，并帮助炎性细胞迁移出血管，并在正常引起炎症反应中起作用。
急性心筋梗塞
据报告急性心肌梗死患者的IL-6水平表现升高。血浆IL-6水平在梗塞发作后数小时内上升，并在24至48小时后达到峰值(图2)。在升高IL-6后，血液中的CRP升高。该过程中的最大IL-6值与最大CRP值显着相关。
巨细胞病毒诱导的冠状动脉疾病
巨细胞病毒和衣原体感染在冠状动脉病变形成中的作用已被注意。
巨细胞病毒直接作用于内皮细胞以增强IL-6的产生。Blankenberg等人提示巨细胞病毒抗体阳性且血液IL-6水平高的患者未来心脏病死亡的风险很高，但即使巨细胞病毒抗体是阳性，而血液IL-6水平也不高，患者也是没有风险的。
眼科血管新生疾病
损害眼部透明度的代表性病变是血管新生。新形成的血管脆弱，容易重复出血和渗出，形成增殖膜，伴有其他细胞增殖，因此视功能明显受损。外伤性，化学烧伤性角膜血管新生，目前没有药物可以治疗，唯一的办法是角膜移植。
眼内血管新生疾病，典型的例子包括糖尿病性视网膜病、视网膜静脉阻塞、早产儿视网膜病、年龄相关性黄斑变性、血管生成性青光眼等，都是导致失明的难治疾病。当视网膜处于血液不足状态时，将发出信号(称为VEGF的化学物质)通过VEGF(新血管形成)产生新的血管以滋养视网膜。这种“新生血管”非常易于破裂，是视网膜出血和玻璃体出血的根源。
新生血管性青光眼
新生血管性青光眼的病因包括糖尿病性视网膜病变症候群、视网膜中央静脉阻塞和眼缺血综合征。
视网膜血流恶化的疾病引起的严重且难治型的青光眼。当视网膜的血管被糖尿病损坏时，血液循环受阻，引起严重的视力障害。新生血管可能堵塞房角，闭锁房水的出口，引起突然升高。通常约20mmHg的眼压变为50mmHg或更高。
由恶性淋巴瘤、结膜恶性淋巴瘤的眼内转移，继发症是在眼球内经常发展成血管新生性青光眼。
糖尿病黄斑症
黄斑位于视网膜的中心，是观察事物最重要的部分。由于糖尿病疾患，黄斑附近经常出现毛细血管瘤等，血液成分渗出，黄斑肿胀引起的症状，被称为糖尿病黄斑症。
即使在初期的视网膜病变阶段，也可能发生导致视力下降。
在病情严重进展之前，可能没有自觉症状、早期使用新生血管抑制剂很重要。比如现在临床采用 防止血管新生为目的，无血管领域进行的激光凝固治疗，光凝网膜组织变性的凝固瘢会随着时间增长而面积扩大，以至于影响黄斑视力和视野。
网膜静脉闭塞症
高血压是动脉硬化的原因之一，但即使在没有高血压的情况下，动脉硬化也会发展，如果视网膜静脉的根部被阻塞，它就会变成视网膜中央静脉阻塞并在整个视网膜内出血。导致视力突然下降，视野障碍，变视症，玻璃体出血，视力明显下降。合并血管生成性青光眼。
葡萄膜炎合并的眼内新生血管
原田病引起大量视网膜和脉络膜脱离，视神经乳头水肿，后极部分乳头上新生血管，治疗需要大规模类固醇收缩新血管。
Behcet病引起视网膜和乳头上产生新生血管，治疗需要局部给予类固醇药物和用免疫抑制药物。
高度近视的脉络膜新生血管
近视性视网膜病变的患病率为1.7％，女性的患病率约为男性的两倍。如果眼球向后伸展并且视网膜/脉络膜受损，则会产生病理性血管(脉络膜新生血管)，导致出血和肿胀。
脉络膜肿瘤
脉络膜血管瘤，由血管异常增生引起，经常与全身血管瘤多发的Sturge-Weber综合征同时发症。转移性脉络膜肿瘤是在身体的所有器官中发生的血行转移性恶性肿瘤。女性乳腺癌和男性肺癌的脉络膜转移很多见。
如果肿瘤发生在黄斑区域视力的特别重要的部分，或肿瘤周围的视网膜脱离到达黄斑部，则会发生视力恶化。随着肿瘤的增长程度，视野会欠损以至于消失。
巨细胞病毒性疾患
巨细胞病毒性视网膜炎是在免疫功能低下的患者中，如艾滋病患者和接受抗癌治疗的患者，视网膜炎可以作为机会性感染发生。特别是，当外周血中CD4+T细胞的数量小于50/mm 2时，发病率很高。
眼内的恶性肿瘤
眼部分为眼球和眼附属器(眼睑，结膜，眼窝，泪腺等)，肿瘤可以发生在各个部位。预后不良甚至影响生命生存率。
视网膜母细胞瘤
儿童眼球中出现的恶性肿瘤，治疗外科手术，如果有进行性需摘除眼球。脉络膜恶性黑素瘤是恶性黑素瘤发生在成人的眼球的葡萄膜(脉络膜，睫状体，虹膜)中的恶性肿瘤，如果它有进展并且肿瘤很大，需要眼球摘除。
眼球中出现的恶性淋巴瘤
是中枢神经系统的恶性淋巴瘤，脑的淋巴瘤的亚型。一般进行全身化疗，放射治疗和其他医学治疗，目前很难完全治愈。虽然有些预防的治疗方法的报道，到目前为止还没有可靠的方法实际应用。
眼附属器肿瘤
眼睑肿瘤(皮脂腺癌)眼睑中出现的肿瘤在眼部肿瘤中很常见，复发时需再次手术或放疗。
泪腺癌治疗需手术完全切除肿瘤，然而，即使肿瘤被完全切除，也不可能阻止之后的复发。
眼附属淋巴瘤
眼球肉瘤肿瘤压迫眼球偏位，通常难以完全切除，需结合手术、化疗和放疗。髓膜肿是由包括视神经的鞘产生的肿瘤，切除肿瘤时，通常导致失明。
B.淋巴管新生疾病
淋巴管是淋巴液通过的通道，收集由身体各角落产生的淋巴液，并过滤淋巴液中含有的细菌和病毒，最后将其返回血管，淋巴管相关的疾病很多是疑难病症。
近年来，血管生成在个体发育和各种病理学如肿瘤和炎症中的分子机制得到了阐明，另一方面，关于相同血管器官中淋巴系统形成的分子机制的研究逐渐确定了淋巴管中特异性表达的受体、其配体、转录因子等，但针对它们的特异性抗体难以制备并且难以获得细胞系，这是推进研究的障碍。提示淋巴管不仅可作为细胞外液过多引流的循环系统，而且对免疫系统有很大影响，可明确恶性肿瘤淋巴结转移和淋巴水肿等病理状况是重要的。
淋巴管在维持体内微环境衡定、免疫反应和肿瘤淋巴转移等方面起着重要作用，淋巴管新生与胚胎发育、外伤修复、先天性淋巴水肿、炎症转归和肿瘤转移密切相关。在趋化因子和生长因子的作用下，淋巴管内皮细胞迁移、增殖和构成管腔，形成新的淋巴管。近年来发现淋巴管内皮祖细胞参与淋巴管新生。淋巴管内皮特异表达Prox-1、podopiann、VEGFR-3 LYVE-1等，这些因子和受体调控淋巴管新生。VEGF-C/VEGFR-3或VEGF-D/VEGFR-3信号途径在淋巴管新生过程中起着重要作用。VEGF-C、VEGF-D和VEGFR-3可作为基因治疗的靶点，有望治疗淋巴管新生障碍性疾病以及抗移植后免疫排斥和抗肿瘤淋巴管转移。
淋巴管瘤病/Gorham病(日本指定为难治性疾病277)
是一种难以治愈的疾病，不明原因导致弥漫性异常扩张的淋巴组织侵入全身器官，如骨骼和胸部(肺，纵隔，心脏)，腹部(腹腔，脾脏)，皮肤，皮下组织。许多在儿童和青少年时期引起骨质疏松症，Gorham病在病理学上识别出不规则扩张的淋巴管。淋巴管瘤病/Gorham病也被称为弥漫性淋巴管瘤病，Gorham-Stout综合征，也被称为大量骨质溶解症。
例如食管鳞状细胞癌的淋巴增殖，可以根据淋巴管新生并预测转移，在胃肠道肿瘤中，食管癌与结肠癌和胃癌相比具有高度恶性，是预后不良的肿瘤之一。
近年来，恶性肿瘤的生长和转移涉及血管和淋巴管的形成，推测血管生成与肿瘤生长有关，而肿瘤组织的淋巴管生成与淋巴结转移有关。筑後等对淋巴管分布和淋巴管密度进行了测定，显示了肿瘤区域和非肿瘤区域中淋巴管数量的比率。粘膜固有层在肿瘤边缘淋巴管数量有显著增加。淋巴管占血管数量的百分比也观察到淋巴管显著增加。
巨大的淋巴管畸形
(颈部面部病变)是巨大的肿瘤性淋巴管生成异常，在面部，口腔，咽喉头颈部先天发生。
淋巴管畸形(淋巴管瘤)由大小淋巴囊肿组成的肿块，同时有血管病变。眶内淋巴管瘤可能引起眶内出血或感染导致失明。如果淋巴病变扩展到皮肤和粘膜，表现局部淋巴管瘤，症状出现反复淋巴 瘘，出血，感染。在病程内任何部位病变都可能发生感染和出血，反复急性肿胀和炎症。
在巨大的面部病变中，由于肿瘤形成，变色和变形，表现出高度的丑陋性，影响患者社会生活的适应性，一生的生活质量受到限制。
C.多肽
Semaphorin3A(Sema3A)
由SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列的全长(即Semaphorin3A蛋白)。
氨基酸序列2：
Figure PCTCN2020120360-appb-000001
Figure PCTCN2020120360-appb-000002
Figure PCTCN2020120360-appb-000003
Figure PCTCN2020120360-appb-000004
氨基酸序列4：
Figure PCTCN2020120360-appb-000005
Figure PCTCN2020120360-appb-000006
Figure PCTCN2020120360-appb-000007
Figure PCTCN2020120360-appb-000008
多肽(a)：由序列编号4所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号4中166aa～235aa区域的多肽。
多肽(b)：由序列编号2所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号2中166aa～235aa区域的多肽。
多肽(c)：多肽是其中(a)或(b)的多肽的少量(优选1至几个)氨基酸残基被取代，缺失和/或插入的多肽。肽是与原始序列具有80％或更高，优选90％或更高，更优选95％或更高，还更优选98％或更高的同源性的多肽。
通常，在蛋白质中，即使蛋白质的氨基酸序列中的少量氨基酸残基被取代，缺失或插入，它们也可具有与原始蛋白质几乎相同的功能。这些是本领域技术人员所熟知的。因此，由上述(c)的多肽组成的区域也可以产生Semaphorin3A蛋白的生理活性，类似于上述70aa区域。
氨基酸序列的“同源性”：意味着比对两个氨基酸序列，使得待比较的两个氨基酸序列的氨基酸残基尽可能匹配，并以匹配氨基酸残基的数量除以氨基酸残基总数的数字表示为百分比。在对准时，根据需要将间隙适当地插入两个要比较的序列中的一个或两个中。
序列的这种比对可以使用已知程序进行，例如BLAST，FASTA，CLUSTAL W等。当插入缺口时，氨基酸残基的总数是将一个缺口计为一个氨基酸残基的残基数。当以这种方式计数的氨基酸残基总数在待比较的两个序列之间不同时，同源性(％)是较长序列中氨基酸残基的总数，它是通过除以计算得出的。
构成天然蛋白质的20种氨基酸是具有低极性侧链的中性氨基酸(Gly，Ile，Val，Leu，Ala，Met，Pro)和具有亲水侧链的中性氨基酸(Asn，Gln，Thr，Ser，Tyr Cys)，酸性氨基酸(Asp，Glu)，碱性氨基酸(Arg，Lys，His)，芳香族氨基酸(Phe，Tyr，Trp)已知它们可以组合在一起并且它们之间的取代通常不会改变多肽的性质。
因此，当用上述多肽(a)或(b)中的氨基酸残基取代时，可以通过在这些各自的基团之间取代来获得用作预防、抑制或治疗结膜中的角膜疾病的活性成分的多肽。或者，增加了维持角膜损伤的预防、抑制或治疗活性的可能性。
具体地，上述多肽(c)是与由SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列的全长(即Semaphorin3A蛋白)组成的多肽具有80％或更高序列同源性的多肽，或者与含有70个氨基酸区域的Sema3A蛋白质片段具有80％或更高序列同源性的多肽。
因此，活性成分多肽c是仅由这些氨基酸组成的多肽，或者是通过向多肽(c)的一端或两端添加氨基酸或多肽而获得的多肽，和/或具有角膜疾病或角膜损伤改成血管等的预防、抑制或治疗活性的多肽。
这些具体实例类似于上述活性成分多肽a和b，并且没有特别限制，例如，由SEQ ID NO：2或4的全长(即Sema3A蛋白)组成的多肽和具有80％或更多的序列同源性的多肽，与具有角膜疾病或角膜疾病或角膜损伤或预防、抑制或治疗活性的Sema3A蛋白片段具有80％或更高序列同源性的多肽，与包括70个氨基酸区域的80％或更多序列同源性。并且，通过将具有分泌结构域的区域与除 Semaphorin3A之外的分泌的信号素的其他区域融合而获得的嵌合蛋白质。
作为活性成分多肽c，特别优选的是在70aa区域中的同源性高于由(c)的多肽组成整个区域的同源性的多肽。例如，当活性成分多肽c是与具有SEQ ID NO：2的氨基酸序列的多肽具有80％同源性的多肽时，优选在70aa区域具有超过80％同源性的多肽，特别是70aa。该区域的同源性为90％以上，更优选为95％以上，进一步优选为98％以上，更优选地，其中70aa区域中的序列相同的多肽是优选的。另外，例如，活性成分多肽c是嵌合蛋白，其中与人Sema3A蛋白的Sema结构域具有80％序列同源性的区域和除Sema3A之外的分泌的Semaphorin的Sema结构域以外的区域是融合的。在一些情况下，优选Sema结构域中70aa区域的同源性大于80％，特别是70aa区域中的同源性为90％或更高，更优选为95％或更高，还更优选为98％或更高，更优选为90％或更高。优选的是嵌合蛋白，其中70aa区中的序列是相同的。
在上述活性成分多肽a至c中，具有SEQ ID NO：2或SEQ ID NO：4所示氨基酸序列的多肽特别是80％或更多，优选90％或更多，更优选95％或更多，更优选地，具有98％或更高同源性的多肽，并且其中，如上所述，优选在70aa区域中具有高于多肽整体的同源性的多肽。最优选地，在本发明的预防、抑制剂或治疗剂中作为活性成分含有的多肽是具有SEQ ID NO：2或SEQ ID NO：4所示氨基酸序列的多肽。
通常，在包含多肽的药物中，为了增强多肽在体内的稳定性，将糖链或聚乙二醇(PEG)链添加至多肽，或至少一种构成多肽的氨基酸。使用D-型氨基酸作为一部分并添加Fc结构域的技术是众所周知的并且使用的技术。通过添加糖链或PEG链或使用D-型氨基酸作为构成多肽的氨基酸的至少一部分，在体内变得难以被肽酶降解，并且多肽在体内减半的时间会更长。
本发明中使用的多肽可以是已经经历这些已知的体内稳定化修饰的多肽，只要它对角膜疾病或角膜损伤具有预防、抑制或治疗活性即可。除非上下文另有明确说明，否则说明书和权利要求书中的术语“多肽”在某种意义上使用，其还包括经修饰用于体内稳定化的那些。
糖基转化为多肽是众所周知的，例如，糖链能够与N-末端，C-末端或它们之间的氨基酸结合，但优选与N-末端或C-末端结合，以便不抑制多肽的活性。要添加的糖链的数量优选为一个或两个，并且优选为一个。糖链优选为单糖至四糖，更优选为二糖或三糖。糖链可以直接或通过间隔结构如具有约1-10个碳原子的亚甲基链与多肽的游离氨基或羧基连接。
PEG链可以与N-末端，C-末端或它们之间的氨基酸连接，并且通常一个或两个PEG链与多肽上的游离氨基或羧基连接。PEG链的分子量没有特别限制，但通常为约3000至约7000，优选约5000。
其中构成多肽的至少一部分氨基酸是D-形式也是众所周知的，例如虽然构成多肽的一些氨基酸可以是D-形式，但构成多肽的所有氨基酸都是D-型氨基酸而不是仅仅形成D-形式的氨基酸，因为多肽的活性不受抑制。最好是向多肽添加Fc结构域的方法也是公知的，并且可以例如使用市售的Fc融合蛋白表达载体如pFUSEN-Fc(InvivoGen制造)来生产。
用作本发明活性成分的Semaphorin 3A可以通过常规方法使用例如市售的肽合成仪容易地制备。而且，它可以使用已知的基因工程技术容易地制备。例如，通过RT-PCR从表达Semaphorin3A基因的组织中提取的RNA制备Semaphorin基因的cDNA，将全长或所需部分的cDNA整合到表达载体中， 可以引入它以获得靶多肽。可以通过已知方法进行RNA的提取，RT-PCR，将cDNA整合到载体中，以及将载体导入宿主细胞中。
此外，待使用的载体和宿主细胞也是众所周知的，并且各种载体和宿主细胞是可商购的。用于产生嵌合蛋白的方法也是本领域熟知的，并且嵌合蛋白可以例如通过上述Koppel等人的文章中描述的方法产生。而且，上述稳定化修改可以通过上述各个文献中描述的已知方法容易地进行。
发明详述
本发明涉及一种用于预防、抑制和/或者治疗血管新生疾病、淋巴管新生疾病的多肽，其中，所述多肽选自以下(a)至(c)中的任何一种多肽：
(a)由序列编号4所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号4中166aa～235aa区域的多肽；
(b)由序列编号2所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号2中166aa～235aa区域的多肽；
(c)与(a)或(b)的多肽具有80％或更高序列同源性的多肽。
在一个实施方案中，上述(c)的多肽是其中(a)或(b)的多肽的少量(优选1至几个)氨基酸残基被取代，缺失和/或插入的多肽。肽是与原始序列具有80％或更高，优选90％或更高，更优选95％或更高，还更优选98％或更高的同源性的多肽。
在一个实施方案中，本发明还涉及一种多肽(为方便起见，下文称为“活性成分多肽c”)，其具有包含上述(c)的多肽的区域，并且对角膜疾病或角膜损伤具有预防、抑制或治疗活性。与上述活性成分多肽a和b类似，它们可用于制备本发明的预防、抑制剂或治疗剂。
在一个实施方案中，当用上述(a)或(b)的多肽中的氨基酸残基取代时，可以通过本文中定义的基团之间相互取代来获得用作活性成分的多肽(c)，作为本发明的预防、抑制剂或治疗剂。
具体地，上述的多肽(c)是与由SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列的全长(即Semaphorin3A蛋白)组成的多肽具有80％或更高序列同源性的多肽，或者与含有70个氨基酸区域的Sema3A蛋白质片段具有80％或更高序列同源性的多肽。
在一个实施方案中，活性成分多肽c是仅由这些氨基酸组成的多肽，或者是通过向(c)的多肽的一端或两端添加氨基酸或多肽而获得的多肽，和/或具有角膜疾病或角膜损伤的预防、抑制或治疗活性的多肽。
在一个实施方案中，活性成分多肽c类似于上述活性成分多肽a和b，并且没有特别限制，例如，由SEQ ID NO：2或4的全长(即Sema3A蛋白)组成的多肽或者具有80％或更高序列同源性的序列。具有同源性的多肽，与具有角膜疾病角膜损伤或预防、抑制或治疗活性的Sema3A蛋白片段具有80％或更高序列同源性的多肽，其与包括70个氨基酸区域的80％或更多序列具有同源性，并且通过将具有分泌结构域的区域与除Semaphorin3A之外的分泌的信号素的其他区域融合而获得的嵌合蛋白质。
在一个实施方案中，作为活性成分多肽c，特别优选的是在70aa区域中的同源性高于由(c)的多肽组成整个区域的同源性的多肽。例如，当活性成分多肽c是与具有SEQ ID NO：2的氨基酸序列 的多肽具有80％同源性的多肽时，优选在70aa区域具有超过80％同源性的多肽，特别是70aa。该区域的同源性为90％以上，更优选为95％以上，进一步优选为98％以上，
更优选地，其中70aa区域中的序列相同的多肽是优选的。另外，例如，活性成分多肽c是嵌合蛋白，其中与人Sema3A蛋白的Sema结构域具有80％序列同源性的区域和除Sema3A之外的分泌的Semaphorin的Sema结构域以外的区域是融合的。在一些情况下，优选Sema结构域中70aa区域的同源性大于80％，特别是70aa区域中的同源性为90％或更高，更优选为95％或更高，还更优选为98％或更高，最优选为90％或更高。优选的是嵌合蛋白，其中70aa区中的序列是相同的。
在一个实施方案中，在上述活性成分多肽a至c中，具有SEQ ID NO：2或SEQ ID NO：4所示氨基酸序列的多肽特别是具有80％或更多，优选90％或更多，更优选95％或更多，更优选具有98％或更高同源性的多肽，并且其中，如上所述，优选在70aa区域中具有高于多肽整体的同源性的多肽。最优选地，在本发明的预防、抑制剂或治疗剂中作为活性成分含有的多肽是具有SEQ ID NO：2或SEQ ID NO：4所示氨基酸序列的多肽。
在一个实施方案中，本发明中使用的多肽可以是已经经历已知的体内稳定化修饰的多肽，只要它具有本发明的预防、抑制或治疗活性即可。优选的，其为糖基化的多肽，例如，糖链能够与N-末端，C-末端或它们之间的氨基酸结合，但优选与N-末端或C-末端结合，以便不抑制多肽的活性。要添加的糖链的数量优选为一个或两个，并且优选为一个。糖链优选为单糖至四糖，更优选为二糖或三糖。糖链可以直接或通过间隔结构如具有约1-10个碳原子的亚甲基链与多肽的游离氨基或羧基连接。
在一个实施方案中，本发明中使用的多肽还优选PEG化的多肽，PEG链可以与N-末端，C-末端或它们之间的氨基酸连接，并且通常一个或两个PEG链与多肽上的游离氨基或羧基连接。PEG链的分子量没有特别限制，但通常为约3000至约7000，优选约5000。
在一个实施方案中，其中构成本发明中使用的多肽的至少一部分氨基酸是D形式；优选向多肽添加Fc结构域，更优选例如使用市售的Fc融合蛋白表达载体如pFUSEN-Fc(InvivoGen制造)来生产多肽。
在一个实施方案中，用作本发明活性成分的Semaphorin 3A可以通过常规方法使用例如市售的肽合成仪容易地制备。而且，它可以使用已知的基因工程技术容易地制备。
例如，通过RT-PCR从表达Semaphorin3A基因的组织中提取的RNA制备Semaphorin基因的cDNA，将全长或所需部分的cDNA整合到表达载体中，可以引入它以获得靶多肽。可以通过已知方法进行RNA的提取，RT-PCR，将cDNA整合到载体中，以及将载体导入宿主细胞中。
Semaphorin3A是本发明的预防、抑制或治疗剂中的活性成分，对预防、抑制或治疗血管/淋巴管新生疾患是有效的。
本发明还涉及一种预防剂、抑制剂或治疗剂，其包含上述多肽(优选Semaphorin 3A)或适合于每种给药形式的药理学上可接受的赋形剂，稳定剂，防腐剂，缓冲剂。任选的，还可以适当地混合添加剂如增溶剂，乳化剂，稀释剂，张力剂等以制备制剂。上述可以使用的制剂方法和添加剂在药物制剂领域是公知的，并且可以使用任何方法和添加剂。
本发明还涉及前述多肽在制备用于预防、抑制和/或者治疗血管新生疾病、淋巴管新生疾病的药 物中的用途，优选的，还涉及前述多肽在制备用于预防、抑制和/或者治疗癌症相关的疾病、动脉硬化、阿尔茨海默病、瘢痕形成、老化相关的药物中的用途。
优选的，所述多肽选自以下(a)至(c)中的任何一种多肽：
(a)由序列编号4所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号4中166aa～235aa区域的多肽；
(b)由序列编号2所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号2中166aa～235aa区域之多肽；
(c)与(a)或(b)的多肽具有80％或更高序列同源性的多肽，优选90％或更高，更优选95％或更高，还更优选98％或更高的同源性的多肽，尤其优选的是嵌合蛋白，其中70aa区中的序列是相同的。
更优选的，所述多肽为糖基化的多肽；
还优选的，所述多肽为PEG化的多肽；任选的，PEG链的分子量为约3000至约7000，优选约5000。
在一个实施方案中，其中，上述血管新生疾病或者淋巴管新生疾病为癌症。
在一个实施方案中，其中，上述癌症为选自以下的一种或多种疾病：乳腺癌、(特别是转移性乳腺癌)、大肠癌、结直肠癌、进行性结肠直肠癌、食道癌、浅表型食管癌、早期食道癌、喉头癌、副鼻腔恶性肿瘤、胃肠道间质瘤(GIST)、肾(肾细胞)癌、肝脏(成人原发性)癌症(特别是不能实施手术的肝癌)、淋巴瘤、淋巴管瘤病/Gorham病、巨大的淋巴管畸形、黑色素瘤(恶性黑色素瘤)、肺癌、非小细胞肺癌(NSCLC)、转移性非小细胞肺癌、卵巢癌、上皮性卵巢癌、胰腺癌、前列腺癌、胃癌、鼻腔肿瘤、妇科恶性肿瘤、子宫颈癌(特别是早期浸润癌)、子宫内膜癌、膀胱癌(浅表型膀胱癌、浸润性癌)中。
在一个实施方案中，其中，上述血管新生疾病或者淋巴管新生疾病为眼科血管新生疾病或者淋巴管新生疾病。
在一个实施方案中，其中，上述眼科血管新生疾病或者淋巴管新生疾病选自以下的一种或多种疾病：糖尿病性血管增值性眼底病变、网膜血管肿、von Hippel-Lindau病、体表软血管瘤、黄斑变性、近视性脉络膜新生血管、糖尿病网膜症、未成熟儿视网膜症、新生血管性青光眼、糖尿病黄斑症、网膜静脉闭塞症、葡萄膜炎合并的眼内新生血管、Behcet病、结核性葡萄膜炎、高度近视的脉络膜新生血管、恶性淋巴瘤，结膜恶性淋巴瘤、脉络膜肿瘤、巨细胞病毒性疾患例如巨细胞病毒性视网膜炎、眼内的恶性肿瘤例如视网膜母细胞瘤、脉络膜恶性黑素瘤、眼球中出现的恶性淋巴瘤、眼附属器肿瘤例如视神经肿瘤。
在一个实施方案中，其中，上述新生血管性青光眼，包括伴随全身糖尿病引起的眼底血管新生及继发性血管新生性青光眼的血管、淋巴管机能减退。
在一个实施方案中，其中，上述血管新生疾病或者淋巴管新生疾病为选自以下的一种或多种疾病：瘢痕瘤、类风湿性关节炎(RA)、银屑病(牛皮癣)、动脉硬化、结节性硬化症、阿尔茨海默病如血管性阿尔茨海默、老化、急性心筋梗塞、巨细胞病毒和衣原体感染诱发的冠状动脉病变。
在一个实施方案中，其中，上述血管新生疾病或者淋巴管新生疾病为瘢痕组织形成相关疾病。
在一个实施方案中，其中，上述瘢痕组织形成相关疾病为选自以下的一种或多种疾病：伴随疤痕、溃疡，伤口，梗塞引起的坏死引起的疾病，各种器官的组织缺陷。
在一个实施方案中，其中，上述血管新生疾病或者淋巴管新生疾病为选自以下的一种或多种疾病：老化性器官老化、伴随血管机能下降引起虚血状态老化性器官老化的血管、淋巴管机能减退。
在一个实施方案中，其中，上述器官老化疾病包括：血管性痴呆和阿尔茨海默病。
在本发明的实施方案中，血管/淋巴管新生疾患的原因没有特别限制，可以是局部发生的疾患或全身疾患。
在一个实施方案中，本发明的预防和/或治疗剂可以由单独的Semaphorin 3A组成，或者由适合于每种剂型的药理学上可接受的赋形剂，稳定剂，防腐剂，缓冲剂组成。此外，可以适当地混合诸如增溶剂，乳化剂，稀释剂，张度剂之类的添加剂以制备制剂。并且配制方法可以使用任何方法和添加剂。
本发明的预防、抑制或治疗剂的给药目标是哺乳动物，实例包括人，狗，猫，兔，仓鼠等。来自与待预防、抑制或治疗的患者相同的物种的本发明的前述多肽(优选为Semaphorin 3A)的使用被认为对于预防、抑制或治疗它更有效，并且从临床应用的安全性的观点来看也是期望的。因此，例如，当本发明的预防、抑制或治疗剂的给药目标是人时，特别优选包含上述活性成分多肽b作为活性成分的预防、抑制或治疗剂。
本发明的预防、抑制或治疗剂眼科疾患可以通过给予要预防的角膜或待治疗的角膜病变来使用。作为给药方法，可以提及局部给药(角膜上的滴眼剂给药，结膜下注射给药等)等。剂型包括隐形眼镜类型、眼软膏、注射剂、滴眼剂等。
在隐形眼镜镜片类型中，本发明的前述多肽(优选为Semaphorin 3A)可以包含在具有隐形眼镜状球形表面的透明薄膜中，并且从薄膜释放的本发明的前述多肽(优选为Semaphorin 3A)可以施用于角膜。
本发明的前述多肽(优选为Semaphorin 3A)的剂量可以根据症状，年龄，体重，给药方法等适当选择，并没有特别限制。
本发明的前述多肽(优选为Semaphorin 3A)单位剂量为约6000至60000U，优选约4000至40000U，更优选为约500至5000U。
在一个实施方案中，在滴眼剂的情况下，滴眼剂剂量可为约1至3000ng，约20至900ng，约50至900ng，约50至500ng，约80至500ng。优选的，在一个实施方案中，滴眼剂浓度为约10ng/ml至约1000ng/ml，例如约10ng/ml、约100ng/ml、约300ng/ml、约1000ng/ml。以上剂型及用量优选每日一次或数次，或每隔几天，持续数天至数月，具体取决于症状的改善程度。
在结膜下注射的情况下，本发明的前述多肽(优选为Semaphorin 3A)的量通常为每天约500至5000U，优选约500至5000U，并且对于受试动物，可施用一次或数次分开。
在一个实施方案中，结膜下注射的量可以是约0.001至10ng/天，约0.05至5ng/天。优选的，在一个实施方案中，浓度为约10ng/ml至1000ng/ml，例如，约10ng/ml、约100ng/ml、约300ng/ml、约1000ng/ml。优选地，根据症状改善的程度，给药可以定期进行，每日一次或数次，或每天一次或几天，持续数天至数月。
本发明的前述多肽(优选为Semaphorin 3A)还可以制备为以下剂型：
在一个实施方案中，所述剂型为滴眼液，其用于治疗干眼症，角膜炎，结膜炎，结膜淋巴肿瘤，血管新生抑制、角膜化学烧伤，角膜上皮愈合，睑板腺癌：其中，
活性成份为：前述多肽(优选为Semaphorin 3A)；
活性成份浓度为：约100～300ng/ml。
在另一个实施方案中，所述剂型为滴眼液，其用于治疗血管新生抑制、角膜化学烧伤，结膜淋巴肿瘤，角膜上皮愈合，睑板腺癌：其中，
活性成份为：前述多肽(优选为Semaphorin 3A)；
活性成份浓度为：约100ng/ml；
规格：5ml/瓶，含有活性成份：500ng/瓶；
共计125滴/瓶；0.040ml/滴。
在另一个实施方案中，所述剂型为滴眼液，其用于治疗干眼症，角膜炎，结膜炎，结膜淋巴肿瘤，血管新生抑制、角膜化学烧伤，角膜上皮愈合，睑板腺癌：其中，
滴眼液活性成份浓度为：约300ng/ml；
规格：5ml/瓶，含有前述多肽(优选为Sema3A)1500ng/瓶；
共计125滴/瓶；0.040ml/滴。
在一个实施方案中，所述剂型为眼膏，其用于治疗顽固性干眼症，角膜炎，结膜炎，结膜淋巴肿瘤，角膜上皮愈合，血管新生抑制、角膜化学烧伤，睑板腺癌：
活性成份为：前述多肽(优选为Semaphorin 3A)；
活性成分浓度为：约100～300ng/ml；
规格：3.5g/只；含有前述多肽(优选为Sema3A)0.15％/只。
在一个实施方案中，所述剂型为角膜软性接触药膜，其用于抑制炎症和病理性血管、淋巴管新生并使其机能正常化，修复角膜化学烧伤瘢痕，维持角膜机能正常化；
所述角膜软性接触药膜还用于治疗慢性结膜炎、顽固性干眼症、电光性眼炎、强膜炎、角膜化学烧伤、角膜激光灼伤、史蒂文斯·约翰逊病炎症、蚕食性角膜炎、睑板腺癌等难治性角膜、结膜、强膜眼睑疾患：
活性成份为：前述多肽(优选为Semaphorin 3A)；
活性成份浓度为：约100～300ng/ml；
含有前述多肽(优选为Sema3A)0.05％～0.15％/枚；
规格：直径:13.5mm-14.5mm；厚度:0.01mm-0.09mm。
所述角膜软性接触药膜具有优良的保湿性及吸水性物质和黏弹性、帮助本角膜软性接触药膜发挥速效和长效的作用。
在一个实施方案中，所述剂型为注射液，其用于抑制炎症和病理性血管，淋巴管新生并使其机能正常化，修复角膜化学烧伤瘢痕：
活性成份为：前述多肽(优选为Semaphorin 3A)；
活性成份浓度为：约100～300ng/ml；
规格：0.5ml/管；含有前述多肽(优选为Sema3A)50ng～150ng/管。
所述注射液用于炎症细胞减少，症状得到控制，组织机能恢复正常，病性血管/淋巴管新生抑制，血管/淋巴管机能正常化。优选的，所述注射液还用于以下炎症相关疾病：
眼科术后炎症，脊髓水肿，输卵管手术后预防粘连，预防手术后腹膜粘连，Senear-Usher综合症，亚急性甲状腺炎，甲状腺中毒症，剧症肝炎甲状腺中毒，亚急性湿疹，过敏体质性皮炎，过敏性鼻炎，阴茎硬结，溃疡性结肠炎，角膜炎对症治疗，花粉症，货币湿疹，眼眶假瘤的对症治疗，睑缘炎对症治疗，关节炎牛皮癣，癌症末期的全身状况改善，支气管痉挛，急性湿疹，急性中耳炎，急性白血病，出血性凝血因子紊乱的倾向，硬皮病，巩膜炎症状性治疗，格林-巴利综合征，突然发生肢体皮炎，血管舒缩鼻炎，结节性红斑，结节性动脉周围炎，结膜炎对症治疗，预防瘢痕疙瘩，局灶性肠炎，虹膜睫状体炎的对症治疗，嗜酸性肉芽肿，喉炎，喉头水肿，红斑，红皮症，，网状细胞肉瘤病，自家感作性皮炎，咽鼓管狭窄，视束性脊髓炎，湿疹/皮炎组，紫癜，耳鼻喉科手术后，幼年型类风湿性关节炎，改善严重衰弱性疾病的一般状况，儿童哮喘性支气管炎，脂溢性皮炎，史蒂文斯-约翰逊病，脊髓视网膜炎，接触性皮炎，哮喘性支气管炎，全身系统性红斑狼疮，全身性血管炎，前列腺癌，早期瘢痕疙瘩，增生性天疱疮，主动脉炎综合征，多发性肌炎，多发性硬化症，多发性骨髓瘤，多动脉炎，Vegena肉芽肿病，中毒疹，痛风性关节炎，手指皮炎Düring疱疹性皮炎，难治性口腔炎，耐火性舌炎，乳腺癌复发转移，肾病，肾病综合征，粘膜皮肤综合症，脑炎，脑脊髓炎，脑水肿，脓疱性牛皮癣，肺纤维化，鼻褶，非感染性慢性关节炎，皮肌炎，皮肤口炎，皮肤网状病，皮肤白血病，弥漫性间质性肺炎，鼻窦炎，Fuchs综合症，Hebra红色皮疹，骨性关节炎，放射性肺炎，霍奇金病，慢性湿疹，慢性中耳炎，免疫性溶血性贫血，视网膜血管炎对症治疗，脉络膜炎症状性治疗，药物过敏，药疹，Reiter综合征，落叶天疱疮，风湿性心脏炎，风湿性脊椎炎，类风湿性脊椎炎的肢体关节炎，类风湿性多肌痛，风湿热，Lipschutz急性外阴溃疡，慢性盘状红斑狼疮，器官移植，生殖器湿疹，化学过敏，化学成瘾，外耳道湿疹/皮炎，喉息肉，喉结节，耳炎湿疹/皮炎，组织移植，鼻前庭湿疹/皮炎，鼻翼周围有湿疹/皮炎，吸毒成瘾，肛门湿疹，渗出性中耳炎，对症治疗葡萄膜炎，腐蚀性食管炎，恶性秃头症，非传染性滑囊炎，非感染性肩周炎，非感染性肌腱炎，非感染性腹膜炎，非感染性肌腱炎，活动性肝硬化，掌跖脓疱病，Behcet病，对症治疗外眼炎症，眼前段炎症性疾病症状性治疗，内眼炎症对症治疗，眼眶炎性疾病的对症治疗，对症治疗眼部炎症性疾病，牛奶/婴儿/小儿湿疹，肝硬化伴顽固性腹水，充血性心力衰竭，肝硬化伴胆汁淤积，慢性淋巴细胞白血病，淋巴肉瘤病，恶性淋巴肿。
在一个实施方案中，所述剂型为口服液，其用于治疗呼吸道过敏及炎症，恢复粘膜正常功能：
活性成分成份为：Sema3A；
活性成分浓度为：约100～300ng/ml；
规格(1)10ml或(2)100ml；
前述多肽(优选为Sema3A)0.05％～0.15％/只。
在一个实施方案中，所述剂型为漱口液，其用于治疗口腔，呼吸道过敏及炎症，恢复粘膜正常功能：
活性成分成份为：前述多肽(优选为Semaphorin 3A)；
活性成分浓度为：约100～300ng/ml；
规格(1)5.0ml/只；
前述多肽(优选为Sema3A)0.05％～0.15％/只。
在一个实施方案中，所述剂型为药膜黏附片，其用于粘膜疾患，皮肤疾患，烧伤/创伤等创面的治疗：
活性成分浓度为：约100～300ng/ml；
规格：0.05g/枚，前述多肽(优选为Sema3A)0.05％～0.15％/枚；
其中，所述药膜黏附片包括：①口腔黏附片、②妇科粘膜黏附片；③机械外伤，化学外伤，溃疡等黏附片。
优选的，口腔黏附片通过以下方法制备：采用黏膜粘着剂卡波姆(CP934)、海藻酸钠、低黏度羧甲基纤维素钠(SCMC)，用直接压片法压片制备Sema3A口腔黏附片。
在一个实施方案中，所述剂型为乳膏，其用于治疗粘膜疾患，皮肤疾患，烧伤/创伤等创面：
活性成分成份为：前述多肽(优选为Semaphorin 3A)；
活性成分浓度为：约100～300ng/ml；
规格：5.0g/只，前述多肽(优选为Sema3A)0.05％～0.15％/只。
在一个实施方案中，所述剂型为栓剂，其用于治疗妇科疾患，痔疮：
活性成分成份为：前述多肽(优选为Semaphorin 3A)；
活性成分浓度为：约100～300ng/ml；
规格：0.15g/枚；前述多肽(优选为Sema3A)0.05％～0.15％/枚。
在一个实施方案中，所述剂型为伤口护理用敷料：
活性成分成份为：前述多肽(优选为Semaphorin 3A)；
活性成分浓度为：约100～300ng/ml；
规格：5.0g/只；前述多肽(优选为Sema3A)0.05％～0.15％/只。
上述敷料可以分为以下种类：
①水凝胶伤口敷料：治疗激光灼伤和烧烫伤的水凝胶伤口敷料、抑菌促愈合功能的医用胶体敷料；
②液体伤口敷料；
③伤口护理用敷料。
在一个实施方案中，所述剂型为喷雾剂，其中：
活性成分成份为：前述多肽(优选为Semaphorin 3A)；
活性成分浓度为：约100～300ng/ml；
规格：5.0ml/只前述多肽(优选为Sema3A)0.05％～0.15％/只。
所述喷雾剂可以分为以下种类：鼻喷雾剂；口腔喷剂；妇科喷剂：一种用于治疗妇科炎症的生物制剂。
在一个实施方案中，所述剂型为涂膜剂，其用于粘膜疾患，皮肤疾患，烧伤/创伤等创面的治疗：
活性成分成份为：前述多肽(优选为Semaphorin 3A)；
规格：5.0g/只；含有前述多肽(优选为Sema3A)0.05％～0.15％/只；
活性成分浓度为：100～300ng/ml。
本发明的预防剂、抑制剂或治疗剂可以单独使用或与其他预防、抑制剂或治疗剂组合使用。其他预防、抑制或治疗剂包括抗生素，抗炎剂，抗病毒剂，细胞生长促进剂，伤口愈合剂，细胞外基质组分，维生素等。
以上述眼膏为例，例如Semaphorin 3A/透明质酸软膏，Semaphorin 3A/neomedrol EE软膏，Semaphorin 3A/Oders Linderon软膏，Semaphorin 3A/脱欧麻眼膏0.1％。Semaphorin 3A/透明质酸软膏可用作透明质酸眼科手术的制剂。neomedrol EE软膏是抗生素和合成肾上腺皮质激素药的组合药物，通过与Semaphorin 3A组合使用可以预期高的抗炎作用。对于上述Linderon A软膏也是如此。肾上腺皮质激素的组分可以从neomedrol EE软膏中的组分适当地减少并施用。
已知通过反馈作用稳定体内Semaphorin的浓度。因此，Semaphorin在体内缓和地起作用。另外，由于Semaphorin的给药可以激活分裂能力而不引起角膜内皮细胞的显着形态变化，因此本发明的预防、抑制或治疗剂不太可能引起副作用。
在实施方案中，本发明提供预防、抑制或治疗角膜疾病或角膜损伤的方法，包括给哺乳动物施用Semaphorin的步骤。
在实施方案中，本发明提供了Semaphorin 3A，用于预防、抑制或治疗角膜疾病或角膜损伤。
在实施方案中，本发明提供了Semaphorin 3A在制备预防、抑制或治疗角膜疾病或角膜损伤的药物中的用途。
附图说明
为了使本发明的内容得到更充分的理解，下面通过本发明的具体实施例并结合附图，对本发明作进一步详细地说明，其中：
图1为本发明所述的用于动物模型生体荧光素染色的示意图，其中上方两图为角膜损伤的观察图像，下方为模式图，其中(a)为角膜碱烧伤模型的动物生体荧光素染色在室内光下的照片；(b)为裂隙灯蓝色自由滤光器下的照片。
图2为碱性角膜烧伤模型制成后，对全角膜上皮搔爬处理、荧光素染色的照片。
图3为上皮欠损临床症状比较照片。
图4为治疗14天后PBS给药对照组显微镜下照片放大图。
图5为角膜上皮欠损评价，浓度100ng/ml和1000ng/ml治疗组症状明显好转。
图6为角膜血管生成评价，结果显示Sema3A以浓度依赖性方式抑制新血管形成。
图7为清创术后14天，角膜平铺片的CD31抗体和LYVE-1抗体免疫染色照片。
图8表明Sema3A有效抑制了角膜碱烧伤后病理性血管生成。
图9表明Sema3A有效抑制了角膜碱烧伤后病理性淋巴管生成。
图10为角膜组织炎症细胞浸润的免疫染色照片。(角膜组织的βⅢtubulin和F4/80免疫染色照片)。
图11为F4/80抗体阳性细胞(巨噬细胞)数评价。
图12为角膜切片神经纤维免疫染色照片。
图13为角膜βⅢtubulin阳性神经纤维密度评价。
为了进一步理解本发明，下面结合实施例对本发明的具体实施方案进行详细描述。但是应当理解，这些描述的目的只是为进一步说明本发明的特征和优点，而不构成对本发明的权利要求的任何限制。
实施例
实施例1
本实施例的目的是验证本发明所述的多肽，尤其是Semaphorin 3A的血管新生/淋巴管新生，及血管内皮机能异常相关疾病，尤其是癌症、眼科疾病、动脉硬化、心脑肾血管疾患、阿尔茨海默病、老化性疾病、瘢痕形成等疾病的预防、抑制和/或者治疗作用。
1、 实验方法：
通过滴入2ml 0.15M NaOH制作角膜碱损伤模型，并进行以下检查。药物每隔1天给药1次，并从产生碱性损伤的那天起持续7天。实验组在术后第0、3、6和9天结膜下给予Semaphorin 3A(2μl)，LSBio公司制。对照组结膜下给予PBS液。
2、 实验动物分组及治疗：
BALB/c雄性8周龄
将实验动物随机分为以下各组，每组8只：
(1)PBS作为给药对照组(n＝8)
(2)Semaphorin3A(10ng/ml 2μl/结膜下注射)给药组(n＝8)；
(3)Semaphorin3A(100ng/ml 2μl/结膜下注射)给药组(n＝8)；
(4)Semaphorin3A(1000ng/ml 2μl/结膜下注射)给药组(n＝8)；
3、 动物模型生体染色：
使用裂隙灯蓝色自由滤光器(蓝色无滤光器)，其仅选择性地透射由荧光素发射的绿色荧光颜色，因此可以获得角膜损伤的观察图像。
4、 荧光素染色试验：用荧光素染色观察角膜的状况。如果有染色，则意味着角膜糜烂和角膜上皮的状况不稳定。
该试验用于通过使用渗透性上皮缺陷的特性和上皮细胞之间的弱粘附区域来检查角膜溃疡(角膜伤口)。
5、 评价指标：
记录角膜所见结果：在碱性损伤，角膜混浊，创伤后3、6、9、14天在裂隙灯显微镜下观察角膜，如下所述对角膜新血管形成和角膜上皮缺损的程度进行半定量评估：
角膜混浊：按程度统计得分为0-4的五个等级。
角膜内新生血管分级：侵袭到角膜内的长度和范围按程度统计分为0-4的五个等级。
角膜上皮缺损范围：由于上皮缺损通常呈椭圆形，因此通过缺损范围的长轴与角膜直径的比率来评价。
给药物组和对照组，使用unpaired Student's t test.检验进行比较。**P<0.01，***P<0.001；ns：无显著性差异。
临床症状评点：
每天用裂隙灯显微镜检查动物模型的眼睛临床症状变化。角膜混浊评分系统是：新血管形成(NV)和角膜上皮缺损区域。定义评分系统以半定量术语描述角膜混浊(0-4)和NV(0-4)的程度。在角膜损伤模型做成后第2、5、6、10、14天，盲检观察者对于每个角膜的混浊度指定混浊评分：
0＝透明角膜；
1＝最小的表面(非角膜基质)不透明度；
2＝最小深度(角膜基质)不透明度；
3＝中度角膜基质不透明；
4＝强度的角膜基质不透明。
NV评分系统：
0＝角膜无血管侵袭；
1＝在不到两个象限的情况下，血管长度小于角膜半径的一半；
2＝血管长度超过角膜半径的一半且小于两个象限；
3＝血管长度小于角膜半径的一半在两个以上的象限中；
4＝血管长度超过角膜半径的一半在两个以上的象限。
计算角膜上皮缺损的面积，作为缺陷面积的比例。
长轴：角膜直径。
统计分析
所有数据均表示为平均值±SEM。
低于检测限的值计为0pg/ml。使用unpaired Student's t test.检验进行比较。**P<0.01，***P<0.001；ns：无显着差异。
检验来统计比较未治疗的受损角膜(对照)和Sema3A治疗的角膜每个时间点的临床评分。
6. 实验结果：
上皮欠损模型
图1(a)为角膜碱烧伤模型在室内光下摄影得到的照片。如图所示，图1(a)中白色破线内为角膜区域，角膜碱烧伤后，角膜上皮脱落。黑色实线框内为烧伤后变性脱落的上皮组织堆积域。图1(b)为碱性角膜烧伤后，荧光素染色并在裂隙灯蓝色自由滤光器下摄影得到的照片。如图所示，图中白色破线内为角膜区域，箭头指向白色部分荧光素附着的角膜上皮缺损区域。
图2展示了对全角膜上皮搔爬处理后荧光染色的照片。如图2所示，白色破线范围内为角膜区域，清创后角膜表面上皮组织完全剥离，全角膜呈现荧光素着色，整个角膜像毛玻璃一样变浑浊。
上皮欠损临床症状比较
图3展示了角膜上皮欠损临床症状的比较。在碱性角膜外伤后第3、6、9、14天观察治疗组和对照组角膜修复变化，对上皮欠损进行荧光素染色，显微镜下的照片。其中：角膜碱烧伤动物模型经过2周的观察，第1、3、5、7行照片为室内光下第3、6、9、14天的角膜上皮临床症状变化；第2、4、6、8行照片为荧光素染色后，裂隙灯蓝色自由滤光器(蓝色无滤光器下)第3、6、9、14天的角膜上皮临床症状变化。
根据实验结果可见，烧伤后2周，对照组和治疗组10ng/ml，1000ng/ml眼睛表面如结膜和角膜仍然有荧光素染色区域，炎症没有痊愈。角膜变得浑浊，伴有炎症和血管侵犯超过2周，100ng/ml对血管生成，上皮缺陷和伤口愈合有很明显影响。角膜几乎没有荧光素染色，泪液的储留充足。基本恢复正常眼状态。
图4为图3中治疗14天后PBS给药对照组显微镜下照片的放大图。其中，图4(a)中白色破线范围内为角膜组织领域，箭头指示为碱烧伤后角膜新生血管。血管粗大，影响角膜透视度，动物模型视力急剧下降。图4(b)中白色破线范围内为角膜组织领域，白色箭头指示为碱烧伤后全角膜变性呈现白斑，不透明状态。白色实线范围内为角膜变性瘢痕增殖领域，黑色箭头指示为组织变性、增生肥厚，呈现不透明状态。动物模型视力高度下降或失明。
上皮欠损评价
图5展示了对角膜上皮欠损的评价。在制造成碱性外伤模型后第2、3、4、6、10、14天观察角膜修复变化，荧光素染色，临床采点(图5)。
将角膜上皮缺损的程度评分为五级：
纵轴评点方法(参考：Den等人，Efficacy of early systemic betamethasone or cyclosporin A after corneal alkali injury via inflammatory cytokine reduction)
0＝透明角膜；
1＝最小的表面(非角膜基质)不透明度；
2＝最小深度(角膜基质)不透明度；
3＝中度角膜基质不透明；
4＝强度的角膜基质不透明。
从第6天起浓度依存临床症状的变化出现差异，浓度100ng/ml和1000ng/ml治疗组症状明显好转。浓度100ng/ml治疗组在第6天效果显著(**P<0.01)；在第14天的有显著性差异(*P<0.05)。
通过给药，本治疗角膜烧伤组织显著修复，角膜上皮愈合良好，浓度100ng/ml治疗组角膜透明与正常角膜无差异。
血管生成评价
图6展示了对角膜血管生成的评价。在碱性创伤后2、5、6、10、14天，根据新生血管生成的长 度和程度分级标准分五个阶段中评估。
用抗CD31抗体(血管内皮标记物)在14天后对角膜进行免疫染色，利用平铺角膜片量化血管区域。在手术显微镜下测量角膜中的新生血管长度。其中，小鼠每组8只(n＝8)，检定为mean±S.D。
新血管形成：其侵入角膜长度和范围以0至4的五个等级评分。
新生血管，新生淋巴管评分系统方法如下:
0＝无
1＝在不到两个象限的情况下，血管/淋巴管长度小于角膜半径的一半；
2＝血管/淋巴管长度超过角膜半径的一半且小于两个象限；
3＝血管/淋巴管长度小于角膜半径的一半在两个以上的象限中；
4＝血管/淋巴管长度超过角膜半径的一半，在两个以上的象限。
计算角膜上皮缺损的面积作为缺陷面积的比例
长轴：角膜直径
观察2周的结果，Sema3A以浓度依赖性方式抑制新血管形成。
其中，与PBS对照组相比，100ng/ml的Sema3A在第4天呈现出对心血管形成的抑制；在第10天有显著性差异(**P<0.01)，通过给药本治疗显著抑制了角膜中新血管产生和生长形成。
血管，淋巴管，角膜平铺片免疫染色评价
图7显示清创术后14天，用CD31抗体(血管内皮标记物)，LYVE-1抗体(淋巴管内皮标记物)对角膜平铺片进行免疫染色。
治疗组给药Sema3A后，对Sema3A浓度为10ng/ml，100ng/ml，1000ng/ml的治疗组及对照组的血管、淋巴管进行免疫染色。利用血管、淋巴区域面积的百分比进行定量化评价。
根据图7可见，PBS治疗对照组有大面积的血管和淋巴管新生，与PBS组相比，Sema3A浓度100ng/ml治疗组明显抑制了血管和淋巴管的新生，角膜中心部分保持了正常的透明度。表明Sema3A能有效促进上皮形成。
血管/淋巴管生成领域评价
图8展示了对角膜血管新生区域的评价(以血管新生区域面积(％)表示)。清创术后14天，治疗组Sema3A给药后，浓度100ng/ml的Sema3A治疗组的血管新生面积明显缩小，与对照组的有显著性差异：**P＝0.014；小鼠各组为8只(n＝8)；检定为mean±S.D。上述结果表明Sema3A有效抑制了角膜碱烧伤后病理血管生成。
图9展示了对角膜淋巴管新生区域的评价(以淋巴管新生面积(％)表示)。清创术后14天，治疗组Sema3A给药后，浓度100ng/ml的Sema3A治疗组的淋巴管新生面积明显缩小，与对照组的有显著性差异：**P＝0.02；小鼠各组为8只(n＝8)；检定为mean±S.D。表明Sema3A有效抑制了角膜碱烧伤后病理性淋巴管生成。
角膜组织炎症细胞浸润
图10为角膜组织的βⅢtubulin和F4/80免疫染色照片(清创术后第14天)。其展示了角膜组织炎症细胞的浸润。
三角白色箭头指示免疫染色阳性炎症细胞；
A左：神经纤维标记物βIII微管蛋白免疫染色；
B中：抗F4/80抗体用于小鼠巨噬细胞检测，浓度100ng/ml的Sema3A治疗组角膜组织F4/80阳性细胞(巨噬细胞)浸润和对照组相比，明显减少。表明Sema3A抑制角膜碱性创伤后发生的炎症反应。
F4/80抗体阳性细胞(巨噬细胞)数评价
图11展示了对F4/80抗体阳性细胞(巨噬细胞)数的评价。
清创术后第14天，治疗组Sema3A给药后，浓度100nng/ml的Sema3A治疗组F4/80抗体阳性细胞数明显减少，与对照组相比有显著性差异：**P<0.0001；小鼠各组为15只(n＝15)，检定为mean±S.D。
角膜切片神经纤维免疫染色
图12展示了角膜切片神经纤维的免疫染色照片。清创术后第14天，用抗βIII微管蛋白抗体，PGP9.5(神经标记物)对角膜切片进行免疫染色。白色箭头指示部分为神经纤维，对照组和治疗组的各浓度角膜及分布在角膜组织中角膜神经纤维免疫荧光染色。
从图12中可见，浓度100ng/ml和高浓度1000ng/ml的Sema3A投与角膜组织中的神经丛未见破坏。
图13对角膜感觉神经(三叉神经)的影响进行了定量。用βⅢtubulin阳性神经纤维密度评价。
清创术后第14天，定量化角膜神经纤维区域。
角膜神经纤维被量化为图中所示的代表性共聚焦图像中β-微管蛋白III染色阳性阈值区域的百分比。
N.S.:各治疗组与正常和对照组没有显著性差异；每组小鼠15只(n＝15)；检定为mean±S.D。
该结果证明给药Sema3A，对角膜组织中的神经纤维没有影响。
总之，我们使用小鼠角膜碱性损伤模型检查了Semaphorin3A对角膜上皮缺损，角膜新生血管形成和淋巴管生成的影响。
尤其在100ng/ml的Semaphorin3A给药组中，角膜上皮缺损，角膜新生血管形成，角膜新生淋巴管生成和炎性细胞浸润均被显著抑制，并且与β-III微管蛋白阳性神经纤维正常小鼠角膜相比没有减少。
根据我们在体内的研究表明这些结果表明Semaphorin3A有效促进上皮形成，抑制病理性血管生成和抑制角膜碱性创伤后发生的炎症。
已经通过上述实施例对本发明的各个方面进行了例示。显然，上述实施例仅仅是为清楚地说明所 作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (20)

  1. 一种多肽在制备用于预防、抑制和/或者治疗血管新生疾病、淋巴管新生疾病的药物中的用途，
    其中，所述多肽选自以下(a)至(c)中的任何一种多肽：
    (a)由序列编号4所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号4中166aa～235aa区域的多肽；
    (b)由序列编号2所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号2中166aa～235aa区域的多肽；
    (c)与(a)或(b)的多肽具有80％或更高序列同源性的多肽，优选90％或更高，更优选95％或更高，还更优选98％或更高的同源性的多肽，尤其优选的是嵌合蛋白，其中70aa区中的序列是相同的；
    更优选的，所述多肽为糖基化的多肽；
    还优选的，所述多肽为PEG化的多肽；任选的，PEG链的分子量为约3000至约7000，优选约5000。
  2. 一种治疗用于预防、抑制和/或者治疗血管新生疾病、淋巴管新生疾病的方法，所述方法包括向所需的受试者给予有效量的多肽，
    其中，所述多肽选自以下(a)至(c)中的任何一种多肽：
    (a)由序列编号4所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号4中166aa～235aa区域的多肽；
    (b)由序列编号2所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号2中166aa～235aa区域的多肽；
    (c)与(a)或(b)的多肽具有80％或更高序列同源性的多肽，优选90％或更高，更优选95％或更高，还更优选98％或更高的同源性的多肽，尤其优选的是嵌合蛋白，其中70aa区中的序列是相同的；
    更优选的，所述多肽为糖基化的多肽；
    还优选的，所述多肽为PEG化的多肽；任选的，PEG链的分子量为约3000至约7000，优选约5000。
  3. 一种多肽，其用于预防、抑制和/或者治疗血管新生疾病、淋巴管新生疾病，
    其中，所述多肽选自以下(a)至(c)中的任何一种多肽：
    (a)由序列编号4所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号4中166aa～235aa区域的多肽；
    (b)由序列编号2所示氨基酸序列中的连续70个以上的氨基酸构成，且含有序列编号2中166aa～235aa区域的多肽；
    (c)与(a)或(b)的多肽具有80％或更高序列同源性的多肽，优选90％或更高，更优选95％或更高，还更优选98％或更高的同源性的多肽，尤其优选的是嵌合蛋白，其中70aa区中的序列是相同的；
    更优选的，所述多肽为糖基化的多肽；
    还优选的，所述多肽为PEG化的多肽；任选的，PEG链的分子量为约3000至约7000，优选约 5000。
  4. 权利要求1-3所述的任一项所述的用途、方法或多肽，其中，所述多肽的单位剂量为约6000至60000U，优选约4000至40000U，或约500至5000U。
  5. 权利要求1-4所述的任一项的用途、方法或多肽，其中，所述多肽的单位剂量为约1至3000ng、约20至900ng、约50至900ng、约50至500ng、约80至500ng。
  6. 权利要求1-5所述的任一项的用途、方法或多肽，其中，所述多肽的浓度为约10ng/ml至约1000ng/ml，例如约10ng/ml、约100ng/ml、约300ng/ml、约1000ng/ml，优选约100ng/ml。
  7. 权利要求1-6所述的任一项的用途、方法或多肽，其中，所述药物为滴眼剂、眼膏、注射液、角膜软性接触药膜、口服液、漱口液、药膜黏附片、乳膏、栓剂、喷雾剂。
  8. 权利要求1-7所述的任一项的用途、方法或多肽，其中，所述血管新生疾病或者淋巴管新生疾病为癌症。
  9. 权利要求1-8所述的任一项的用途、方法或多肽，其中，所述癌症为选自以下的一种或多种疾病：乳腺癌(特别是转移性乳腺癌)、大肠癌、结直肠癌、进行性结肠直肠癌、食道癌、浅表型食管癌、早期食道癌、喉头癌、副鼻腔恶性肿瘤、胃肠道间质瘤(GIST)、肾(肾细胞)癌、肝脏(成人原发性)癌症(特别是不能实施手术的肝癌)、淋巴瘤、淋巴管瘤病/Gorham病、巨大的淋巴管畸形、黑色素瘤(恶性黑色素瘤)、肺癌、非小细胞肺癌(NSCLC)、转移性非小细胞肺癌、卵巢癌、上皮性卵巢癌、胰腺癌、前列腺癌、胃癌、鼻腔肿瘤、妇科恶性肿瘤、子宫颈癌(特别是早期浸润癌)、子宫内膜癌、膀胱癌(浅表型膀胱癌、浸润性癌)。
  10. 权利要求1-9所述的任一项的用途、方法或多肽，其中，所述血管新生疾病或者淋巴管新生疾病为眼科血管新生疾病或者淋巴管新生疾病。
  11. 权利要求1-10所述的任一项的用途、方法或多肽，其中，所述眼科血管新生疾病或者淋巴管新生疾病选自以下的一种或多种疾病：糖尿病性血管增值性眼底病变、网膜血管肿、von Hippel-Lindau病、体表软血管瘤、黄斑变性、近视性脉络膜新生血管、糖尿病网膜症、未成熟儿视网膜症、新生血管性青光眼、糖尿病黄斑症、网膜静脉闭塞症、葡萄膜炎合并的眼内新生血管、Behcet病、结核性葡萄膜炎、高度近视的脉络膜新生血管、恶性淋巴瘤，结膜恶性淋巴瘤、脉络膜肿瘤、巨细胞病毒性疾患例如巨细胞病毒性视网膜炎、眼内的恶性肿瘤例如视网膜母细胞瘤、脉络膜恶性黑素瘤、眼球中出现的恶性淋巴瘤、眼附属器肿瘤例如视神经肿瘤。
  12. 权利要求1-11所述的任一项的用途、方法或多肽，其中，所述新生血管性青光眼，包括伴随全身糖尿病引起的眼底血管新生及继发性血管新生性青光眼的血管、淋巴管机能减退。
  13. 权利要求1-12所述的任一项的用途、方法或多肽，其中，所述血管新生疾病或者淋巴管新生疾病为选自以下的一种或多种疾病：瘢痕瘤、类风湿性关节炎(RA)、银屑病(牛皮癣)、动脉硬化、结节性硬化症、阿尔茨海默病如血管性阿尔茨海默、老化、急性心筋梗塞、巨细胞病毒和衣原体感染诱发的冠状动脉病变。
  14. 权利要求1-13所述的任一项所述的用途、方法或多肽，其中，所述血管新生疾病或者淋巴管新生疾病为瘢痕组织形成相关疾病。
  15. 权利要求1-14所述的任一项所述的用途、方法或多肽，其中，所述瘢痕组织形成相关疾病为选自以下的一种或多种疾病：伴随疤痕、溃疡，伤口，梗塞引起的坏死引起的疾病，各种器官的组织缺陷。
  16. 权利要求1-15所述的任一项的用途、方法或多肽，其中，所述血管新生疾病或者淋巴管新生疾病为选自以下的一种或多种疾病：老化性器官老化、伴随血管机能下降引起虚血状态老化性器官老化的血管、淋巴管机能减退。
  17. 权利要求1-16所述的任一项的用途、方法或多肽，其中，所述器官老化性疾病包括：血管性痴呆和阿尔茨海默病。
  18. 权利要求1-17所述的任一项的用途、方法或多肽，其中，所述血管新生疾病或者淋巴管新生疾病为、心脑肾血管疾患相关疾病。
  19. 权利要求1-18所述的任一项的用途、方法或多肽，其中，所述多肽为Semaphorin。
  20. 权利要求1-19所述的任一项的用途、方法或多肽，其中，所述多肽与其他预防、抑制剂或治疗剂组合，例如Semaphorin与透明质酸组合，Semaphorin与neomedrol EE组合，Semaphorin和Oders Linderon组合，Semaphorin与脱欧麻组合。
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