Polypeptide for angiogenesis and lymphangiogenesis related diseases and application thereof

Abstract

The invention relates to application of polypeptide in preparation of drugs for preventing, inhibiting and / or treating angiogenesis diseases and lymphangiogenesis diseases, in particular to diseases such as cancer, ophthalmic diseases, arteriosclerosis, heart, brain and kidney vascular diseases, Alzheimer's disease, aging diseases and scar formation.

Description

[0001] This application is a divisional application of the invention patent application with application date of October 12, 2020, application number 202080068534.1, and invention name “A polypeptide for angiogenesis and lymphangiogenesis related diseases and its use”. Technical Field

[0002] The present invention relates to a preventive, inhibitory and / or therapeutic agent containing Semaphorin protein (Semas) and pharmaceutically acceptable components thereof as active ingredients, and its use in preparing a drug for preventing, inhibiting and / or treating angiogenesis diseases and lymphangiogenesis-related diseases, especially in the prevention and treatment of cancer-related diseases, scar formation, cardiovascular diseases, organ aging diseases, Alzheimer's disease, and especially in the prevention and treatment of ophthalmic angiogenesis-related diseases. Background Art

[0003] Human cells maintain their activities and functions by taking in nutrients and oxygen supplied by blood vessels around the cells. The number of necessary cells is also strictly controlled by the original functions of humans, however, cancer cells cannot control this and proliferate very actively. Since cancer cells need a lot of nutrients and oxygen to carry out such activities compared to normal cells, they begin to create new blood vessels, namely neovascularization. The new blood vessels produced by cancer cells have become a source of support for their activities.

[0004] Angiogenesis is the formation of new blood vessels using pre-existing vasculature and plays a vital role in many physiological processes and pathological conditions, including wound healing, cancer, age-related macular degeneration, tumor growth and diabetic retinopathy.

[0005] The following cancers are associated with angiogenesis: breast cancer (especially metastatic breast cancer), colorectal cancer, progressive colorectal cancer, esophageal cancer, superficial esophageal cancer, early esophageal cancer, laryngeal cancer, paranasal malignancies, gastrointestinal stromal tumors (GIST), kidney (renal cell) cancer, liver (adult primary) cancer (especially inoperable liver cancer), lymphoma, lymphangiomatosis / Gorham disease, giant lymphatic malformation, 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 tumors, gynecological malignancies, cervical cancer (especially early invasive cancer), endometrial cancer, bladder cancer (superficial bladder cancer, invasive cancer).

[0006] Recent studies have shown that the expression of these angiogenesis factors has been identified in breast cancer, endometrial cancer, colon cancer, and bladder cancer, and there is a direct correlation between their expression level and prognosis. In addition, the density of angiogenesis in breast cancer tumor tissue is related to metastasis, while angiogenesis is an important factor affecting the prognosis of cancer patients in esophageal cancer, colon cancer, etc. The probability of breast cancer metastasizing to lymph nodes is the highest. Therefore, inhibiting lymphatic proliferation plays an important role in inhibiting breast cancer metastasis. Esophageal cancer may spread to lymph nodes regardless of the degree of progression. Prostate cancer most commonly metastasizes to the lymph nodes around the prostate.

[0007] If cancer cells metastasize to lymph nodes, they may metastasize to distant sites through lymphatic vessels. The difficulty with metastasis to the liver is that there are almost no subjective symptoms at first, and it is very easy to become aggravated. Cancer that has already metastasized is difficult to treat, so early prevention and early treatment are extremely important.

[0008] Currently, several U.S. Food and Drug Administration (FDA)-approved angiogenesis inhibitors for human cancer treatment have anti-angiogenic activity that can delay tumor growth, but recent findings in animal studies have shown the potential for congenital anomalies, which has clarified the potential for complications.

[0009] Side effects of angiogenesis inhibitor therapy include bleeding, arterial thrombosis (causing stroke or heart attack), hypertension, and proteinuria (3-5). Gastrointestinal perforation and gastrointestinal fistulas are also considered side effects of some angiogenesis inhibitors.

[0010] For example, the anti-VEGF antibody Bevacizumab (trade name AVASTIN) cannot be used for patients with squamous cell carcinoma and patients diagnosed with tumor infiltration into large blood vessels due to the risk of bleeding. It cannot be used for patients with brain metastases due to the risk of bleeding in tumor lesions.

[0011] In addition, the existing combination therapy of anti-VEGF preparations and chemotherapy drugs for maintenance treatment still has the financial problem of high drug prices. Even for maintenance treatment, many patients cannot afford the combination of high-priced drugs Avastin and pemetrexed.

[0012] Angiogenic diseases also include: keloid, rheumatoid arthritis (RA), psoriasis, atherosclerosis, tuberous sclerosis, Alzheimer's disease such as vascular Alzheimer's, aging, acute myocardial infarction, cytomegalovirus and chlamydia infection-induced coronary artery disease.

[0013] Due to lifestyle diseases and the super-aging society, arteriosclerosis-related diseases are increasing rapidly. According to the important statistics of the Ministry of Health, Labor and Welfare, arteriosclerosis-related diseases (stroke, myocardial infarction) account for about one-third of the mortality rate, which is comparable to the cause of death from malignant tumors. However, since there are no symptoms until the onset of the disease in most cases, it is too late to find it, so it is extremely important to prevent the occurrence of the disease by taking this drug early.

[0014] Endothelial dysfunction is initially caused by lifestyle diseases (obesity, stress, smoking, lack of exercise, etc.), hypertension, dyslipidemia, diabetes, sleep apnea syndrome, chronic kidney disease, and other diseases, as well as aging, gender, menopause, genetics, etc. that damage endothelial cells. Vascular dysfunction promotes the occurrence of cardiovascular diseases, obesity and other factors make blood vessels narrow and harden, and cholesterol and neutral fat in the blood increase and deposit, aggravating the development of arteriosclerosis. It causes angina pectoris and renal dysfunction, thrombosis causes cerebral infarction and myocardial infarction, and vascular rupture causes cerebral hemorrhage. Arteries begin to harden as aging progresses, causing severe cerebral infarction and myocardial infarction.

[0015] Among these diseases, psoriasis, for example, is not easy to improve despite long-term treatment, and there is currently no basic treatment. The current treatment of RA also relies on angiogenesis inhibitors. When tuberous sclerosis and LAM disease occur together, a drug called sirolimus is currently used, mainly in the United States, but there is no definite opinion on its effectiveness due to various side effects. Severe cases of kidney tumors and lung LAM usually involve life prognosis.

[0016] Ophthalmic angiogenesis diseases include: diabetic fundus angiogenesis, retinal angioma, von Hippel-Lindau disease, superficial soft hemangioma, macular degeneration, myopic choroidal neovascularization, diabetic retinopathy, immature infantile retinopathy, neovascular glaucoma, diabetic maculopathy, retinal vein occlusive disease, intraocular neovascularization associated with uveitis, Behcet's disease, tuberculous uveitis, choroidal neovascularization in high myopia, malignant lymphoma, conjunctival malignant lymphoma, choroidal tumors, cytomegalovirus diseases such as cytomegalovirus retinitis, intraocular malignant tumors such as retinoblastoma, choroidal malignant melanoma, malignant lymphoma occurring in the eyeball, and ocular adnexal tumors such as optic nerve tumors.

[0017] There is currently no drug that can treat traumatic and chemical burn corneal angiogenesis. The only way is corneal transplantation. Angiogenic glaucoma develops as the formation of new retinal blood vessels progresses and spreads throughout the eyeball. The trabecular bundles are covered with new blood vessels, resulting in resistance to the outflow of aqueous humor and increased intraocular pressure. Methods for lowering intraocular pressure include intravenous drip and medical treatment, but the effect is only temporary in any case. Laser treatment leads to a decrease in the amount of blood required by the retina, resulting in a condition of insufficient blood. If the new blood vessels blocking the angle of the chamber are not cured, the intraocular pressure will hardly drop. At this stage, only glaucoma surgery can be performed, but the surgical effect of neovascular glaucoma is not good.

[0018] Tuberculous uveitis is treated with anti-tuberculosis therapy and oral corticosteroids to treat neovascularization. The side effects of corticosteroids cause high intraocular pressure, glaucoma, cataracts, corneal fungal disease, etc., and affect the development of children.

[0019] The treatment of choroidal neovascularization in pathological myopia is retinal photocoagulation, but the effect is poor and complications are frequent. There is no effective data on the efficacy of surgical treatments such as macular transposition and neovascularization. The current clinical anti-VEGF injection drug treatment may be accompanied by worsening vision. Moreover, side effects are difficult to avoid, such as endophthalmitis, stroke, cataract, retinal detachment, retinal pigment epithelial tear, etc. Stroke has been reported in clinical trials at home and abroad. High treatment costs are also a disadvantage of current anti-VEGF injection drugs.

[0020] Treatment for CMV retinitis is oral and intravenous medication, but existing drugs often cause bone marrow suppression and renal insufficiency, making treatment difficult.

[0021] Neoplasia / Gorham disease is clinically treated with steroids, interferon α, propranolol, chemotherapy (vincristine), etc., but the treatment effect is limited. If there is a chest lesion, such as the breast, the prognosis is not good. If the lesion spreads to multiple organs, it causes various symptoms and 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.

[0022] Giant lymphatic malformations (cervical facial lesions) are difficult to recover from any treatment. Not only are they resistant to treatment, but they also cause persistent functional impairment (breathing disorders, eating / swallowing disorders, visual impairment, hearing impairment, etc.) from the moment of birth, requiring lifelong medical attention.

[0023] However, to date, there is still no report clearly indicating the role and function of Sema3A in diseases related to angiogenesis and lymphangiogenesis, especially in cancer, scar formation and ophthalmic diseases. Summary of the invention

[0024] The present invention provides a polypeptide or a fragment containing the polypeptide as an active ingredient for preventing, inhibiting and / or treating angiogenesis-related diseases, especially cancer, ophthalmic diseases, arteriosclerosis, cardiovascular diseases, aging diseases, scar formation, Alzheimer's disease and other diseases, and its use in preparing a drug for preventing, inhibiting and / or treating angiogenesis-related diseases and lymphangiogenesis-related diseases, especially in the prevention and treatment of cancer-related diseases, arteriosclerosis, Alzheimer's disease, scar formation, and especially in the prevention and treatment of ophthalmic angiogenesis-related diseases.

[0025] The present invention also relates to a method for using a polypeptide to prevent, inhibit and / or treat diseases related to angiogenesis and lymphangiogenesis, especially cancer, ophthalmic diseases, arteriosclerosis, cardiovascular diseases, aging diseases, scar formation, Alzheimer's disease and the like.

[0026] The present invention also relates to a polypeptide for preventing, inhibiting and / or treating diseases related to angiogenesis and lymphangiogenesis, especially cancer, ophthalmic diseases, arteriosclerosis, cardiovascular diseases, aging diseases, scar formation, Alzheimer's disease and the like.

[0027] In one embodiment, the polypeptide is selected from any one of the following polypeptides (a) to (c):

[0028] (a) a polypeptide consisting of 70 or more consecutive amino acids in the amino acid sequence of SEQ ID NO: 4, and containing the region 166aa to 235aa in SEQ ID NO: 4;

[0029] (b) a polypeptide consisting of more than 70 consecutive amino acids in the amino acid sequence of SEQ ID NO: 2, and containing the region 166aa to 235aa in SEQ ID NO: 2;

[0030] (c) A polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b).

[0031] The specific implementation manner and meaning of the technical solution of the present invention will be described in detail below.

[0032] definition

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; however, in the event of any conflict, the definitions in this specification shall prevail.

[0034] As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0035] Unless otherwise specified, all percentages (%) in this specification are weight percentages (wt %).

[0036] All numerical values ​​or expressions used in the specification and claims referring to component amounts, process conditions, etc., should be understood to be modified by "about" in all cases. The term "about" when referring to a quantity or a numerical range means that the quantity or numerical range referred to is an approximate value within the experimental variability (or within the statistical experimental error), so that the quantity or numerical range can be within the stated quantity or numerical range, for example. + Varies between 5.

[0037] All ranges involving the same component or property include endpoints, which can be independently combined. Since these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range cited in the application is intended to include all subranges within the range.

[0038] When the invention is directed to physical properties such as molecular weight or to ranges of chemical properties, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "comprising" (and related terms such as "containing" or "including" or "having" or "including") includes embodiments such as any combination of substances, compositions, methods or processes, etc., which "consist of" or "consist essentially of" the described features.

[0039] "And / or" as used in this specification and claims should be understood as "either or both" of the associated components, that is, the components exist jointly in some cases and separately in other cases. Multiple components listed with "and / or" should be understood in the same way, that is, "one or more" associated components. In addition to the components specifically identified by the "and / or" clause, other components may optionally be present, whether related or unrelated to those specifically identified components. Therefore, as a non-limiting example, reference to "A and / or B", when used in conjunction with open-ended text such as "comprising", in one embodiment, may refer to only A (optionally including components other than B); in another embodiment, may refer to only B (optionally including components other than A); in yet another embodiment, refers to A and B (optionally including other components), etc.

[0040] "Or" as used in this specification and claims should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be translated as inclusive, that is, including at least one of a plurality or listed components, but also including more than one, and optionally other unlisted items. Only terms that clearly point to opposites, such as "only one" or "exactly one", or "consisting of..." used in the claims should refer to including exactly one of a plurality or listed components. Generally, the term "or" as used herein is considered to point to exclusive choices (i.e., one or the other but not both) only when there is an exclusive antecedent such as "either", "one of", "only one" or "exactly one".

[0041] It should be understood that in any method claimed herein that includes more than one step or act, the order of the method steps and acts is not necessarily limited to the order of the method steps and acts recited unless explicitly indicated to the contrary.

[0042] The abbreviations used in the present invention have their usual meanings in the fields of food, biology and chemistry.

[0043] A. Angiogenesis Disease

[0044] Angiogenesis: New capillaries are generated from pre-existing blood vessels in the form of sprouts or non-sprouts (or intussusception) mainly through the proliferation and migration of vascular endothelial cells on the basis of existing capillaries and / or venules.

[0045] Specific diseases are listed below:

[0046] Rheumatoid arthritis (RA)

[0047] RA is a chronic inflammatory disease that often invades all joints. The inflammatory synovium of RA has significant new growth of early capillaries and small blood vessels such as post-capillary venules, which are the sites of inflammatory cell infiltration. The new blood vessels also serve as nutrient vessels for the synovium and are deeply involved in the disease pathology. Currently, the treatment of RA also relies on angiogenesis inhibitors.

[0048] Psoriasis

[0049] Nickoloff et al. observed that the production of IL-8 (angiogenic factor) and TSP-1 was reduced in the lesions of psoriasis patients compared with normal skin. Based on the method of culturing epidermal cells established from lesion tissue, they confirmed enhanced angiogenesis from rat corneas.

[0050] arteriosclerosis

[0051] Arteriosclerosis is accompanied by hyperlipidemia, hypertension, aging and other diseases. New blood vessels can be observed near atherosclerotic plaques. When atherosclerotic lesions are formed, PDGF, TNF-α, FGF and TGF-β factors are secreted to induce angiogenesis.

[0052] After endothelial dysfunction occurs, arteriosclerosis occurs, blood vessels become narrower and harder, and cholesterol and neutral fat in the blood increase and deposit inside the blood vessels, aggravating the development of arteriosclerosis.

[0053] When blood vessels become narrowed and hardened, it causes angina pectoris and renal dysfunction; when blood clots form, it causes cerebral infarction and myocardial infarction; when blood vessels rupture, it causes cerebral hemorrhage.

[0054] Tuberous sclerosis

[0055] Tuberous sclerosis complex (TSC) is a renal angiomyolipoma;

[0056] Lymphangioleiomyomatosis (LAM) is a disease that causes hamartomas throughout the body, such as angiofibromas on the face and fundus. Renal angiomyolipomas have many new blood vessels and sometimes rupture due to enlargement. As the number of tumor cells increases, they may become malignant.

[0057] When tuberous sclerosis complex diseases occur with LAM disease, most of them occur in women of childbearing age, and diseases other than LAM are likely to occur in many organs (brain, skin, heart, lungs, kidneys, etc.), accompanied by seizures, skin lesions and other tuberous sclerosis complex symptoms.

[0058] cancer

[0059] Nasal tumors

[0060] Sinus tumors are tumors arising from sinus tissue (mainly mucosal epithelium). Representative benign 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 high recurrence rate and the possibility of malignant transformation. The recurrence rate is about 30%, and most recur within 2 years after surgery. Malignant transformation is considered to be about 10%, usually transformed into squamous cell carcinoma.

[0061] Gynecological malignancies

[0062] The most common gynecological malignancies are cervical cancer, placental hemangioma, and cervical cavernous hemangioma complicated by pregnancy.

[0063] In cerebrospinal cavernous hemangiomas, angiogenesis is deeply involved in the growth of metastatic lesions, which has the greatest impact on prognosis.

[0064] Alzheimer's disease

[0065] Vascular Alzheimer's disease

[0066] Dementia is closely related to blood vessels. Vascular obstruction leads to vascular dementia and Alzheimer's disease. Symptom improvement requires restoration of cerebral blood flow.

[0067] Alzheimer's disease (AD) is one of the dementias. The cause is believed to be the accumulation of amyloid beta (Aβ) in the brain. Immunotherapy in basic and clinical trials has shown that reducing amyloid deposition is useful for improving cognitive function. Antibody or vaccine immunotherapy against Aβ is considered promising for removing Aβ from the brain, but side effects such as angiogenic edema and cerebral microbleeds are evident. The permeability of the blood-brain barrier (BBB) ​​between the brain and blood is closely related to cerebral vascular endothelial cells, and it has been found that the vascular density is also increased in AD patients.

[0068] aging

[0069] Blood vessels throughout the body not only deliver oxygen and nutrients to every corner of the body, but also move inflammatory cells to the site when abnormalities such as tissue damage or inflammation occur, promoting tissue repair. In this process, vascular endothelial cells covering the lumen of blood vessels are activated by TNFα (an inflammatory cytokine) and help inflammatory cells migrate out of the blood vessels and play a role in causing normal inflammatory responses.

[0070] acute myocardial infarction

[0071] Elevated IL-6 levels have been reported in patients with acute myocardial infarction. Plasma IL-6 levels rise within hours after the onset of infarction and peak after 24 to 48 hours ( Figure 2 ). After the increase of IL-6, the CRP in the blood increased. The maximum IL-6 value in the process was significantly correlated with the maximum CRP value.

[0072] Cytomegalovirus-induced coronary artery disease

[0073] The role of cytomegalovirus and chlamydia infection in the development of coronary artery lesions has been noted.

[0074] CMV acts directly on endothelial cells to enhance IL-6 production. Blankenberg et al. suggest that patients with positive CMV antibodies and high blood IL-6 levels are at high risk of future cardiac death, but even if CMV antibodies are positive and blood IL-6 levels are not high, patients are not at risk.

[0075] Ophthalmic angiogenesis diseases

[0076] The representative lesion that damages the transparency of the eye is angiogenesis. The newly formed blood vessels are fragile and prone to repeated bleeding and exudation, forming a proliferative membrane accompanied by the proliferation of other cells, so the visual function is significantly impaired. There is currently no drug that can treat traumatic and chemical burn corneal angiogenesis, and the only way is corneal transplantation.

[0077] Typical examples of intraocular angiogenesis diseases include diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration, and angiogenic glaucoma, which are intractable diseases that lead to blindness. When the retina is in a state of blood deficiency, it will send a signal (a chemical substance called VEGF) to generate new blood vessels through VEGF (neovascularization) to nourish the retina. This "neovascularization" is very easy to rupture and is the root cause of retinal hemorrhage and vitreous hemorrhage.

[0078] Neovascular glaucoma

[0079] Causes of neovascular glaucoma include diabetic retinopathy syndrome, central retinal vein occlusion, and ocular ischemic syndrome.

[0080] A serious and difficult-to-treat type of glaucoma caused by a disease that deteriorates retinal blood flow. When the blood vessels of the retina are damaged by diabetes, blood circulation is blocked, causing severe visual impairment. New blood vessels may block the angle of the chamber, blocking the outlet of the aqueous humor, causing a sudden increase in intraocular pressure, which is usually about 20 mmHg, to 50 mmHg or higher.

[0081] The secondary symptom of intraocular metastasis of malignant lymphoma and conjunctival malignant lymphoma is often the development of angiogenic glaucoma in the eyeball.

[0082] Diabetic Maculopathy

[0083] The macula is located in the center of the retina and is the most important part for observing things. Due to diabetes, capillary hemangiomas often appear near the macula, blood components seep out, and the symptoms caused by macular swelling are called diabetic maculopathy.

[0084] Even in the early stages of retinopathy, vision loss may occur.

[0085] Before the disease progresses seriously, there may be no subjective symptoms, and it is important to use angiogenesis inhibitors early. For example, laser coagulation treatment is currently used in the clinic to prevent angiogenesis in the avascular field. The coagulation scar of photocoagulation retinal tissue degeneration will expand over time, thus affecting macular vision and visual field.

[0086] Omental venous occlusive disease

[0087] Hypertension is one of the causes of arteriosclerosis, but even in the absence of hypertension, arteriosclerosis can develop, and if the root of the retinal vein is blocked, it can become central retinal vein occlusion and cause bleeding throughout the retina. This can lead to sudden visual acuity loss, visual field impairment, degeneration of vision, vitreous hemorrhage, and significant visual acuity loss. Combined with angiogenic glaucoma.

[0088] Uveitis complicated by intraocular neovascularization

[0089] Harada disease causes massive retinal and choroidal detachment, papilledema, and neovascularization of some papillae at the posterior pole, and treatment requires massive steroid therapy to shrink the new vessels.

[0090] Behcet's disease causes neovascularization of the retina and papilla; treatment requires topical steroids and immunosuppressive drugs.

[0091] Choroidal neovascularization in high myopia

[0092] The prevalence of myopic retinopathy is 1.7%, and the incidence in women is about twice that in men. If the eyeball stretches backward and the retina / choroid is damaged, pathological blood vessels (choroidal neovascularization) will develop, causing bleeding and swelling.

[0093] Choroidal tumors

[0094] Choroidal hemangiomas, caused by abnormal proliferation of blood vessels, often occur together with Sturge-Weber syndrome, a systemic disease characterized by multiple hemangiomas. Metastatic choroidal tumors are hematogenous metastatic malignancies that occur in all organs of the body. Choroidal metastases are common in female breast cancer and male lung cancer.

[0095] If the tumor occurs in the macula, a particularly important part of vision, or if the retina around the tumor detaches and reaches the macula, vision may deteriorate. As the tumor grows, the field of vision may be reduced or even lost.

[0096] Cytomegalovirus disease

[0097] CMV retinitis is an opportunistic infection that can occur in immunocompromised patients, such as AIDS patients and patients receiving anticancer therapy. In particular, when the number of CD4+ T cells in the peripheral blood is less than 50 / mm 2 The incidence rate is high.

[0098] Malignant tumors in the eye

[0099] The eye is divided into the eyeball and eye appendages (eyelids, conjunctiva, eye sockets, lacrimal glands, etc.), and tumors can occur in various parts. Poor prognosis may even affect life survival rate.

[0100] Retinoblastoma

[0101] Malignant tumors that appear in the eye of children are treated surgically, and if they progress, the eye needs to be removed. Choroidal melanoma is a malignant tumor that occurs in the uveal tract (choroid, ciliary body, iris) of the eye of adults. If it progresses and the tumor is large, the eye needs to be removed.

[0102] Malignant lymphoma in the eye

[0103] It is a malignant lymphoma of the central nervous system, a subtype of brain lymphoma. It is generally treated with systemic chemotherapy, radiotherapy and other medical treatments, and it is difficult to be completely cured at present. Although some preventive treatment methods have been reported, there is no reliable method for practical application so far.

[0104] Ocular adnexal tumors

[0105] Eyelid tumors (sebaceous gland carcinoma) Tumors that occur in the eyelids are very common among eye tumors, and recurrence requires further surgery or radiotherapy.

[0106] Treatment for lacrimal gland cancer requires complete surgical removal of the tumor; however, even if the tumor is completely removed, it is not possible to prevent subsequent recurrence.

[0107] Ocular adnexal lymphoma

[0108] Ocular sarcomas are tumors that compress the eyeball and are often difficult to remove completely, requiring a combination of surgery, chemotherapy, and radiation therapy. Meningiomas are tumors that arise from the sheath that covers the optic nerve and often result in blindness when the tumor is removed.

[0109] B. Lymphangiogenesis

[0110] Lymphatic vessels are channels through which lymph passes. They collect lymph produced from all corners of the body, filter out bacteria and viruses contained in the lymph, and finally return it to the blood vessels. Many diseases related to lymphatic vessels are difficult to treat.

[0111] In recent years, the molecular mechanisms of angiogenesis in individual development and various pathologies such as tumors and inflammation have been elucidated. On the other hand, studies on the molecular mechanisms of lymphatic system formation in the same vascular organs have gradually identified receptors, their ligands, transcription factors, etc. specifically expressed in lymphatic vessels, but it is difficult to prepare specific antibodies against them and obtain cell lines, which is an obstacle to advancing research. It is suggested that lymphatic vessels not only serve as a circulatory system for excessive drainage of extracellular fluid, but also have a great influence on the immune system, which is important for clarifying pathological conditions such as lymph node metastasis of malignant tumors and lymphedema.

[0112] Lymphatic vessels play an important role in maintaining the homeostasis of the microenvironment in the body, immune response and tumor lymphatic metastasis. Lymphangiogenesis 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 luminal structures to form new lymphatic vessels. In recent years, it has been found that lymphatic endothelial progenitor cells are involved in lymphangiogenesis. Lymphatic endothelial cells specifically express Prox-1, podopiann, VEGFR-3LYVE-1, etc. These factors and receptors regulate lymphangiogenesis. VEGF-C / VEGFR-3 or VEGF-D / VEGFR-3 signaling pathways play an important role in the process of lymphangiogenesis. VEGF-C, VEGF-D and VEGFR-3 can be used as targets for gene therapy, which is expected to treat lymphangiogenesis disorders, as well as anti-post-transplant immune rejection and anti-tumor lymphatic metastasis.

[0113] Lymphangiomatosis / Gorham disease (designated as refractory disease 277 in Japan)

[0114] It is an incurable disease with unknown causes of diffuse abnormally dilated lymphatic tissue that invades organs throughout the body, such as bones and the chest (lungs, mediastinum, heart), abdomen (abdomen, spleen), skin, and subcutaneous tissue. Many cause osteoporosis in children and adolescents. Gorham disease is pathologically identified by irregular dilated lymphatic vessels. Lymphangiomatosis / Gorham disease is also known as diffuse lymphangiomatosis, Gorham-Stout syndrome, and massive osteolysis.

[0115] For example, lymphatic proliferation in esophageal squamous cell carcinoma can predict metastasis based on lymphangiogenesis. Among gastrointestinal tumors, esophageal cancer is highly malignant compared with colon cancer and gastric cancer and is one of the tumors with a poor prognosis.

[0116] 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, while lymphangiogenesis in tumor tissue is related to lymph node metastasis. Chikugo 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. The number of lymphatic vessels in the mucosal lamina propria increased significantly at the edge of the tumor. A significant increase in lymphatic vessels was also observed in the percentage of lymphatic vessels to the number of blood vessels.

[0117] Giant lymphatic malformation

[0118] (Neck and Facial Lesions) are giant neoplastic lymphangiogenic abnormalities that occur congenitally in the face, mouth, pharynx, and head and neck.

[0119] Lymphatic malformation (lymphangioma) is a mass composed of large and small lymphatic cysts, with vascular lesions. Intraorbital lymphangioma may cause intraorbital bleeding or infection leading to blindness. If the lymphatic lesions extend to the skin and mucous membranes, it manifests as localized lymphangioma, with symptoms of repeated lymphatic fistulas, bleeding, and infection. Infection and bleeding, repeated acute swelling and inflammation may occur in any part of the lesion during the course of the disease.

[0120] In giant facial lesions, due to tumor formation, discoloration and deformation, they show a high degree of ugliness, affecting the patient's adaptability to social life and limiting the quality of life throughout life.

[0121] C. Peptide

[0122] Semaphorin3A (Sema3A)

[0123] The full length of the amino acid sequence shown by SEQ ID NO: 2 or SEQ ID NO: 4 (i.e., Semaphorin 3A protein).

[0124] Amino acid sequence 2:

[0125]

[0126]

[0127]

[0128]

[0129] Amino acid sequence 4:

[0130]

[0131]

[0132]

[0133]

[0134] Polypeptide (a): A polypeptide consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 4, and containing the region from 166aa to 235aa in SEQ ID NO: 4.

[0135] Polypeptide (b): A polypeptide consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2, and containing the region from 166aa to 235aa in SEQ ID NO: 2.

[0136] Polypeptide (c): The polypeptide is a polypeptide in which a small number (preferably 1 to several) of amino acid residues of the polypeptide of (a) or (b) are substituted, deleted and / or inserted. The peptide is a polypeptide having 80% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more homology with the original sequence.

[0137] Generally, in a protein, even if a small number of amino acid residues in the amino acid sequence of the protein are replaced, deleted or inserted, they can have almost the same function as the original protein. This is well known to those skilled in the art. Therefore, the region consisting of the polypeptide of (c) above can also produce the physiological activity of Semaphorin3A protein, similar to the above 70aa region.

[0138] "Homology" of amino acid sequences means aligning 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 is expressed as a percentage by dividing the number of matching amino acid residues by the total number of amino acid residues. During alignment, gaps are appropriately inserted into one or both of the two sequences to be compared as needed.

[0139] This alignment of sequences can be performed using known programs, such as BLAST, FASTA, CLUSTAL W, etc. When gaps are inserted, the total number of amino acid residues is the number of residues for which one gap is counted as one amino acid residue. When the total number of amino acid residues counted in this manner is different between the two sequences to be compared, the homology (%) is the total number of amino acid residues in the longer sequence, which is calculated by dividing.

[0140] The 20 amino acids that constitute natural proteins are neutral amino acids with low polar 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) It is known that they can be combined together and substitutions between them generally do not change the properties of the polypeptide.

[0141] Therefore, when the amino acid residues in the above polypeptide (a) or (b) are substituted, a polypeptide useful as an active ingredient for preventing, inhibiting or treating corneal diseases in the conjunctiva can be obtained by substituting between these respective groups. Alternatively, the possibility of maintaining the preventive, inhibitory or therapeutic activity of corneal damage is increased.

[0142] Specifically, the above-mentioned polypeptide (c) is a polypeptide having 80% or higher sequence homology with a polypeptide consisting of the full length of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 (i.e., Semaphorin3A protein), or a polypeptide having 80% or higher sequence homology with a Sema3A protein fragment containing a 70 amino acid region.

[0143] Therefore, the active ingredient polypeptide c is a polypeptide consisting only of these amino acids, or a polypeptide obtained by adding amino acids or polypeptides to one or both ends of polypeptide (c), and / or a polypeptide having preventive, inhibitory or therapeutic activity for corneal diseases or corneal damage converted into blood vessels, etc.

[0144] These specific examples are similar to the above-mentioned active ingredient polypeptides a and b, and are not particularly limited, for example, a polypeptide consisting of the full length of SEQ ID NO: 2 or 4 (i.e., Sema3A protein) and a polypeptide having a sequence homology of 80% or more, a polypeptide having a sequence homology of 80% or more with a Sema3A protein fragment having corneal disease or corneal disease or corneal damage or preventive, inhibitory or therapeutic activity, and a region including 70 amino acids having a sequence homology of 80% or more. And, a chimeric protein obtained by fusing a region having a secretion domain with other regions of a secreted semaphorin other than Semaphorin3A.

[0145] As the active ingredient polypeptide c, a polypeptide having a higher homology in the 70aa region than the homology of the entire region consisting of the polypeptide of (c) is particularly preferred. For example, when the active ingredient polypeptide c is a polypeptide having 80% homology with a polypeptide having an amino acid sequence of SEQ ID NO: 2, a polypeptide having more than 80% homology in the 70aa region is preferred, especially 70aa. The homology in this region is 90% or more, more preferably 95% or more, further preferably 98% or more, and more preferably, a polypeptide having the same sequence in the 70aa region is preferred. In addition, for example, the active ingredient polypeptide c is a chimeric protein in which a region having 80% sequence homology with the Sema domain of the human Sema3A protein and a region other than the Sema domain of a secreted Semaphorin other than Sema3A are fused. In some cases, it is preferred that the homology in the 70aa region of the Sema domain is greater than 80%, particularly 90% or higher homology in the 70aa region, more preferably 95% or higher, still more preferably 98% or higher, more preferably 90% or higher. Preferred are chimeric proteins, wherein the sequences in the 70aa region are identical.

[0146] Among the above-mentioned active ingredient polypeptides a to c, the polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is particularly a polypeptide having 80% or more, preferably 90% or more, more preferably 95% or more, more preferably, 98% or more homology, and among them, as described above, a polypeptide having a homology higher than that of the entire polypeptide in a 70aa region is preferred. Most preferably, 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.

[0147] Typically, in a drug comprising a polypeptide, in order to enhance the stability of the polypeptide in vivo, a sugar chain or polyethylene glycol (PEG) chain is added to the polypeptide, or at least one amino acid constituting the polypeptide. The technology of using D-amino acids as a part and adding an Fc domain is a well-known and used technology. By adding a sugar chain or a PEG chain or using a D-amino acid as at least a part of the amino acids constituting the polypeptide, it becomes difficult to be degraded by peptidases in vivo, and the polypeptide is halved in vivo for a longer time.

[0148] The polypeptide used in the present invention may be one 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. Unless the context clearly indicates otherwise, the term "polypeptide" in the specification and claims is used in a sense that also includes those modified for in vivo stabilization.

[0149] The conversion of glycosyl groups to polypeptides is well known, for example, sugar chains can be bound to the N-terminus, the C-terminus or an amino acid therebetween, but preferably to the N-terminus or the 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. Sugar chains are preferably monosaccharides to tetrasaccharides, more preferably disaccharides or trisaccharides. Sugar chains can be directly or through a spacer structure such as a methylene chain having about 1-10 carbon atoms and attached to the free amino group or carboxyl group of the polypeptide.

[0150] The PEG chain can be linked to the N-terminus, the C-terminus or an amino acid therebetween, and typically one or two PEG chains are linked to a free amino group or carboxyl group on the polypeptide. The molecular weight of the PEG chain is not particularly limited, but is typically about 3000 to about 7000, preferably about 5000.

[0151] It is also well known that at least a part of the amino acids constituting the polypeptide is in the D-form, for example, although some of the amino acids constituting the polypeptide may be in the D-form, all the amino acids constituting the polypeptide are D-type amino acids rather than only amino acids forming the D-form, because the activity of the polypeptide is not inhibited. It is also well known that a method for adding an Fc domain to a polypeptide is preferably used, and can be produced, for example, using a commercially available Fc fusion protein expression vector such as pFUSEN-Fc (manufactured by InvivoGen).

[0152] Semaphorin 3A used as the active ingredient of the present invention can be easily prepared by conventional methods using, for example, a commercially available peptide synthesizer. Moreover, it can be easily prepared using known genetic engineering techniques. For example, the cDNA of the Semaphorin gene is prepared by RT-PCR from RNA extracted from tissues expressing the Semaphorin3A gene, and the cDNA of the full length or a desired portion is integrated into an expression vector, which can be introduced to obtain the target polypeptide. Extraction of RNA, RT-PCR, integration of cDNA into a vector, and introduction of the vector into a host cell can be performed by known methods.

[0153] In addition, the vectors and host cells to be used are also well known, and various vectors and host cells are commercially available. Methods for producing chimeric proteins are also well known in the art, and chimeric proteins can be produced, for example, by the methods described in the above-mentioned article by Koppel et al. Moreover, the above-mentioned stabilization modification can be easily performed by known methods described in the above-mentioned various documents. DETAILED DESCRIPTION OF THE INVENTION

[0155] 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):

[0156] (a) a polypeptide consisting of 70 or more consecutive amino acids in the amino acid sequence of SEQ ID NO: 4, and containing the region 166aa to 235aa in SEQ ID NO: 4;

[0157] (b) a polypeptide consisting of more than 70 consecutive amino acids in the amino acid sequence of SEQ ID NO: 2, and containing the region 166aa to 235aa in SEQ ID NO: 2;

[0158] (c) A polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b).

[0159] In one embodiment, the polypeptide of (c) above is a polypeptide in which a small amount (preferably 1 to several) of amino acid residues of the polypeptide of (a) or (b) are substituted, deleted and / or inserted. The peptide is a polypeptide having 80% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more homology with the original sequence.

[0160] In one embodiment, the present invention also relates to a polypeptide (hereinafter referred to as "active ingredient polypeptide c" for convenience), which has a region comprising the polypeptide of (c) above and has preventive, inhibitory or therapeutic activity against corneal diseases or corneal damage. Similar to the above-mentioned active ingredient polypeptides a and b, they can be used to prepare the preventive, inhibitory or therapeutic agents of the present invention.

[0161] In one embodiment, when the amino acid residues in the polypeptide of (a) or (b) above are substituted, the polypeptide (c) used as an active ingredient can be obtained by mutual substitution between the groups defined herein, as a preventive, inhibitory or therapeutic agent of the present invention.

[0162] Specifically, the above-mentioned polypeptide (c) is a polypeptide having 80% or higher sequence homology with a polypeptide consisting of the full length of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 (i.e., Semaphorin3A protein), or a polypeptide having 80% or higher sequence homology with a Sema3A protein fragment containing a 70 amino acid region.

[0163] In one embodiment, the active ingredient polypeptide c is a polypeptide consisting 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 a polypeptide having preventive, inhibitory or therapeutic activity for corneal diseases or corneal damage.

[0164] In one embodiment, the active ingredient polypeptide c is similar to the above-mentioned active ingredient polypeptides a and b, and is not particularly limited, for example, a polypeptide consisting of the full length of SEQ ID NO: 2 or 4 (i.e., Sema3A protein) or a sequence having 80% or more sequence homology. A polypeptide having homology, a polypeptide having 80% or more sequence homology with a Sema3A protein fragment having corneal damage or preventive, inhibitory or therapeutic activity for corneal diseases, having homology with 80% or more of a sequence including a 70 amino acid region, and a chimeric protein obtained by fusing a region having a secretion domain with other regions of a secreted semaphorin other than Semaphorin3A.

[0165] In one embodiment, as the active ingredient polypeptide c, a polypeptide having a higher homology in the 70aa region than the homology of the entire region consisting of the polypeptide of (c) is particularly preferred. For example, when the active ingredient polypeptide c is a polypeptide having 80% homology with the polypeptide having the amino acid sequence of SEQ ID NO: 2, a polypeptide having more than 80% homology in the 70aa region is preferred, especially 70aa. The homology in this region is 90% or more, more preferably 95% or more, and further preferably 98% or more.

[0166] More preferably, a polypeptide in which the sequences in the 70aa region are identical is preferred. In addition, for example, the active ingredient polypeptide c is a chimeric protein in which a region having 80% sequence homology with the Sema domain of the human Sema3A protein and a region other than the Sema domain of a secreted Semaphorin other than Sema3A are fused. In some cases, it is preferred that the homology in the 70aa region in the Sema domain is greater than 80%, and in particular, the homology in the 70aa region is 90% or higher, more preferably 95% or higher, still more preferably 98% or higher, and most preferably 90% or higher. Preferred is a chimeric protein in which the sequences in the 70aa region are identical.

[0167] In one embodiment, among the above-mentioned active ingredient polypeptides a to c, the polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is particularly a polypeptide having 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 98% or more homology, and among them, as described above, it is preferred that the polypeptide has a higher homology in the 70aa region than the entire polypeptide. Most preferably, 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.

[0168] In one embodiment, the polypeptide used in the present invention may be a polypeptide that has undergone a known in vivo stabilization modification, as long as it has the preventive, inhibitory or therapeutic activity of the present invention. Preferably, it is a glycosylated polypeptide, for example, the sugar chain can be bound to the N-terminus, the C-terminus or an amino acid therebetween, but preferably to the N-terminus or the 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, more preferably a disaccharide or a trisaccharide. The sugar chain can be directly or through a spacer structure such as a methylene chain having about 1-10 carbon atoms and connected to the free amino group or carboxyl group of the polypeptide.

[0169] In one embodiment, the polypeptide used in the present invention is also preferably a PEGylated polypeptide, and the PEG chain can be connected to the N-terminus, the C-terminus or an amino acid therebetween, and usually one or two PEG chains are connected to a free amino group or a carboxyl group on the polypeptide. The molecular weight of the PEG chain is not particularly limited, but is usually about 3000 to about 7000, preferably about 5000.

[0170] In one embodiment, wherein at least a portion of the amino acids constituting the polypeptide used in the present invention is in D form; preferably, an Fc domain is added to the polypeptide, and more preferably, the polypeptide is produced using a commercially available Fc fusion protein expression vector such as pFUSEN-Fc (manufactured by InvivoGen).

[0171] In one embodiment, Semaphorin 3A used as an active ingredient of the present invention can be easily prepared by conventional methods using, for example, a commercially available peptide synthesizer. Furthermore, it can be easily prepared using known genetic engineering techniques.

[0172] For example, cDNA of the Semaphorin gene is prepared by RT-PCR from RNA extracted from a tissue expressing the Semaphorin3A gene, and the full-length or desired portion of the cDNA is integrated into an expression vector, which can be introduced to obtain the target polypeptide. Extraction of RNA, RT-PCR, integration of cDNA into a vector, and introduction of the vector into a host cell can be performed by known methods.

[0173] Semaphorin 3A is an active ingredient in the preventive, inhibitory or therapeutic agent of the present invention, and is effective for preventing, inhibiting or treating angiogenesis / lymphangiogenesis diseases.

[0174] The present invention also relates to a preventive, inhibitory or therapeutic agent, which comprises the above polypeptide (preferably Semaphorin3A) or a pharmacologically acceptable excipient, stabilizer, preservative, buffer suitable for each administration form. Optionally, additives such as solubilizers, emulsifiers, diluents, tonicity agents, etc. can also be appropriately mixed to prepare the preparation. The above-mentioned preparation methods and additives that can be used are well known in the field of pharmaceutical preparations, and any method and additive can be used.

[0175] The present invention also relates to the use of the aforementioned polypeptide in the preparation of a drug for preventing, inhibiting and / or treating angiogenesis diseases and lymphangiogenesis diseases. Preferably, it also relates to the use of the aforementioned polypeptide in the preparation of a drug for preventing, inhibiting and / or treating cancer-related diseases, arteriosclerosis, Alzheimer's disease, scar formation, and aging-related diseases.

[0176] Preferably, the polypeptide is selected from any one of the following polypeptides (a) to (c):

[0177] (a) a polypeptide consisting of 70 or more consecutive amino acids in the amino acid sequence of SEQ ID NO: 4, and containing the region 166aa to 235aa in SEQ ID NO: 4;

[0178] (b) a polypeptide consisting of more than 70 consecutive amino acids in the amino acid sequence of SEQ ID NO: 2, and containing the region 166aa to 235aa in SEQ ID NO: 2;

[0179] (c) A polypeptide having 80% or more sequence homology with the polypeptide of (a) or (b), preferably 90% or more, more preferably 95% or more, still more preferably 98% or more homology, and particularly preferably a chimeric protein, wherein the sequences in the 70aa region are identical.

[0180] More preferably, the polypeptide is a glycosylated polypeptide;

[0181] Also preferably, the polypeptide is a PEGylated polypeptide; optionally, the molecular weight of the PEG chain is from about 3000 to about 7000, preferably about 5000.

[0182] In one embodiment, the angiogenesis disease or lymphangiogenesis disease is cancer.

[0183] In one embodiment, the cancer is one or more selected from the following diseases: breast cancer, (especially metastatic breast cancer), colon cancer, colorectal cancer, progressive colorectal cancer, esophageal cancer, superficial esophageal cancer, early esophageal cancer, laryngeal cancer, paranasal malignancies, gastrointestinal stromal tumors (GIST), kidney (renal cell) cancer, liver (adult primary) cancer (especially inoperable liver cancer), lymphoma, lymphangiomatosis / Gorham disease, giant lymphatic malformation, 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 tumors, gynecological malignancies, cervical cancer (especially early invasive cancer), endometrial cancer, bladder cancer (superficial bladder cancer, invasive cancer).

[0184] In one embodiment, the angiogenesis disease or lymphangiogenesis disease is an ophthalmic angiogenesis disease or lymphangiogenesis disease.

[0185] In one embodiment, the above-mentioned ophthalmic angiogenesis disease or lymphangiogenesis disease is selected from one or more of the following diseases: diabetic angiogenic fundus disease, retinal angioma, von Hippel-Lindau disease, superficial soft hemangioma, macular degeneration, myopic choroidal neovascularization, diabetic retinopathy, immature infantile retinopathy, neovascular glaucoma, diabetic maculopathy, retinal vein occlusive disease, intraocular neovascularization associated with uveitis, Behcet's disease, tuberculous uveitis, choroidal neovascularization in high myopia, malignant lymphoma, conjunctival malignant lymphoma, choroidal tumors, cytomegalovirus diseases such as cytomegalovirus retinitis, intraocular malignant tumors such as retinoblastoma, choroidal malignant melanoma, malignant lymphoma occurring in the eyeball, and ocular adnexal tumors such as optic nerve tumors.

[0186] In one embodiment, the neovascular glaucoma includes retinal angiogenesis caused by systemic diabetes and vascular and lymphatic hypofunction caused by secondary angiogenic glaucoma.

[0187] In one embodiment, the angiogenesis disease or lymphangiogenesis disease is one or more diseases selected from the following: keloid, rheumatoid arthritis (RA), psoriasis, arteriosclerosis, tuberous sclerosis, Alzheimer's disease such as vascular Alzheimer's, aging, acute myocardial infarction, cytomegalovirus and chlamydia infection-induced coronary artery disease.

[0188] In one embodiment, the angiogenesis disease or lymphangiogenesis disease is a disease associated with scar tissue formation.

[0189] In one embodiment, the above-mentioned diseases associated with scar tissue formation are one or more diseases selected from the following: diseases associated with scars, ulcers, wounds, diseases caused by necrosis caused by infarction, and tissue defects of various organs.

[0190] In one embodiment, the angiogenesis disease or lymphangiogenesis disease is one or more diseases selected from the following: aging of organs, ischemic state caused by decreased vascular function, aging of blood vessels and lymphatic vessels, aging of organs.

[0191] In one embodiment, the above-mentioned organ aging diseases include: vascular dementia and Alzheimer's disease.

[0192] In the embodiments of the present invention, the cause of the angiogenesis / lymphangiogenesis disorder is not particularly limited, and may be a locally occurring disorder or a systemic disorder.

[0193] In one embodiment, the preventive and / or therapeutic agent of the present invention can be composed of Semaphorin 3A alone, or composed of pharmacologically acceptable excipients, stabilizers, preservatives, buffers suitable for each dosage form. In addition, additives such as solubilizers, emulsifiers, diluents, tonicity agents, etc. can be appropriately mixed to prepare the preparation. And the preparation method can use any method and additives.

[0194] The administration target of the preventive, inhibitory or therapeutic agent of the present invention is a mammal, and examples include humans, dogs, cats, rabbits, hamsters, etc. The use of the aforementioned polypeptide of the present invention (preferably Semaphorin 3A) from the same species as the patient to be prevented, inhibited or treated is considered to be more effective for preventing, inhibiting or treating it, and is also desirable from the perspective of safety in clinical applications. Therefore, for example, when the administration target of the preventive, inhibitory or therapeutic agent of the present invention is human, a preventive, inhibitory or therapeutic agent comprising the above-mentioned active ingredient polypeptide b as an active ingredient is particularly preferred.

[0195] The preventive, inhibitory or therapeutic agent for ophthalmic diseases of the present invention can be used by administering to the cornea to be prevented or the corneal lesion to be treated. As the administration method, local administration (eye drop administration on the cornea, subconjunctival injection administration, etc.) and the like can be mentioned. The dosage form includes contact lens type, eye ointment, injection, eye drops, etc.

[0196] In the contact lens type, the aforementioned polypeptide of the present invention (preferably Semaphorin 3A) may 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 may be applied to the cornea.

[0197] The dosage of the aforementioned polypeptide (preferably Semaphorin 3A) of the present invention can be appropriately selected according to symptoms, age, body weight, administration method, etc., and is not particularly limited.

[0198] The unit dose of the aforementioned polypeptide of the present invention (preferably Semaphorin 3A) is about 6000 to 60000 U, preferably about 4000 to 40000 U, and more preferably about 500 to 5000 U.

[0199] In one embodiment, in the case of eye drops, the eye drop dosage 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. Preferably, in one embodiment, the eye drop concentration is about 10 ng / ml to about 1000 ng / ml, for example, about 10 ng / ml, about 100 ng / ml, about 300 ng / ml, about 1000 ng / ml. The above dosage form and dosage 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 the symptoms.

[0200] In the case of subconjunctival injection, the amount of the aforementioned polypeptide of the present invention (preferably Semaphorin 3A) is generally about 500 to 5000 U, preferably about 500 to 5000 U per day, and can be administered once or dividedly several times to a subject animal.

[0201] In one embodiment, the amount of subconjunctival injection can be about 0.001 to 10 ng / day, about 0.05 to 5 ng / day. Preferably, in one embodiment, 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. Preferably, administration can be performed regularly, once or several times a day, or once or several days a day, for several days to several months, depending on the degree of improvement of the symptoms.

[0202] The aforementioned polypeptide of the present invention (preferably Semaphorin 3A) can also be prepared into the following dosage forms:

[0203] In one embodiment, the dosage form is an eye drop for treating dry eye, keratitis, conjunctivitis, conjunctival lymphoma, angiogenesis inhibition, corneal chemical burns, corneal epithelial healing, meibomian gland carcinoma: wherein,

[0204] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0205] The concentration of active ingredients is about 100-300 ng / ml.

[0206] In another embodiment, the dosage form is an eye drop for treating angiogenesis inhibition, corneal chemical burns, conjunctival lymphoma, corneal epithelial healing, and meibomian gland carcinoma: wherein,

[0207] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0208] Active ingredient concentration: about 100ng / ml;

[0209] Specification: 5ml / bottle, containing active ingredients: 500ng / bottle;

[0210] A total of 125 drops / bottle; 0.040ml / drop.

[0211] In another embodiment, the dosage form is an eye drop for treating dry eye, keratitis, conjunctivitis, conjunctival lymphoma, angiogenesis inhibition, corneal chemical burns, corneal epithelial healing, meibomian gland carcinoma: wherein,

[0212] The concentration of active ingredients in eye drops is: about 300ng / ml;

[0213] Specification: 5ml / bottle, containing the aforementioned polypeptide (preferably Sema3A) 1500ng / bottle;

[0214] A total of 125 drops / bottle; 0.040ml / drop.

[0215] In one embodiment, 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 burns, and meibomian gland carcinoma:

[0216] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0217] Active ingredient concentration: about 100-300 ng / ml;

[0218] Specifications: 3.5 g / piece; contains 0.15% of the aforementioned polypeptide (preferably Sema3A) / piece.

[0219] In one embodiment, the dosage form is a corneal soft contact film, which is used to inhibit inflammation and pathological angiogenesis and lymphangiogenesis and normalize their functions, repair corneal chemical burn scars, and maintain normal corneal function;

[0220] The corneal soft contact film is also used to treat refractory corneal, conjunctival, and eyelid diseases such as chronic conjunctivitis, intractable dry eye, photoelectric conjunctivitis, corneal chemical burns, corneal laser burns, Stevens Johnson disease inflammation, erosive keratitis, and meibomian gland cancer:

[0221] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0222] Active ingredient concentration: about 100-300ng / ml;

[0223] Contains the aforementioned polypeptide (preferably Sema3A) 0.05% to 0.15% per tablet;

[0224] Specifications: Diameter: 13.5mm-14.5mm; Thickness: 0.01mm-0.09mm.

[0225] The corneal soft contact medicine film has excellent moisture retention, water-absorbing substances and viscoelasticity, which help the corneal soft contact medicine film to exert rapid and long-lasting effects.

[0226] In one embodiment, the dosage form is an injection solution, which is used to inhibit inflammation and pathological blood vessels, normalize lymphangiogenesis and repair corneal chemical burn scars:

[0227] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0228] Active ingredient concentration: about 100-300ng / ml;

[0229] Specifications: 0.5 ml / tube; contains 50 ng to 150 ng of the aforementioned polypeptide (preferably Sema3A) / tube.

[0230] The injection is used to reduce inflammatory cells, control symptoms, restore normal tissue function, inhibit pathological angiogenesis / lymphangiogenesis, and normalize vascular / lymphatic function. Preferably, the injection is also used for the following inflammation-related diseases:

[0231] Postoperative inflammation in ophthalmology, spinal edema, prevention of adhesion after fallopian tube surgery, prevention of peritoneal adhesion after surgery, Senear-Usher syndrome, subacute thyroiditis, thyrotoxicosis, severe hepatitis thyroid poisoning, subacute eczema, allergic dermatitis, allergic rhinitis, penile induration, ulcerative colitis, symptomatic treatment of keratitis, hay fever, numismatic eczema, symptomatic treatment of orbital pseudotumor, symptomatic treatment of blepharitis, arthritis psoriasis, improvement of systemic condition in terminal cancer, bronchospasm, acute eczema, acute otitis media, acute leukemia, tendency of hemorrhagic coagulation factor disorder, scleroderma, symptomatic treatment of scleritis, Guillain-Barré syndrome, sudden onset of acrodermatitis, vasomotor rhinitis, erythema nodosum, periarteritis nodosa, Symptomatic treatment of meningitis, Prevention of keloids, Focal enteritis, Symptomatic treatment of iridocyclitis, Eosinophilic granuloma, Laryngitis, Laryngeal edema, Erythema, Erythroderma,, Reticulocyte sarcomatosis, Autosomal dermatitis, Eustachian tube stenosis, Optic myelitis, Eczema / dermatitis group, Purpura, After ENT surgery, Juvenile rheumatoid arthritis, Improvement of general condition in severe debilitating diseases, Asthmatic bronchitis in children, Seborrheic dermatitis, Stevens-Johnson disease, Myeloretinitis, Contact dermatitis, Asthmatic bronchitis, Systemic systemic lupus erythematosus, Systemic vasculitis, Prostate cancer, Early keloids, Hyperplastic pemphigus, Aortitis syndrome, Polymyositis, Multiple sclerosis, Multiple myeloma, Polyarteritis, Vegena Granulomatous disease, toxic eruption, gouty arthritis, finger dermatitis Düring herpetic dermatitis, refractory stomatitis, refractory glossitis, recurrence and metastasis of breast cancer, nephropathy, nephrotic syndrome, mucocutaneous syndrome, encephalitis, encephalomyelitis, cerebral edema, pustular psoriasis, pulmonary fibrosis, nasal folds, non-infectious chronic arthritis, dermatomyositis, cutaneous stomatitis, cutaneous reticular disease, cutaneous leukemia, diffuse interstitial pneumonia, sinusitis, Fuchs syndrome, Hebra red rash, osteoarthritis, radiation pneumonitis, Hodgkin's disease, chronic eczema, chronic otitis media, immune hemolytic anemia, symptomatic treatment of retinal vasculitis, symptomatic treatment of choroiditis, drug allergy, drug rash, Reiter syndrome, pemphigus foliaceus, rheumatic carditis, rheumatoid arthritis Rheumatic spondylitis, Limb arthritis in rheumatoid spondylitis, Polymyalgia rheumatoid, Rheumatic fever, Lipschutz acute vulvar ulcer, Chronic discoid lupus erythematosus, Organ transplant, Genital eczema, Chemical allergy, Chemical addiction, Eczema / dermatitis of the external auditory canal, Laryngeal polyps, Laryngeal nodules, Otitis eczema / dermatitis, Tissue transplant, Eczema / dermatitis of the nasal vestibule, Eczema / dermatitis around the nasal wing, Drug addiction, Anal eczema, Otitis media with exudation, Symptomatic treatment of uveitis, Corrosive esophagitis, Malignant alopecia, Non-infectious bursitis, Non-infectious frozen shoulder, Non-infectious tendinitis, Non-infectious peritonitis, Non-infectious tendinitis, Active cirrhosis, Palmoplantar pustulosis, Behcet's disease, Symptomatic treatment of external eye inflammation, Symptomatic treatment of anterior segment inflammatory diseases,Symptomatic treatment of intraocular inflammation, Symptomatic treatment of inflammatory diseases of the orbit, Symptomatic treatment of inflammatory diseases of the eye, Milk / infantile / pediatric eczema, Cirrhosis with refractory ascites, Congestive heart failure, Cirrhosis with cholestasis, Chronic lymphocytic leukemia, Lymphosarcoma, Malignant lymphoma. ,

[0232] In one embodiment, the dosage form is an oral solution, which is used to treat respiratory allergies and inflammation and restore normal mucosal function:

[0233] The active ingredients are: Sema3A;

[0234] Active ingredient concentration: about 100-300 ng / ml;

[0235] Specifications: (1) 10 ml or (2) 100 ml;

[0236] The aforementioned polypeptide (preferably Sema3A) is 0.05% to 0.15% per mouse.

[0237] In one embodiment, the dosage form is a mouthwash, which is used to treat oral and respiratory allergies and inflammation and restore the normal function of the mucosa:

[0238] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0239] Active ingredient concentration: about 100-300 ng / ml;

[0240] Specifications (1) 5.0ml / piece;

[0241] The aforementioned polypeptide (preferably Sema3A) is 0.05% to 0.15% per mouse.

[0242] In one embodiment, the dosage form is a drug film adhesive sheet, which is used for the treatment of mucosal diseases, skin diseases, burns / trauma and other wounds:

[0243] Active ingredient concentration: about 100-300 ng / ml;

[0244] Specification: 0.05g / piece, the aforementioned polypeptide (preferably Sema3A) 0.05% to 0.15% / piece;

[0245] Among them, the drug film adhesive sheets include: ① oral adhesive sheets, ② gynecological mucosal adhesive sheets; ③ mechanical trauma, chemical trauma, ulcer and other adhesive sheets.

[0246] Preferably, 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) to prepare Sema3A oral adhesive tablets by direct tableting.

[0247] In one embodiment, the dosage form is a cream, which is used to treat mucosal diseases, skin diseases, burns / trauma and other wounds:

[0248] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0249] Active ingredient concentration: about 100-300 ng / ml;

[0250] Specification: 5.0 g / piece, the aforementioned polypeptide (preferably Sema3A) 0.05% to 0.15% / piece.

[0251] In one embodiment, the dosage form is a suppository for the treatment of gynecological disorders, hemorrhoids:

[0252] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0253] Active ingredient concentration: about 100-300 ng / ml;

[0254] Specifications: 0.15 g / piece; the aforementioned polypeptide (preferably Sema3A) 0.05% to 0.15% / piece.

[0255] In one embodiment, the dosage form is a wound care dressing:

[0256] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0257] Active ingredient concentration: about 100-300 ng / ml;

[0258] Specification: 5.0 g / piece; the aforementioned polypeptide (preferably Sema3A) 0.05% to 0.15% / piece.

[0259] The above dressings can be divided into the following categories:

[0260] ① Hydrogel wound dressings: hydrogel wound dressings for treating laser burns and scalds, and medical colloid dressings with antibacterial and healing functions;

[0261] ②Liquid wound dressing;

[0262] ③Dressings for wound care.

[0263] In one embodiment, the dosage form is a spray, wherein:

[0264] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0265] Active ingredient concentration: about 100-300 ng / ml;

[0266] Specification: 5.0 ml / piece, the aforementioned polypeptide (preferably Sema3A) 0.05% to 0.15% / piece.

[0267] The spray can be divided into the following categories: nasal spray; oral spray; gynecological spray: a biological preparation used to treat gynecological inflammation.

[0268] In one embodiment, the dosage form is a film-coating agent, which is used for the treatment of mucosal diseases, skin diseases, burns / trauma and other wounds:

[0269] The active ingredient is: the aforementioned polypeptide (preferably Semaphorin 3A);

[0270] Specification: 5.0 g / piece; containing 0.05% to 0.15% of the aforementioned polypeptide (preferably Sema3A) / piece;

[0271] The concentration of active ingredients is: 100-300ng / ml.

[0272] The preventive, inhibitory or therapeutic agent of the present invention can be used alone or in combination with other preventive, inhibitory 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, etc.

[0273] Take the above-mentioned eye ointment as an example, such as Semaphorin 3A / hyaluronic acid ointment, Semaphorin 3A / neomedrolEE ointment, Semaphorin 3A / Oders Linderon ointment, Semaphorin 3A / Oders Linderon 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 adrenocortical hormone drugs, and a high anti-inflammatory effect can be expected by combining it with Semaphorin 3A. The same is true for the above-mentioned Linderon A ointment. The component of adrenocortical hormone can be appropriately reduced from the component in neomedrol EE ointment and applied.

[0274] It is known that the concentration of Semaphorin in the body is stabilized by a feedback effect. Therefore, Semaphorin acts gently in the body. In addition, since the administration of Semaphorin can activate the ability to divide without causing significant morphological changes in corneal endothelial cells, the preventive, inhibitory or therapeutic agent of the present invention is unlikely to cause side effects.

[0275] In an embodiment, the present invention provides a method for preventing, inhibiting or treating corneal diseases or corneal damage, comprising the step of administering Semaphorin to a mammal.

[0276] In an embodiment, the present invention provides Semaphorin 3A for use in preventing, inhibiting or treating corneal diseases or corneal damage.

[0277] In an embodiment, the present invention provides use of Semaphorin 3A in preparing a medicament for preventing, inhibiting or treating corneal diseases or corneal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0278] In order to make the content of the present invention more fully understood, the present invention is further described in detail below through specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0279] Figure 1 Schematic diagram of the fluorescein staining of animal models according to the present invention, wherein the upper two figures are observation images of corneal damage, and the lower one is a schematic diagram, wherein (a) is a photograph of the animal fluorescein staining of the corneal alkali burn model under room light; (b) is a photograph under the blue free filter of a slit lamp.

[0280] Figure 2 These are photos of the entire corneal epithelium being scratched and stained with fluorescein after the alkaline corneal burn model was made.

[0281] Figure 3 The following are photos comparing the clinical symptoms of epithelial loss.

[0282] Figure 4 This is an enlarged photo of the PBS control group under a microscope after 14 days of treatment.

[0283] Figure 5 For the evaluation of corneal epithelial damage, the symptoms of the treatment groups with concentrations of 100ng / ml and 1000ng / ml were significantly improved.

[0284] Figure 6 For corneal angiogenesis evaluation, the results showed that Sema3A inhibited new blood vessel formation in a concentration-dependent manner.

[0285] Figure 7 These are photos of corneal flat mounts immunostained with antibodies for CD31 and LYVE-1 14 days after debridement.

[0286] Figure 8 These results suggest that Sema3A effectively inhibits pathological angiogenesis after corneal alkali burn.

[0287] Fig. 9 These results suggest that Sema3A effectively inhibits pathological lymphangiogenesis after corneal alkali burn.

[0288] Fig.10 Immunostaining photos of inflammatory cell infiltration in corneal tissue (photos of βⅢtubulin and F4 / 80 immunostaining in corneal tissue).

[0289] Fig.11 Evaluation of the number of F4 / 80 antibody-positive cells (macrophages).

[0290] Fig.12 These are photos of corneal sections with immunostaining of nerve fibers.

[0291] Fig.13 To evaluate the density of corneal βⅢtubulin-positive nerve fibers.

[0292] In order to further understand the present invention, the specific embodiments of the present invention are described in detail below in conjunction with examples. However, it should be understood that the purpose of these descriptions is only to further illustrate the features and advantages of the present invention, and does not constitute any limitation to the claims of the present invention. Example

[0293] Example 1

[0294] The purpose of this example is to verify the preventive, inhibitory and / or therapeutic effects of the polypeptides described in the present invention, especially Semaphorin 3A, on angiogenesis / lymphangiogenesis, and diseases related to endothelial dysfunction, especially cancer, ophthalmic diseases, arteriosclerosis, cardiovascular, cerebrovascular and renal diseases, Alzheimer's disease, aging diseases, scar formation and the like.

[0295] 1. Experimental methods :

[0296] The corneal alkali injury model was prepared by dripping 2 ml of 0.15 M NaOH, and the following examinations were performed. The drug was administered once every other day and continued for 7 days from the day of alkaline injury. The experimental group was given Semaphorin 3A (2 μl) manufactured by LSBio under the conjunctiva on days 0, 3, 6, and 9 after surgery. The control group was given PBS solution under the conjunctiva.

[0297] 2. Experimental animal grouping and treatment :

[0298] BALB / c male 8 weeks old

[0299] The experimental animals were randomly divided into the following groups, with 8 animals in each group:

[0300] (1) PBS as the control group (n=8)

[0301] (2) Semaphorin3A (10 ng / ml 2 μl / subconjunctival injection) administration group (n=8);

[0302] (3) Semaphorin3A (100 ng / ml 2 μl / subconjunctival injection) administration group (n=8);

[0303] (4) Semaphorin3A (1000 ng / ml 2 μl / subconjunctival injection) administration group (n=8);

[0304] 3. Animal model biological staining :

[0305] A slit lamp blue free filter (blue no filter) is used, which selectively transmits only the green fluorescence color emitted by fluorescein, so that an observation image of corneal damage can be obtained.

[0306] 4. Fluorescein staining test :Observe the condition of the cornea with fluorescein staining. If there is staining, it means corneal erosion and the condition of the corneal epithelium is unstable.

[0307] This test is used to detect corneal ulcers (corneal wounds) by using the properties of permeable epithelial defects and areas of poor adhesion between epithelial cells.

[0308] 5. Evaluation indicators :

[0309] Record corneal findings: The cornea was observed under a slit lamp microscope 3, 6, 9, and 14 days after alkaline injury, corneal opacity, and the extent of corneal neovascularization and corneal epithelial defect was semiquantitatively evaluated as follows:

[0310] Corneal opacity: The degree of corneal opacity is statistically scored into five levels from 0 to 4.

[0311] Grading of corneal neovascularization: The length and range of invasion into the cornea are divided into five grades from 0 to 4 according to the degree.

[0312] Corneal epithelial defect extent: Since epithelial defects are usually elliptical, the extent of the defect is evaluated by the ratio of the long axis of the defect to the corneal diameter.

[0313] The drug group and the control group were compared using the unpaired Student's t test. **P<0.01, ***P<0.001; ns: no significant difference.

[0314] Clinical symptom review:

[0315] The eyes of the animal model were examined daily for changes in clinical symptoms using a slit lamp microscope. The corneal opacity scoring system is: neovascularization (NV) and corneal epithelial defect area. The defined scoring system describes the degree of corneal opacity (0-4) and NV (0-4) in semiquantitative terms. On days 2, 5, 6, 10, and 14 after the corneal injury model was created, a blind observer assigned a turbidity score for the opacity of each cornea:

[0316] 0 = clear cornea;

[0317] 1 = minimal surface (non-corneal stromal) opacity;

[0318] 2 = minimum depth (corneal stroma) opacity;

[0319] 3 = moderate corneal stromal opacity;

[0320] 4 = Intensity of corneal stroma opacity.

[0321] NV Scoring System:

[0322] 0 = no corneal vascular invasion;

[0323] 1 = vessel length less than half the corneal radius in less than two quadrants;

[0324] 2 = Vessel length exceeds half of the corneal radius and is less than two quadrants;

[0325] 3 = Vessel length less than half the corneal radius in more than two quadrants;

[0326] 4 = Vessel length exceeds half of the corneal radius in more than two quadrants.

[0327] The area of ​​corneal epithelial defect was calculated as a proportion of the defect area.

[0328] Long axis: diameter of the cornea.

[0329] Statistical analysis

[0330] All data are expressed as mean ± SEM.

[0331] The values ​​below the detection limit were counted as 0 pg / ml. The comparison was performed using the unpaired Student's t test. **P < 0.01, ***P < 0.001; ns: no significant difference.

[0332] The PCR test was used to statistically compare the clinical scores at each time point between untreated damaged corneas (control) and Sema3A-treated corneas.

[0333] 6. Experimental Results :

[0334] Epithelial defect model

[0335] Figure 1 (a) is a photo of a corneal alkali burn model taken under indoor light. Figure 1 The white dashed line in (a) is the corneal area, where the corneal epithelium is detached after corneal alkali burn. The black solid line frame is the accumulation area of ​​epithelial tissue that is degenerated and detached after the burn. Figure 1 (b) is a photo of the cornea after alkaline corneal burn, stained with fluorescein and photographed under the blue free filter of a slit lamp. As shown in the figure, the white broken line is the corneal area, and the arrow points to the corneal epithelial defect area where the fluorescein in the white part is attached.

[0336] Figure 2 The following pictures show the fluorescent staining of the whole corneal epithelium after scratching. Figure 2 As shown, the area within the white broken line is the corneal area. After debridement, the corneal epithelial tissue is completely peeled off, the entire cornea is stained with fluorescein, and the entire cornea becomes turbid like frosted glass.

[0337] Comparison of clinical symptoms of epithelial damage

[0338] Figure 3 The comparison of clinical symptoms of corneal epithelial deficiency is shown. The corneal repair changes of the treatment group and the control group were observed on the 3rd, 6th, 9th and 14th days after alkaline corneal trauma, and the epithelial deficiency was stained with fluorescein and photographed under a microscope. Among them: the corneal alkali burn animal model was observed for 2 weeks, and the 1st, 3rd, 5th and 7th rows of photos were the changes in clinical symptoms of corneal epithelium under indoor light on the 3rd, 6th, 9th and 14th days; the 2nd, 4th, 6th and 8th rows of photos were the changes in clinical symptoms of corneal epithelium under the blue free filter of the slit lamp (blue filter-free) on the 3rd, 6th, 9th and 14th days after fluorescein staining.

[0339] According to the experimental results, 2 weeks after the burn, the control group and the treatment group 10ng / ml, 1000ng / ml still had fluorescein staining areas on the surface of the eye such as the conjunctiva and cornea, and the inflammation had not healed. The cornea became turbid, accompanied by inflammation and vascular invasion for more than 2 weeks, and 100ng / ml had a significant effect on angiogenesis, epithelial defects and wound healing. There was almost no fluorescein staining on the cornea, and the retention of tears was sufficient. The eye basically returned to normal state.

[0340] Figure 4 for Figure 3 The enlarged image of the microscope photo of the PBS control group after 14 days of treatment. Figure 4 The white broken line in (a) is the corneal tissue area, and the arrow indicates the corneal neovascularization after alkali burn. The blood vessels are enlarged, affecting the corneal perspective, and the animal model's vision drops sharply. Figure 4(b) The white broken line in the corneal tissue area indicates that the whole cornea is degenerated after alkali burn and presents white spots and opacity. The solid white line indicates the corneal degeneration and scar proliferation area, and the black arrow indicates tissue degeneration, hyperplasia and hypertrophy, presenting an opaque state. The animal model has a severe visual impairment or blindness.

[0341] Epithelial defect assessment

[0342] Figure 5 Evaluation of corneal epithelial damage was demonstrated. Corneal repair changes were observed on days 2, 3, 4, 6, 10, and 14 after the alkaline trauma model was created. Fluorescein staining and clinical sampling ( Figure 5 ).

[0343] The degree of corneal epithelial defect is scored into five grades:

[0344] Vertical axis evaluation method (reference: Den et al., Efficacy of early systemic betamethasone orcyclosporin A after corneal alkaliinjury via inflammatory cytokine reduction)

[0345] 0 = clear cornea;

[0346] 1 = minimal surface (non-corneal stromal) opacity;

[0347] 2 = minimum depth (corneal stroma) opacity;

[0348] 3 = moderate corneal stromal opacity;

[0349] 4 = Intensity of corneal stroma opacity.

[0350] From the 6th day, the concentration-dependent clinical symptoms showed differences, and the symptoms of the 100ng / ml and 1000ng / ml treatment groups were significantly improved. The 100ng / ml treatment group had a significant effect on the 6th day (**P<0.01); there was a significant difference on the 14th day (*P<0.05).

[0351] Through administration, the corneal burn tissue was significantly repaired and the corneal epithelium healed well. The cornea of ​​the treatment group with a concentration of 100ng / ml was transparent and had no difference from the normal cornea.

[0352] Angiogenesis Assessment

[0353] Figure 6Evaluation of corneal angiogenesis was presented. The angiogenesis was evaluated in five stages according to the grading criteria of the length and extent of neovascularization at 2, 5, 6, 10, and 14 days after alkaline wounding.

[0354] The cornea was immunostained with anti-CD31 antibody (vascular endothelial marker) after 14 days, and the vascular area was quantified by flat-mounting corneal slices. The length of new blood vessels in the cornea was measured under an operating microscope. There were 8 mice in each group (n=8), and the assay was mean±SD.

[0355] Neovascularization: The length and extent of its invasion into the cornea are scored on a five-point scale of 0 to 4.

[0356] The scoring system for neovascularization and neolymphatic development is as follows:

[0357] 0 = None

[0358] 1 = blood / lymphatic vessel length less than half the corneal radius in less than two quadrants;

[0359] 2 = blood vessel / lymphatic vessel length exceeds half of the corneal radius and is less than two quadrants;

[0360] 3 = blood vessel / lymphatic vessel length less than half the corneal radius in more than two quadrants;

[0361] 4 = Blood vessels / lymphatic vessels longer than half the corneal radius in more than two quadrants.

[0362] Calculate the area of ​​corneal epithelial defect as a proportion of the defect area

[0363] Long axis: corneal diameter

[0364] After 2 weeks of observation, Sema3A inhibited neovascularization in a concentration-dependent manner.

[0365] Among them, compared with the PBS control group, 100ng / ml Sema3A showed an inhibition of angiogenesis on the 4th day; there was a significant difference on the 10th day (**P<0.01). The administration of this treatment significantly inhibited the generation and growth of new blood vessels in the cornea.

[0366] Immunostaining evaluation of blood vessels, lymphatic vessels, and corneal flat mounts

[0367] Figure 7 Shown are corneal flat mounts immunostained with CD31 antibody (vascular endothelial marker) and LYVE-1 antibody (lymphatic endothelial marker) 14 days after debridement.

[0368] After the treatment group was given Sema3A, the blood vessels and lymphatic vessels of the treatment group and the control group with Sema3A concentrations of 10 ng / ml, 100 ng / ml, and 1000 ng / ml were immunostained. The percentage of blood vessel and lymphatic area was used for quantitative evaluation.

[0369] according to Figure 7 It can be seen that the PBS-treated control group had large-area angiogenesis and lymphangiogenesis. Compared with the PBS group, the Sema3A concentration of 100 ng / ml treatment group significantly inhibited the angiogenesis and lymphangiogenesis, and the central part of the cornea maintained normal transparency, indicating that Sema3A can effectively promote epithelial formation.

[0370] Angiogenesis / lymphangiogenesis field evaluation

[0371] Figure 8 The evaluation of corneal angiogenesis area (expressed as angiogenesis area (%)) is shown. 14 days after debridement, after administration of Sema3A in the treatment group, the angiogenesis area in the Sema3A treatment group at a concentration of 100ng / ml was significantly reduced, with a significant difference from that in the control group: **P=0.014; each group of mice consisted of 8 mice (n=8); the assay was mean±SD. The above results indicate that Sema3A effectively inhibits pathological angiogenesis after corneal alkali burn.

[0372] Fig. 9 The evaluation of corneal lymphangiogenesis (expressed as lymphangiogenesis area (%)) is shown. 14 days after debridement, after administration of Sema3A in the treatment group, the lymphangiogenesis area in the Sema3A treatment group at a concentration of 100 ng / ml was significantly reduced, which was significantly different from that in the control group: **P=0.02; each group of mice consisted of 8 mice (n=8); the assay was mean±SD. This indicates that Sema3A effectively inhibits pathological lymphangiogenesis after corneal alkali burn.

[0373] Inflammatory cell infiltration of corneal tissue

[0374] Fig.10 The βⅢtubulin and F4 / 80 immunostaining photos of corneal tissue (14 days after debridement) show the infiltration of inflammatory cells in corneal tissue.

[0375] The triangular white arrows indicate immunostaining-positive inflammatory cells;

[0376] A Left: Immunostaining of the neurofibrillary marker βIII tubulin;

[0377] In B, anti-F4 / 80 antibody was used for mouse macrophage detection. The infiltration of F4 / 80 positive cells (macrophages) in corneal tissue of the Sema3A treatment group at a concentration of 100 ng / ml was significantly reduced compared with the control group, indicating that Sema3A inhibits the inflammatory response after corneal alkaline trauma.

[0378] Evaluation of the number of F4 / 80 antibody positive cells (macrophages)

[0379] Fig.11 Evaluation of the number of F4 / 80 antibody-positive cells (macrophages) is shown.

[0380] On the 14th day after debridement, after the administration of Sema3A to the treatment group, the number of F4 / 80 antibody-positive cells in the Sema3A treatment group at a concentration of 100 nng / ml was significantly reduced, and there was a significant difference compared with the control group: **P<0.0001; each group of mice consisted of 15 mice (n=15), and the test was mean±SD.

[0381] Immunostaining of nerve fibers in corneal sections

[0382] Fig.12 The immunostaining photos of nerve fibers in corneal sections are shown. On the 14th day after debridement, corneal sections were immunostained with anti-βIII tubulin antibody, PGP 9.5 (neural marker). The white arrows indicate the nerve fibers. Immunofluorescence staining of corneal nerve fibers distributed in corneal tissue at each concentration in the control group and the treatment group.

[0383] from Fig.12 As can be seen, no damage was observed to the nerve plexus in the corneal tissue when Sema3A at a concentration of 100 ng / ml and a high concentration of 1000 ng / ml was administered.

[0384] Fig.13 The effects on the corneal sensory nerve (trigeminal nerve) were quantified, as assessed by the density of βⅢtubulin-positive nerve fibers.

[0385] The corneal nerve fiber area was quantified 14 days after debridement.

[0386] Corneal nerve fibers were quantified as the percentage of thresholded areas positive for β-tubulin III staining in representative confocal images shown in the figure.

[0387] NS: There was no significant difference between the treatment groups and the normal and control groups; 15 mice in each group (n=15); the test was mean±SD.

[0388] This result demonstrates that administration of Sema3A has no effect on nerve fibers in corneal tissue.

[0389] In conclusion, we examined the effects of Semaphorin3A on corneal epithelial defects, corneal neovascularization and lymphangiogenesis using a mouse corneal alkaline injury model.

[0390] Especially in the 100ng / ml Semaphorin3A administration group, corneal epithelial defects, corneal neovascularization, corneal neolymphangiogenesis and inflammatory cell infiltration were significantly inhibited, and there was no reduction in β-III tubulin-positive nerve fibers compared with normal mouse corneas.

[0391] Together with our in vivo studies, these results suggest that Semaphorin3A effectively promotes epithelialization, inhibits pathological angiogenesis, and suppresses inflammation following corneal alkaline trauma.

[0392] Various aspects of the present invention have been illustrated through the above embodiments. Obviously, the above embodiments are only examples for clear explanation, and are not limitations on the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the protection scope of the invention.

[0393] In summary, the present invention relates to the following technical solutions:

[0394] 1. Use of a polypeptide in the preparation of a drug for preventing, inhibiting and / or treating angiogenesis diseases and lymphangiogenesis diseases,

[0395] Wherein, the polypeptide has:

[0396] (b) The amino acid sequence shown in SEQ ID NO: 2.

[0397] 2. The use described in Technical Solution 1, wherein the polypeptide is a glycosylated polypeptide.

[0398] 3. The use described in Technical Solution 1, wherein the polypeptide is a PEGylated polypeptide.

[0399] 4. The use described in technical solution 1, wherein the polypeptide is a PEGylated polypeptide, and the molecular weight of the PEG chain is 3000 to 7000.

[0400] 5. The use described in Technical Solution 4, wherein the molecular weight of the PEG chain is 5000.

[0401] 6. The use according to any one of technical solutions 1-5, wherein the unit dose of the polypeptide is 6000 to 60000 U.

[0402] 7. The use according to any one of technical solutions 1-5, wherein the unit dose of the polypeptide is 4000 to 40000 U.

[0403] 8. The use according to any one of technical solutions 1-5, wherein the unit dose of the polypeptide is 500 to 5000 U.

[0404] 9. The use according to any one of technical solutions 1-5, wherein the unit dose of the polypeptide is 1 to 3000 ng.

[0405] 10. The use according to any one of technical solutions 1-5, wherein the unit dose of the polypeptide is 20 to 900 ng.

[0406] 11. The use according to any one of technical solutions 1-5, wherein the unit dose of the polypeptide is 50 to 900 ng.

[0407] 12. The use according to any one of technical solutions 1-5, wherein the unit dose of the polypeptide is 50 to 500 ng.

[0408] 13. The use according to any one of technical solutions 1-5, wherein the unit dose of the polypeptide is 80 to 500 ng.

[0409] 14. The use according to any one of technical solutions 1 to 5, wherein the concentration of the polypeptide is 10 ng / ml to 1000 ng / ml.

[0410] 15. The use according to any one of technical solutions 1 to 5, wherein the concentration of the polypeptide is 10 ng / ml, 100 ng / ml, 300 ng / ml or 1000 ng / ml.

[0411] 16. The use described in technical solution 15, wherein the concentration of the polypeptide is 100 ng / ml.

[0412] 17. The use according to any one of technical solutions 1 to 5, wherein the drug is eye drops, eye ointments, injections, corneal soft contact films, oral solutions, mouthwashes, film adhesive sheets, creams, suppositories or sprays.

[0413] 18. The use described in technical solution 17, wherein the drug is eye drops with a dosage of 1 to 3000 ng.

[0414] 19. The use described in Technical Solution 18, wherein the dosage of the eye drops is 20 to 900 ng.

[0415] 20. The use described in Technical Solution 18, wherein the dosage of the eye drops is 50 to 900 ng.

[0416] 21. The use described in Technical Solution 18, wherein the dosage of the eye drops is 50 to 500 ng.

[0417] 22. The use described in Technical Solution 18, wherein the dosage of the eye drops is 80 to 500 ng.

[0418] 23. The use described in Technical Solution 17, wherein the drug is eye drops with a concentration of 10 ng / ml to about 1000 ng / ml.

[0419] 24. The use described in technical solution 23, wherein the concentration of the eye drops is 10 ng / ml, 100 ng / ml, 300 ng / ml or 1000 ng / ml.

[0420] 25. The use described in technical solution 18, wherein the eye drops are used once or several times a day, or once every few days, for several days to several months.

[0421] 26. The use described in technical solution 23, wherein the eye drops are used once or several times a day, or once every few days, for several days to several months.

[0422] 27. The use according to any one of technical solutions 1-5, wherein the drug is injected subconjunctivally in an amount of 500 to 5000 U per day, administered once or several times.

[0423] 28. The use according to any one of technical solutions 1-5, wherein the drug is injected subconjunctivally in an amount of 0.001 to 10 ng / day.

[0424] 29. The use described in technical solution 28, wherein the amount of the drug is 0.05 to 5 ng / day.

[0425] 30. The use according to any one of technical solutions 1 to 5, wherein the drug is injected subconjunctivally at a concentration of 10 ng / ml to 1000 ng / ml.

[0426] 31. The use described in technical solution 30, wherein the concentration of the drug is 10 ng / ml, 100 ng / ml, 300 ng / ml or 1000 ng / ml.

[0427] 32. The use described in technical solution 28, wherein the administration is once or several times a day, or once every few days, for several days to several months.

[0428] 33. The use described in technical solution 30, wherein the administration is once or several times a day, or once every few days, for several days to several months.

[0429] 34. The use according to any one of technical solutions 1 to 5, wherein the drug is eye drops with an active ingredient concentration of 100 to 300 ng / ml.

[0430] 35. The use described in technical solution 34, wherein the concentration of the active ingredient in the eye drops is 100 ng / ml or 300 ng / ml.

[0431] 36. The use according to any one of technical solutions 1 to 5, wherein the drug is an eye ointment and the concentration of the active ingredient is 100 to 300 ng / ml.

[0432] 37. The use according to any one of technical solutions 1 to 5, wherein the drug is a corneal soft contact drug film, and the active ingredient concentration is 100 to 300 ng / ml.

[0433] 38. The use according to any one of technical solutions 1 to 5, wherein the drug is an injection solution with an active ingredient concentration of 100 to 300 ng / ml.

[0434] 39. The use according to any one of technical solutions 1 to 5, wherein the drug is an oral solution with an active ingredient concentration of 100 to 300 ng / ml.

[0435] 40. The use according to any one of technical solutions 1 to 5, wherein the drug is a mouthwash with an active ingredient concentration of 100 to 300 ng / ml.

[0436] 41. The use according to any one of technical solutions 1 to 5, wherein the drug is a drug film adhesive sheet and the active ingredient concentration is 100 to 300 ng / ml.

[0437] 42. The use according to any one of technical solutions 1 to 5, wherein the drug is a cream with an active ingredient concentration of 100 to 300 ng / ml.

[0438] 43. The use according to any one of technical solutions 1 to 5, wherein the drug is a suppository and the active ingredient concentration is 100 to 300 ng / ml.

[0439] 44. The use according to any one of technical solutions 1 to 5, wherein the drug is a wound care dressing and the active ingredient concentration is 100 to 300 ng / ml.

[0440] 45. The use according to any one of technical solutions 1 to 5, wherein the drug is a spray and the concentration of the active ingredient is 100 to 300 ng / ml.

[0441] 46. ​​The use according to any one of technical solutions 1 to 5, wherein the drug is a film coating agent and the concentration of the active ingredient is 100 to 300 ng / ml.

[0442] 47. The use according to any one of technical solutions 1 to 5, wherein the angiogenesis disease or lymphangiogenesis disease is cancer.

[0443] 48. The use described in technical solution 47, wherein the cancer is one or more diseases selected from the following: breast cancer, colon cancer, colorectal cancer, esophageal cancer, superficial esophageal cancer, laryngeal cancer, paranasal malignancies, gastrointestinal stromal tumors (GIST), kidney cancer, liver cancer, lymphoma, lymphangiomatosis / Gorham disease, giant lymphatic malformation, melanoma, lung cancer, ovarian cancer, epithelial ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, nasal tumors, gynecological malignancies, cervical cancer, endometrial cancer and bladder cancer.

[0444] 49. The use described in technical solution 48, wherein the cancer is one or more diseases selected from the following: metastatic breast cancer, progressive colorectal cancer, early esophageal cancer, renal cell carcinoma, adult primary cancer of the liver, inoperable liver cancer, malignant melanoma, non-small cell lung cancer (NSCLC), metastatic non-small cell lung cancer, early invasive cervical cancer, superficial bladder cancer and invasive bladder cancer.

[0445] 50. The use according to any one of technical solutions 1 to 5, wherein the angiogenesis disease or lymphangiogenesis disease is an ophthalmic angiogenesis disease or lymphangiogenesis disease.

[0446] 51. The use described in technical solution 50, wherein the ophthalmic angiogenesis disease or lymphangiogenesis disease is selected from one or more of the following diseases: diabetic angiogenic fundus lesions, retinal angioma, von Hippel-Lindau disease, superficial soft hemangioma, macular degeneration, myopic choroidal neovascularization, diabetic retinopathy, immature infantile retinopathy, neovascular glaucoma, diabetic maculopathy, retinal vein occlusive disease, intraocular neovascularization associated with uveitis, Behcet's disease, tuberculous uveitis, choroidal neovascularization in high myopia, malignant lymphoma, conjunctival malignant lymphoma, choroidal tumor, cytomegalovirus disease, intraocular malignant tumor, choroidal malignant melanoma, malignant lymphoma occurring in the eyeball, and ocular adnexal tumor.

[0447] 52. The use described in technical solution 51, wherein the neovascular glaucoma is selected from the retinal angiogenesis caused by systemic diabetes and the vascular and lymphatic dysfunction of secondary angiogenic glaucoma, the cytomegalovirus disease is cytomegalovirus retinitis, the intraocular malignant tumor is retinoblastoma, and the ocular adnexal tumor is an optic nerve tumor.

[0448] 53. The use of any one of technical solutions 1-5, wherein the angiogenesis disease or lymphangiogenesis disease is one or more diseases selected from the following: keloid, rheumatoid arthritis (RA), psoriasis, arteriosclerosis, tuberous sclerosis, Alzheimer's disease, aging, acute myocardial infarction, coronary artery disease induced by cytomegalovirus and chlamydia infection.

[0449] 54. The use described in technical solution 53, wherein the Alzheimer's disease is vascular Alzheimer's disease.

[0450] 55. The use described in any one of technical solutions 1-5, wherein the angiogenesis disease or lymphangiogenesis disease is a disease related to scar tissue formation.

[0451] 56. The use described in technical solution 54, wherein the disease associated with scar tissue formation is one or more diseases selected from the following: diseases caused by necrosis associated with scars, ulcers, wounds, infarction, and tissue defects of various organs.

[0452] 57. The use of any one of technical solutions 1-5, wherein the angiogenesis disease or lymphangiogenesis disease is one or more diseases selected from the following: aging of organs, ischemic state caused by decreased vascular function, aging of blood vessels and lymphatic dysfunction of aging organs.

[0453] 58. The use described in technical solution 57, wherein the organ aging disease is selected from vascular dementia or Alzheimer's disease.

[0454] 59. The use according to any one of technical solutions 1 to 5, wherein the angiogenesis disease or lymphangiogenesis disease is a disease related to cardiovascular, cerebrovascular and renal diseases.

[0455] 60. The use according to any one of technical solutions 1-5, wherein the polypeptide is Semaphorin.

[0456] 61. The use according to any one of technical solutions 1-5, wherein the polypeptide is combined with other preventive, inhibitory or therapeutic agents.

[0457] 62. The use described in technical solution 61, wherein the combination is selected from the combination of Semaphorin and hyaluronic acid, the combination of Semaphorin and neomedrol EE, the combination of Semaphorin and Oders Linderon, or the combination of Semaphorin and deoxycholate.

[0458] 63. Use of a polypeptide in the preparation of a preparation for preventing, inhibiting and / or treating scar formation and related symptoms,

[0459] Wherein, the polypeptide has:

[0460] (b) The amino acid sequence shown in SEQ ID NO: 2.

[0461] 64. Use of a polypeptide in the preparation of a preparation for preventing, inhibiting and / or treating skin and mucosal related symptoms of neovascularization and lymphatic vessels,

[0462] Wherein, the polypeptide has:

[0463] (b) The amino acid sequence shown in SEQ ID NO: 2.

[0464] 65. The use described in technical solution 63 or 64, wherein the polypeptide is a glycosylated polypeptide.

[0465] 66. The use described in technical solution 63 or 64, wherein the polypeptide is a PEGylated polypeptide.

[0466] 67. The use described in technical solution 63 or 64, wherein the polypeptide is a PEGylated polypeptide, and the molecular weight of the PEG chain is 3000 to 7000.

[0467] 68. The use described in technical solution 67, wherein the molecular weight of the PEG chain is 5000.

Claims

1. Use of a polypeptide in the preparation of a drug for preventing, inhibiting and / or treating angiogenesis diseases and lymphangiogenesis diseases, in, The polypeptide has: (b) The amino acid sequence shown in SEQ ID NO:

2.

2. The use according to claim 1, wherein the polypeptide is a glycosylated polypeptide.

3. The method of claim 1, wherein the polypeptide is a PEGylated polypeptide.

4. The method of claim 1, wherein the polypeptide is a PEGylated polypeptide, and the molecular weight of the PEG chain is 3000 to 7000.

5. The use of claim 4, wherein the molecular weight of the PEG chain is 5000.

6. The use according to any one of claims 1 to 5, wherein The unit dose of the polypeptide is 6000 to 60000U.

7. The use according to any one of claims 1 to 5, wherein The unit dose of the polypeptide is 4000 to 40000 U.

8. The use according to any one of claims 1 to 5, wherein The unit dose of the polypeptide is 500 to 5000U.

9. The use according to any one of claims 1 to 5, wherein The unit dose of the polypeptide is 1 to 3000 ng.

10. The use according to any one of claims 1 to 5, wherein The unit dose of the polypeptide is 20 to 900 ng.

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