CN112641928A

CN112641928A - Polypeptide for angiogenesis and lymphangiogenesis related diseases and application thereof

Info

Publication number: CN112641928A
Application number: CN201910961935.9A
Authority: CN
Inventors: 田中纯美
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-10-11
Filing date: 2019-10-11
Publication date: 2021-04-13
Also published as: WO2021068962A1; CN114466859A

Abstract

The invention provides a polypeptide of an auxiliary preparation for preventing, inhibiting and/or treating or promoting the metabolism and quick-acting property of the existing therapeutic drugs or fragments comprising the polypeptide as an active effective ingredient, which is used for preventing, inhibiting and/or treating diseases related to angiogenesis/lymphangiogenesis and vascular endothelial dysfunction, particularly cancers, ophthalmic diseases, arteriosclerosis, cardiovascular, cerebrovascular and renal vascular diseases, Alzheimer's disease, aging diseases, scar formation and the like, and application thereof in preparation of drugs for preventing, inhibiting and/or treating diseases related to angiogenesis and lymphangiogenesis. Especially in the prevention and treatment of cancer-related diseases, arteriosclerosis, Alzheimer's disease, scarring, ageing-related diseases, in particular in the prevention and treatment of ocular angiogenesis-related diseases.

Description

Polypeptide for angiogenesis and lymphangiogenesis related diseases and application thereof

Technical Field

The present invention relates to a prophylactic, inhibitory and/or therapeutic agent comprising Semaphorin protein (Semas), a pharmaceutically acceptable ingredient thereof as an active effective ingredient, and use thereof for the preparation of a medicament for the prevention, inhibition and/or treatment of angiogenesis diseases and lymphangiogenesis-related diseases. In particular in the prevention and treatment of cancer-related diseases, scarring, cardiovascular diseases, organ aging diseases, alzheimer's disease, in particular in the prevention and treatment of ocular angiogenesis-related diseases.

Background

Human cells maintain their activities and functions by taking up nutrients and oxygen supplied by blood vessels around the cells. The number of essential cells is also strictly controlled by the original function of the human, however, cancer cells are uncontrollable and proliferation is very active. Since cancer cells that perform such activities require a large amount of nutrients and oxygen compared to normal cells, new blood vessels, i.e., new blood vessels, begin to be created. The new blood vessels produced by the cancer cells have become a supporting source of their activity.

Angiogenesis is a physiological process that utilizes existing vasculature to form new blood vessels, and plays a critical role in pathological conditions, including wound healing, cancer disorders, age-related macular degeneration, tumor growth, and diabetic retinopathy.

Cancers associated with angiogenesis are about the following: breast cancer, colorectal cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal (renal cell) cancer, liver (adult primary) cancer, lymphoma, lymphangiomatosis/Gorham's disease, giant lymphatic malformations, melanoma (malignant melanoma), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, nasal tumors, gynecological malignancies, endometrial cancer, bladder cancer.

Recent studies have shown that expression of these angiogenic factors is identified, with a direct correlation between the degree of expression and prognosis, including breast, endometrial, colon, and bladder cancers. In addition, the density of angiogenesis in breast cancer tumor tissue is associated with metastasis, while angiogenesis is an important factor affecting the prognosis of cancer patients in esophageal cancer, colon cancer, and the like. While the probability of metastasis of breast cancer to lymph nodes is highest. Therefore, the inhibition of lymphatic vessel proliferation plays an important role in the inhibition of breast cancer metastasis. Esophageal cancer, regardless of its degree of progression, may spread to lymph nodes. Prostate cancer is most commonly metastasized to lymph nodes around the prostate.

If cancer cells metastasize to lymph nodes, they may metastasize distally through the lymph vessels. However, the transfer to the liver is difficult because subjective symptoms hardly appear at first and the severity of the disease is very high. Cancer that has metastasized is difficult to treat, early prevention, early treatment and its importance.

At present, angiogenesis inhibitors for human cancer treatment are several anti-vascular neolife agents approved by the U.S. Food and Drug Administration (FDA) that can delay tumor growth, but recent findings have shown the possibility of congenital abnormalities in animal studies, with clear potential for complications.

Angiogenesis inhibitor treatment side effects include bleeding, arterial thrombosis (causing stroke or heart attack), hypertension and urine protein (3-5). Digestive tract perforations and gastrointestinal fistulas are also considered side effects of certain angiogenesis inhibitors.

Such as Bevacizumab (trade name AVASTIN) which is an anti-VEGF antibody, cannot be used for patients with squamous cell carcinoma and infiltration of large blood vessels by confirmed tumors, because of the risk of bleeding, nor for patients with a high risk of bleeding in a past history. For patients with brain metastasis, the tumor lesions are at risk of bleeding and cannot be used.

In addition, the existing combination therapy of anti-VEGF preparation and chemotherapeutic agent still has the financial problem of high drug price for maintenance therapy, and even if the maintenance therapy is carried out, the combination therapy of high-price drugs of avastin and pemetrexed cannot bear many patients.

Angiogenic diseases also include: rheumatoid Arthritis (RA), psoriasis (psoriasis), arteriosclerosis, tuberous sclerosis, alzheimer's disease such as vascular alzheimer, aging, acute myocardial infarction, coronary lesions induced by cytomegalovirus and chlamydia infections.

Arteriosclerosis-related diseases are rapidly increasing due to lifestyle diseases and a super-aging society. According to the important statistical data of the ministry of health, labor and welfare, the death cause of arteriosclerosis-related diseases (stroke and myocardial infarction) accounts for about one third of the death rate and is comparable to the death cause of malignant tumors. However, in most cases, there is no symptom until the onset of the disease, and it is late until the onset of the disease, so that it is extremely important to prevent the onset of the disease by, for example, administering the drug early.

The early stage of vascular endothelial dysfunction is caused by diseases such as lifestyle-related diseases (obesity, stress, smoking, lack of exercise, etc.), hypertension, dyslipidemia, diabetes, sleep apnea syndrome, chronic nephropathy, etc., and vascular endothelial cells damaged by aging, sex, menopause, heredity, etc. The paralysis of blood vessel function can promote the occurrence of cardiovascular diseases, obesity can make blood vessel become narrow and hard, cholesterol and neutral fat in blood can be increased and deposited, and the development of arteriosclerosis can be accelerated. Causing angina pectoris and renal dysfunction, cerebral infarction and cardiac infarction after thrombosis, and cerebral hemorrhage due to vascular rupture. Arteries that progress in aging begin to be hard, causing severe cerebral infarction and myocardial infarction.

Among these diseases, psoriasis (psoriasis), for example, although it is treated for a long time, improvement is not easy, and there is no basic treatment method at present; current treatments for RA also rely on angiogenesis inhibitors; when the disease of tuberous sclerosis and LAM disease occur together, a drug named sirolimus is currently used mainly in the united states, and there is no suggestion about its effect because of various side effects. Renal tumors and pulmonary LAM are often implicated in life prognosis when the disease is severe.

Ocular angiogenic disorders include: neovascular glaucoma, diabetic macular disease, retinal vein occlusion, uveitis complicated by intraocular neovascularization, Behcet's disease, tuberculous uveitis, highly myopic choroidal neovascularization, malignant lymphoma, conjunctival malignant lymphoma, choroidal tumors, cytomegaloviral disorders such as cytomegaloviral retinitis, intraocular malignant tumors such as retinoblastoma, choroidal malignant melanoma, malignant lymphoma appearing in the eyeball, ocular adnexal tumors such as optic nerve tumors.

Traumatic and chemical burn corneal angiogenesis, no medicine can be used for treatment at present, and the only method is corneal transplantation; angiogenic glaucoma progresses with the progression of retinal neovascularization and spreads throughout the eyeball, the fiber trabecular bundle is covered with new blood vessels, resulting in increased intraocular pressure due to outflow resistance of aqueous humor. Methods for lowering intraocular pressure include intravenous drip and medical treatment, but the effect is only temporary in any case. Laser treatment results in a condition of reduced blood volume required by the retina, insufficient blood. If the neovascularization that obstructs the angle of the atrium is not cured, the intraocular pressure is hardly lowered. Glaucoma surgery is only available at this stage, but neovascular glaucoma surgery is not effective.

Intraocular inflammation of tuberculous uveitis neovessels are treated by antitubercular therapy and oral corticosteroids. The side effects of corticosteroids cause ocular hypertension, glaucoma, cataract, corneal fungus attacks, etc., and affect pediatric development.

The pathological myopia retina choroids neovascularization treatment has poor curative effect and frequent complications due to retina photocoagulation. There is no effective data on the curative effect of macular displacement, neovascular extirpation and other operations. The current clinical anti-VEGF injection drug treatment is possibly accompanied with the deterioration of vision. And the side effects are difficult to avoid, such as endophthalmitis, apoplexy, cataract, retinal detachment, retinal pigment epithelium fissure and the like, and the apoplexy is reported in clinical tests at home and abroad. High treatment costs are also a disadvantage of current anti-VEGF injectable drugs.

The treatment of cytomegalovirus retinitis is oral and intravenous infusion, but the existing medicines often cause bone marrow suppression, renal insufficiency and other reasons to cause treatment difficulty.

Clinical steroids for neoplastic/Gorham disease, interferon alpha, propranolol, chemotherapy (vincristine), etc., but the therapeutic effect is limited. If there is a breast lesion, such as breast, the prognosis is not good. If the lesion spreads to multiple organs, various symptoms are caused, often leaving permanent diseases such as chronic respiratory failure and motor dysfunction. Many cases require long-term treatment with very low cure rates.

It is difficult to recover the large lymphatic vessel malformation (neck and face lesions) with any treatment. It is not only resistant to treatment, but also causes persistent dysfunctions (respiratory disorders, eating/swallowing disorders, vision disorders, hearing disorders, etc.) that require a lifetime medical attendance from the moment of birth.

However, until now, there has been no report clearly indicating the role or function of Sema3A in angiogenesis and lymphangiogenesis related diseases, especially in cancer, scarring and ophthalmic diseases.

Disclosure of Invention

The invention provides a polypeptide for preventing, inhibiting and/or treating diseases related to angiogenesis and lymphangiogenesis, in particular cancers, ophthalmic diseases, arteriosclerosis, cardiovascular diseases, aging diseases, scar formation, Alzheimer diseases and the like, or a fragment containing the polypeptide as an active effective ingredient, and application of the polypeptide in preparing a medicament for preventing, inhibiting and/or treating the diseases related to angiogenesis and lymphangiogenesis. Especially in the prevention and treatment of cancer-related diseases, arteriosclerosis, Alzheimer's disease, scarring, and in particular in the prevention and treatment of ophthalmic angiogenesis-related diseases.

The invention also relates to a method for preventing, inhibiting and/or treating diseases related to angiogenesis and lymphangiogenesis by the polypeptide, in particular to cancers, ophthalmic diseases, arteriosclerosis, cardiovascular diseases, ageing diseases, scar formation, Alzheimer diseases and the like.

The invention also relates to a polypeptide which is used for preventing, inhibiting and/or treating angiogenesis and lymphangiogenesis related diseases, in particular cancers, ophthalmic diseases, arteriosclerosis, cardiovascular diseases, aging diseases, scar formation, Alzheimer diseases and other diseases.

In one embodiment, the above polypeptide is selected from any one of the following (a) to (c):

(a) a polypeptide consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID No. 4 and including the region from 166aa to 235aa in SEQ ID No. 4;

(b) a polypeptide consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID No. 2 and including the region from 166aa to 235aa in SEQ ID No. 2;

(c) a polypeptide having 80% or more sequence homology to the polypeptide of (a) or (b).

The embodiments and meanings of the technical solution of the present invention will be explained in detail hereinafter.

Definition of

Unless defined otherwise, 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, but in the event of conflict, the definitions set forth herein shall control.

As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

Unless otherwise specified, the percentages (%) in this specification are all weight percentages (% by weight).

All numbers or expressions referring to quantities of ingredients, process conditions, etc. used in the specification and claims are to be understood as modified in all instances by the term "about". The term "about" when referring to a quantity or a numerical range means that the quantity or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the quantity or numerical range may vary between, for example, +5 of the quantity or numerical range.

All ranges directed to the same component or property are inclusive of the endpoints, and independently combinable. Because these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range recited herein is intended to include all sub-ranges within that range.

When the present invention is directed to a physical property, such as molecular weight, or to a range 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 "comprising" or "including" or "having" or "including") includes embodiments that are, for example, any combination of materials, compositions, methods, or processes that "consist of or" consist essentially of the recited features.

As used in this specification and claims, "and/or" should be understood to mean "either or both" of the associated components, i.e., the components may be present in combination in some instances and separately in other instances. A plurality of components listed with "and/or" should be understood in the same way, i.e., "one or more" of the associated component. In addition to the "and/or" clause-specific components, other components may optionally be present, whether related or unrelated to those specifically identified components. Thus, as a non-limiting example, reference to "a and/or B," when used in conjunction with open ended words such as "comprising," may refer in one embodiment to a alone (optionally including components other than B); in another embodiment, reference may be made to B alone (optionally including components other than a); in yet another embodiment, refers to a and B (optionally including other components), and the like.

As used in this specification and the claims, the term "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items are separated in a list, "or" and/or "should be interpreted as being inclusive, i.e., including at least one of more or components of the list, but also including more than one, and optionally other unlisted items. Only terms specifically directed to an opposite face such as "only one" or "exactly one," or "consisting of …" as used in the claims, shall mean including exactly one of the plurality or list of components. In general, the term "or" as used herein is considered to refer to an exclusive choice (i.e., one or the other but not both) only when there is an exclusive antecedent such as "or" one, "" only one, "or" exactly one.

It is to be understood that, unless explicitly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

The abbreviations used in the present invention have the usual meaning in the food, biological and chemical fields.

A. Angiogenesis diseases

Angiogenesis (angiogenesis): new capillaries are generated in a bud or non-bud (or intussusception) form from the pre-existing blood vessels mainly through the proliferation and migration of vascular endothelial cells on the basis of the original capillary blood and (or) venules.

Specific diseases are exemplified below:

rheumatoid Arthritis (RA)

RA is a chronic inflammatory disease, frequently invading all joints, the inflammatory synovium of RA has early capillaries, small vessels such as the posterior capillary venules are significantly new and are sites of inflammatory cell infiltration, and the new vessels are also involved deeply in disease morbidity as the trophoblasts of synovium. Currently, RA treatment also relies on angiogenesis inhibitors.

Psoriasis (psoriasis)

Nickoloff and the like observed a decrease in the production of IL-8 (angiogenic factor) and TSP-1 in lesions of psoriasis patients as compared with normal skin, and enhanced angiogenesis was confirmed from rat corneas by a method of culturing epidermal cells established from the tissues of lesions.

Arteriosclerosis

Arteriosclerosis is accompanied by the progression of diseases such as hyperlipidemia, hypertension, aging, etc. Vascular (vasa Vasorum) neovessel stretching is observed near atherosclerotic plaques. PDGF, TNF-alpha, FGF and TGF-beta factors are secreted to induce angiogenesis when an atherosclerotic lesion is formed.

After endothelial dysfunction of blood vessels occurs, arteriosclerosis occurs, blood vessels become narrow and hard, cholesterol and neutral fat in blood are increased and deposited inside the blood vessels, and the development of arteriosclerosis is accelerated.

Angina and renal dysfunction are caused after the blood vessel becomes narrow and hard, cerebral infarction and cardiac infarction are caused after thrombosis, and cerebral hemorrhage is caused by blood vessel rupture.

Tuberous sclerosis

Tuberous sclerosis (Tuberous sclerosis complex: TSC) is renal vascular muscle lipoma;

lymphangioleiomyomatosis (LAM) is a disease that causes systemic hamartomas, such as facial and fundus angiofibromas. Renal vascular muscle lipomas have many angiogenesis, and enlargement sometimes leads to rupture. As the cells of the tumor increase, malignant changes may occur.

While the disease of tuberous sclerosis is associated with LAM disease, most of which is developed in women of childbearing age, diseases other than LAM are likely to occur in many organs (brain, skin, heart, lung, kidney, etc.), accompanied by epilepsy, skin lesions, and other symptoms of tuberous sclerosis.

Cancer treatment

Tumor of nasal cavity

Sinus tumors are tumors caused by sinus tissue (primarily mucosal epithelium). Representative benign tumors of sinus tumors are papilloma and hemangioma. Among the malignancies, squamous cell carcinoma is the most common, in addition to which the nasal cavity produces malignancies of various pathological tissue types, including olfactory neuroblastoma. Clinical features of, for example, papillomas include a high rate of recurrence and the possibility of malignant transformation. The recurrence rate is about 30%, and most recurrences within 2 years after surgery. Malignant transformation is considered to be about 10%, usually into squamous cell carcinoma.

Malignant tumor of gynecology

The most common gynecological malignant tumors are cervical cancer, placental angioma and uterine neck cavernous hemangioma combined with pregnancy.

It occurs in cerebrospinal spongiform angiomas, where the depth of angiogenesis is involved in the growth of metastatic lesions that have the greatest impact on prognosis.

Alzheimer's disease

Vascular alzheimer's disease

Dementia is closely related to blood vessels, and vascular dementia and alzheimer's disease are caused by blockage of blood vessels, and improvement of symptoms requires restoration of cerebral blood flow.

Alzheimer's Disease (AD) is one of the dementias. The etiology is thought to be the accumulation of amyloid beta (a β) in the brain. Immunotherapy in both basic and clinical trials has shown that reduction of amyloid deposition is useful for improvement of cognitive function. For the removal of a β from the brain, antibody or vaccine immunotherapy against a β is considered promising, but side effects such as angiogenic edema and cerebral microhemorrhage are evident. Hyperfiltration of the blood-brain barrier (BBB) present between the brain and blood is closely related to the cerebral vascular endothelial cells, and it has been found that the vascular density of AD patients is also increasing.

Aging of

Blood vessels throughout the body not only supply oxygen and nutrients to every corner of the body, but also move inflammatory cells to the site when abnormality such as tissue damage or inflammation occurs, thereby promoting tissue repair. In this process, vascular endothelial cells that coat the lumen of the blood vessel are activated by TNF α, an inflammatory cytokine, and help inflammatory cells migrate out of the blood vessel and play a role in normally causing an inflammatory response.

Acute myocardial infarction

It is reported that IL-6 levels in patients with acute myocardial infarction appear to be elevated. Plasma IL-6 levels rose within hours after onset of infarction and peaked after 24 to 48 hours (FIG. 2). CRP in blood is elevated after IL-6 is elevated. The maximum IL-6 value in this process was significantly correlated with the maximum CRP value

Cytomegalovirus induced coronary artery disease

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

Cytomegalovirus acts directly on endothelial cells to enhance IL-6 production. Blakenberg et al suggest that patients who are positive for cytomegalovirus antibodies and have high blood IL-6 levels are at high risk of future heart disease death, but even if cytomegalovirus antibodies are positive, the blood IL-6 levels are not high, and the patient is not at risk.

Angiogenesis disease of ophthalmology

A representative lesion that impairs ocular transparency is angiogenesis. Newly formed blood vessels are fragile and are susceptible to repeated bleeding and exudation, forming a proliferative membrane with proliferation of other cells, and thus visual function is significantly impaired. Traumatic and chemical burn corneal angiogenesis, no medicine can treat the corneal angiogenesis at present, and the only method is corneal transplantation.

Intraocular angiogenesis diseases, typical examples of which include diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration, angiogenic glaucoma, and the like, are all intractable diseases that cause blindness. When the retina is in a blood deficient state, a signal (a chemical substance called VEGF) is sent through VEGF (neovascularization) to generate new blood vessels to nourish the retina. This "neovasculature" is very prone to rupture and is the source of retinal and vitreous hemorrhage.

Neovascular glaucoma

The etiology of neovascular glaucoma includes diabetic retinopathy syndrome, central retinal vein occlusion, and ocular ischemic syndrome.

Severe and refractory glaucoma resulting from diseases in which retinal blood flow is worsened. When blood vessels of the retina are damaged by diabetes, blood circulation is blocked, causing severe visual impairment. The new blood vessels may block the angle of the atrium, blocking the aqueous outflow, causing a sudden elevation. Generally, the intraocular pressure of about 20mmHg becomes 50mmHg or more.

Intraocular metastasis from malignant lymphoma, conjunctival malignant lymphoma, is followed by frequent development of angiogenic glaucoma in the eyeball.

Diabetic macular disease

The macula is located in the center of the retina and is the most important part to observe. Due to diabetic conditions, capillary hemangioma and the like often appear near the macula, blood components exude, and symptoms caused by macular swelling are called diabetic macular disease.

Even in the initial stage of retinopathy, a decline in vision may occur.

It is important that the angiogenesis inhibitor is used early, possibly without subjective symptoms, before the disease progresses severely. For example, in the laser coagulation treatment in the avascular field for the purpose of preventing angiogenesis, the coagulated scar of the photocoagulation omentum tissue degeneration has enlarged area with the time increase, so that the macular vision and the visual field are affected.

Retinal vein occlusion

Hypertension is one of the causes of arteriosclerosis, but even in the absence of hypertension, arteriosclerosis progresses, and if the root of the retinal vein is blocked, it becomes a central retinal vein block and bleeds throughout the retina. Resulting in sudden visual deterioration, visual field disturbance, visual deterioration, vitreous hemorrhage, and obvious visual deterioration. Associated with angiogenic glaucoma.

Uveitis-complicated intraocular neovascularization

The disease causes massive retinal and choroidal detachment, edema of optic nerve head, neovascularization in the posterior pole, and large-scale steroid contraction of new blood vessels.

Behcet's disease causes neovascularization in the retina and nipple, and treatment requires topical administration of steroid drugs and immunosuppressive drugs.

Choroidal neovascularization with high myopia

The prevalence of myopic retinopathy is 1.7%, and women have about twice the prevalence of men. If the eyeball stretches backwards and the retina/choroid is damaged, pathological blood vessels (choroidal neovascularization) can develop, leading to bleeding and swelling.

Choroidal tumors

Choroidal hemangioma, caused by abnormal proliferation of blood vessels, is frequently associated with Sturge-Weber syndrome, which is frequently associated with systemic hemangioma. Metastatic choroidal tumors are haematologic metastatic malignancies that occur in all organs of the body. Choroidal metastasis is common in both female breast cancer and male lung cancer.

Deterioration of vision can occur if a tumor occurs in a particularly important part of the vision in the macular region, or if retinal detachment around the tumor reaches the macula. As the tumor grows, the visual field may be under-lost to disappear.

Cytomegalovirus disease

Cytomegalovirus retinitis is a condition of low immune function in patients, such as AIDS patients and patients receiving anticancer therapy, where retinitis can occur as an opportunistic infection. In particular, when the number of CD4+ T cells in peripheral blood is less than 50/mm²The incidence is high.

Intraocular malignant tumors

The eye is divided into eyeball and eye accessory (eyelid, conjunctiva, eye socket, lacrimal gland, etc.), and tumor can occur in various parts. Poor prognosis even affects life survival.

Retinoblastoma

Malignant tumors appearing in the eyeballs of children are treated by surgical operations, and the eyeballs are removed if the operations are performed. Malignant melanoma of choroid is a malignant tumor in which malignant melanoma occurs in the uveal tract (choroid, ciliary body, iris) of the eyeball of an adult, and if it progresses and the tumor is large, removal of the eyeball is required.

Malignant lymphoma appearing in eyeball

Is a malignant lymphoma of central nervous system, and a subtype of lymphoma of brain. General chemotherapy, radiation therapy and other medical treatments are generally carried out, and complete cure is difficult at present. Although some preventive treatment methods have been reported, no reliable method has been practically used so far.

Tumor of eye appendage

Eyelid tumors (sebaceous adenocarcinomas) tumors that appear in the eyelid are common in ocular tumors and require reoperation or radiation therapy for recurrence.

Treatment of lacrimal cancer requires complete surgical removal of the tumor, however, even if the tumor is completely removed, it is impossible to prevent recurrence thereafter.

Collateral lymphoma of eye

The sarcoma tumor of the eyeball compresses the deviation of the eyeball, and is usually difficult to completely remove, and needs to be combined with surgery, chemotherapy and radiotherapy. A medullary tumor is a tumor that is produced by a sheath that includes the optic nerve and, when the tumor is resected, it often leads to blindness.

B. New born disease of lymphatic vessel

Lymphatic vessels are the channels through which lymphatic fluid passes, collecting the lymph fluid produced from the corners of the body, filtering bacteria and viruses contained in the lymph fluid, and finally returning them to blood vessels, and many diseases related to lymphatic vessels are problematic conditions.

In recent years, molecular mechanisms of angiogenesis in ontogeny and various pathologies such as tumor and inflammation have been elucidated, and on the other hand, studies on molecular mechanisms of lymphatic system formation in the same vascular organ have gradually identified receptors specifically expressed in lymphatic vessels, ligands thereof, transcription factors, and the like, but specific antibodies thereto are difficult to prepare and cell lines are difficult to obtain, which is an obstacle to progress of the studies. The lymphatic vessel is suggested to be used as a circulatory system for extra-cellular fluid drainage, has great influence on the immune system, and is important for determining pathological conditions such as malignant tumor lymph node metastasis, lymphedema and the like.

Lymphatic vessels play an important role in maintaining in vivo microenvironment balance, immune response, tumor lymphatic metastasis and the like, and lymphatic vessel neogenesis is closely related to embryonic development, trauma repair, congenital lymphedema, inflammation outcome and tumor metastasis. Under the action of chemotactic factors and growth factors, lymphatic endothelial cells migrate, proliferate and form a lumen to form a new lymphatic vessel. In recent years, lymphatic endothelial progenitor cells have been found to be involved in lymphatic neogenesis. The lymphatic endothelium specifically expresses Prox-1, podopiann, VEGFR-3LYVE-1, etc., and these factors and receptors regulate the regeneration of lymphatic vessels. The VEGF-C/VEGFR-3 or VEGF-D/VEGFR-3 signaling pathways play an important role in the process of lymphatic neogenesis. VEGF-C, VEGF-D and VEGFR-3 can be used as target spot of gene therapy, and is expected to treat lymphatic newborn disorder disease, immune rejection after transplantation and tumor lymphatic metastasis.

lymphangiomatosis/Gorham disease (Japanese designation as refractory disease 277)

Is a disease which is difficult to cure, and the lymphatic tissue whose diffuse abnormal expansion is caused by unknown causes invades the organs of the whole body, such as bones and breasts (lungs, mediastinum, heart), abdomen (abdominal cavity, spleen), skin, subcutaneous tissue. Many cause osteoporosis in children and adolescents, and Gorham's disease pathologically identifies irregularly distended lymphatic vessels. lymphangiomatosis/Gorham's disease is also known as diffuse lymphangiomatosis, Gorham-Stout syndrome, also known as massive osteolysis.

For example, lymphoid proliferation of esophageal squamous cell carcinoma, which can predict metastasis based on lymphatic neogenesis, esophageal cancer is highly malignant compared to colon cancer and gastric cancer among gastrointestinal tumors, and is one of the tumors with poor prognosis.

In recent years, the growth and metastasis of malignant tumors involve the formation of blood vessels and lymphatic vessels, and angiogenesis is presumed to be associated with tumor growth, while lymphangiogenesis of tumor tissues is associated with lymph node metastasis. The lymphatic distribution and lymphatic density were measured after construction and showed the ratio of the number of lymphatic vessels in the tumor and non-tumor regions. The mucosal lamina propria has a significant increase in the number of lymphatic vessels at the tumor margins. A significant increase in lymphatic vessels was also observed as a percentage of the number of blood vessels.

Large lymphatic malformation

(cervical and facial lesions) are large neoplastic lymphogenic abnormalities that occur in the face, mouth, throat and head and neck.

Lymphangioma (lymphangioma) is a mass consisting of large and small lymphocysts, accompanied by vasculopathy. Intraorbital lymphangiomas may cause intraorbital bleeding or infections leading to blindness. If the lymphatic lesions spread to the skin and mucosa, local lymphangioma is present, and recurrent lymphatic fistulae, bleeding, infection appear as symptoms. Infection and bleeding, repeated acute swelling and inflammation may occur at any site during the course of the disease.

In a huge facial lesion, high ugliness is shown due to tumor formation, discoloration and deformation, adaptability of social life of a patient is affected, and life quality in life is limited.

C. Polypeptides

Semaphorin3A(Sema3A)

Consisting of SEQ ID NO: 2 or SEQ ID NO: 4 (i.e., Semaphorin3A protein).

Amino acid sequence 2:

amino acid sequence 4:

polypeptide (a): a polypeptide consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID No. 4 and including the region from 166aa to 235aa in SEQ ID No. 4.

Polypeptide (b): a polypeptide consisting of 70 or more consecutive amino acids in the amino acid sequence shown in SEQ ID No. 2 and including the 166aa to 235aa regions in SEQ ID No. 2.

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, still more preferably 98% or more homology to the original sequence.

In general, in proteins, even if a small number of amino acid residues in the amino acid sequence of the protein are substituted, deleted or inserted, they can have almost the same function as the original protein. These are well known to those skilled in the art. Therefore, the region consisting of the polypeptide of the above (c) can also exert the physiological activity of Semaphorin3A protein, similarly to the above 70aa region.

"homology" of amino acid sequences: it means that two amino acid sequences are aligned such that the amino acid residues of the two amino acid sequences to be compared match as closely as possible and are expressed as a percentage as the number of matching amino acid residues divided by the total number of amino acid residues. Upon alignment, gaps are appropriately inserted into one or both of the two sequences to be compared as needed.

Such alignment of sequences can be performed using known programs, such as BLAST, FASTA, CLUSTAL W, and the like. When a gap is inserted, the total number of amino acid residues is the number of residues counting one gap as one amino acid residue. When the total number of amino acid residues counted in this manner differs 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.

The 20 amino acids constituting the natural protein are neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro) having low polar side chains and neutral amino acids (Asn, gin, Thr, Ser, Tyr Cys) having hydrophilic side chains, acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), aromatic amino acids (Phe, Tyr, Trp) which are known to be combined together and substitutions between them do not generally change the properties of the polypeptide.

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

Specifically, the polypeptide (c) is a polypeptide consisting of SEQ ID NO: 2 or SEQ ID NO: 4 (i.e., Semaphorin3A protein) or a fragment of Sema3A protein containing 70 amino acid regions.

Therefore, the active ingredient polypeptide c is a polypeptide consisting of only these amino acids, or a polypeptide obtained by adding an amino acid or a polypeptide to one or both ends of the polypeptide (c), and/or a polypeptide having preventive, inhibitory or therapeutic activity against corneal diseases or corneal lesions, such as blood vessels.

These specific examples are similar to the above active ingredient polypeptides a and b, and are not particularly limited, for example, those represented by SEQ ID NOs: 2 or 4 (i.e., Sema3A protein) and polypeptides having 80% or more sequence homology, polypeptides having 80% or more sequence homology to a fragment of Sema3A protein having corneal disease or corneal injury or preventive, inhibitory or therapeutic activity, and polypeptides having 80% or more sequence homology to a fragment comprising 70 amino acid regions. And a chimeric protein obtained by fusing a region having a secretory domain to a region other than Semaphorin3A in which a signal is secreted.

As the active ingredient polypeptide c, a polypeptide having higher homology in the 70aa region than that in the entire region composed of the polypeptide of (c) is particularly preferred. For example, when active ingredient polypeptide c is a polypeptide having the sequence of seq id NO: 2, preferably a polypeptide having more than 80% homology in the 70aa region, in particular 70 aa. The homology of this region is 90% or more, more preferably 95% or more, further preferably 98% or more, and more preferably, among them, a polypeptide having the same sequence in the 70aa region is preferable. 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 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%, particularly that the homology in the 70aa region is 90% or higher, more preferably 95% or higher, still more preferably 98% or higher, more preferably 90% or higher. Preferred are chimeric proteins in which the sequences in the 70aa region are identical.

In the above active ingredient polypeptides a to c, a polypeptide having an amino acid sequence of 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, still more preferably 98% or more homology, and wherein, as described above, a polypeptide having homology higher than the polypeptide as a whole in the 70aa region is preferred. Most preferably, the polypeptide contained as an active ingredient in the prophylactic, inhibitory or therapeutic agent of the present invention is a polypeptide having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

Generally, in a drug containing a polypeptide, in order to enhance the stability of the polypeptide in vivo, a sugar chain or a polyethylene glycol (PEG) chain is added to the polypeptide, or at least one amino acid constituting the polypeptide. Techniques for using D-form amino acids as part and adding Fc domains are well known and used. By adding a sugar chain or a PEG chain or using a D-type amino acid as at least a part of amino acids constituting a polypeptide, it becomes difficult to be degraded by a peptidase in vivo, and the time for halving the polypeptide in vivo becomes longer.

The polypeptide used in the present invention may be a polypeptide that has undergone these known in vivo stabilization modifications as long as it has a prophylactic, inhibitory or therapeutic activity on corneal diseases or corneal damage. Unless the context clearly dictates otherwise, the term "polypeptide" in the specification and claims is used in a sense that it also includes those modified for in vivo stabilization.

The conversion of a sugar group into a polypeptide is well known, and for example, a sugar chain can be bonded to the N-terminus, C-terminus or an amino acid therebetween, but is preferably bonded to the N-terminus or C-terminus so as not to inhibit the activity of the polypeptide. The number of sugar chains to be added is preferably one or two, and preferably one. The sugar chain is preferably a monosaccharide to a tetrasaccharide, more preferably a disaccharide or a trisaccharide. The sugar chain may be attached to the free amino or carboxyl group of the polypeptide directly or through a spacer structure such as a methylene chain having about 1-10 carbon atoms.

The PEG chain may be attached to the N-terminus, C-terminus, or amino acids therebetween, and typically one or two PEG chains are attached to a free amino or carboxyl group on the polypeptide. The molecular weight of the PEG chain is not particularly limited, but is generally about 3000 to about 7000, preferably about 5000.

It is also well known that at least a portion of the amino acids that make up a polypeptide are in the D-form, for example, although some of the amino acids that make up a polypeptide may be in the D-form, all of the amino acids that make up a polypeptide are D-form amino acids rather than amino acids that merely form the D-form, because the activity of the polypeptide is not inhibited. Methods for preferably adding an Fc domain to a polypeptide are also well known and can be produced, for example, using a commercially available Fc fusion protein expression vector such as pFUSEN-Fc (manufactured by InvivoGen).

Semaphorin3A used as the active ingredient of the present invention can be easily prepared by a conventional method using, for example, a commercially available peptide synthesizer. Moreover, it can be easily prepared using known genetic engineering techniques. For example, cDNA of the Semaphorin gene is prepared from RNA extracted from a tissue expressing the Semaphorin3A gene by RT-PCR, and the full-length or desired part 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 carried out by known methods.

In addition, 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 carried out by known methods described in the above-mentioned respective documents

Detailed Description

The 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):

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

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 the above-mentioned (c) and has a preventive, inhibitory or therapeutic activity on corneal diseases or corneal injuries. Like the above-mentioned active ingredient polypeptides a and b, they can be used for the preparation of the prophylactic, inhibitory or therapeutic agent of the present invention.

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

In one embodiment, the active ingredient polypeptide c is a polypeptide consisting of only these amino acids, or a polypeptide obtained by adding an amino acid or a polypeptide to one or both ends of the polypeptide of (c), and/or a polypeptide having preventive, inhibitory or therapeutic activity of corneal diseases or corneal injuries.

In one embodiment, active ingredient polypeptide c is similar to active ingredient polypeptides a and b described above, and is not particularly limited, e.g., as represented by SEQ ID NO: 2 or 4 (i.e., Sema3A protein) or a sequence having 80% or greater sequence homology. A polypeptide having a homology, a polypeptide having a sequence homology of 80% or more with a fragment of Sema3A protein having corneal disease corneal injury or preventive, inhibitory or therapeutic activity, which has a homology with 80% or more of a sequence comprising a 70 amino acid region, and a chimeric protein obtained by fusing a region having a secretory domain with other region of a secreted signal element other than Semaphorin 3A.

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

more preferably, a polypeptide wherein the sequence in the 70aa region is 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 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%, particularly that 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 are chimeric proteins in which the sequences in the 70aa region are identical.

In one embodiment, among the above active ingredient polypeptides a to c, a polypeptide having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, in particular a polypeptide having 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 98% or more homology, and wherein, as described above, a polypeptide having homology higher than that of the polypeptide as a whole in the 70aa region is preferred. Most preferably, the polypeptide contained as an active ingredient in the prophylactic, inhibitory or therapeutic agent of the present invention is a polypeptide having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

In one embodiment, the polypeptide used in the present invention may be a polypeptide that has undergone known in vivo stabilization modifications, so long as it has the prophylactic, inhibitory or therapeutic activity of the present invention. Preferably, it is a glycosylated polypeptide, for example, a sugar chain can be bonded to the N-terminus, C-terminus or an amino acid therebetween, but is preferably bonded to the N-terminus or C-terminus so as not to inhibit the activity of the polypeptide. The number of sugar chains to be added is preferably one or two, and preferably one. The sugar chain is preferably a monosaccharide to a tetrasaccharide, more preferably a disaccharide or a trisaccharide. The sugar chain may be attached to the free amino or carboxyl group of the polypeptide directly or through a spacer structure such as a methylene chain having about 1-10 carbon atoms.

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

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

In one embodiment, Semaphorin3A used as the active ingredient of the present invention can be easily prepared by a conventional method using, for example, a commercially available peptide synthesizer. Moreover, it can be easily prepared using known genetic engineering techniques.

For example, cDNA of the Semaphorin gene is prepared from RNA extracted from a tissue expressing the Semaphorin3A gene by RT-PCR, and the full-length or desired part 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 carried out by known methods.

Semaphorin3A is an active ingredient in the prophylactic, inhibitory or therapeutic agent of the present invention, and is effective for the prevention, inhibition or treatment of angiogenesis.

The present invention also relates to a prophylactic, inhibitory or therapeutic agent comprising the above-mentioned polypeptide (preferably Semaphorin 3A) or a pharmacologically acceptable excipient, stabilizer, preservative, buffer suitable for each administration form. Optionally, additives such as solubilizing agents, emulsifiers, diluents, tonicity agents and the like may also be appropriately mixed to prepare a formulation. The above-mentioned formulation methods and additives which can be used are well known in the field of pharmaceutical formulation, and any methods and additives can be used.

The invention also relates to application of the polypeptide in preparing medicaments for preventing, inhibiting and/or treating angiogenesis diseases and lymphangiogenesis diseases, and preferably relates to application of the polypeptide in preparing medicaments for preventing, inhibiting and/or treating cancer-related diseases, arteriosclerosis, Alzheimer disease, scar formation and aging-related diseases.

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

(c) polypeptides 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, particularly preferred are chimeric proteins in which the sequences in the 70aa region are identical.

More preferably, the polypeptide is a glycosylated polypeptide;

also preferably, the polypeptide is a pegylated polypeptide; optionally, the PEG chain has a molecular weight of about 3000 to about 7000, preferably about 5000.

In one embodiment, wherein the angiogenesis disease or lymphangiogenesis disease is cancer.

In one embodiment, wherein the cancer is one or more diseases selected from the group consisting of: breast cancer, colorectal cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal (renal cell) cancer, liver (adult primary) cancer, lymphoma, lymphangiomatosis/Gorham's disease, giant lymphatic malformations, melanoma (malignant melanoma), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, nasal tumors, gynecological malignancies, endometrial cancer, bladder cancer.

In one embodiment, the angiogenesis disease or lymphangiogenesis disease is ophthalmic angiogenesis disease or lymphangiogenesis disease.

In one embodiment, wherein the ocular angiogenic or lymphatic angiogenic disease is selected from one or more of the following: neovascular glaucoma, diabetic macular disease, retinal vein occlusion, uveitis complicated by intraocular neovascularization, Behcet's disease, tuberculous uveitis, highly myopic choroidal neovascularization, malignant lymphoma, conjunctival malignant lymphoma, choroidal tumors, cytomegaloviral disorders such as cytomegaloviral retinitis, intraocular malignant tumors such as retinoblastoma, choroidal malignant melanoma, malignant lymphoma appearing in the eyeball, ocular adnexal tumors such as optic nerve tumors.

In one embodiment, the neovascular glaucoma includes hypovascularity of the vascular and lymphatic vessels accompanying the ocular fundus angiogenesis and secondary angiogenic glaucoma caused by systemic diabetes.

In one embodiment, the angiogenesis disease or lymphangiogenesis disease is one or more diseases selected from the group consisting of: rheumatoid Arthritis (RA), psoriasis (psoriasis), arteriosclerosis, tuberous sclerosis, alzheimer's disease such as vascular alzheimer, aging, acute myocardial infarction, coronary lesions induced by cytomegalovirus and chlamydia infections.

In one embodiment, wherein the angiogenesis disease or lymphangiogenesis disease is a scar tissue formation related disease.

In one embodiment, wherein the above scar tissue formation related diseases are one or more diseases selected from the group consisting of: accompanied by scars, ulcers, wounds, diseases caused by necrosis due to infarction, and tissue defects of various organs.

In one embodiment, the angiogenesis disease or lymphangiogenesis disease is one or more diseases selected from the group consisting of: aging of aging organs, and hypofunction of vessels and lymphatic vessels caused by aging of aging organs in a deficient blood state due to hypofunction of vessels.

In one embodiment, wherein the organ aging disease comprises: vascular dementia and alzheimer's disease.

In the embodiment of the present invention, the cause of the neovascular/lymphatic disorder is not particularly limited, and may be a locally occurring disorder or a systemic disorder.

In one embodiment, the prophylactic and/or therapeutic agent of the present invention may consist of Semaphorin3A alone or pharmacologically acceptable excipients, stabilizers, preservatives, buffers suitable for each dosage form. In addition, additives such as solubilizing agents, emulsifiers, diluents, tonicity agents and the like may be appropriately mixed to prepare a formulation. And the formulation method may use any method and additive.

The object of administration of the prophylactic, inhibitory or therapeutic agent of the present invention is mammals, and examples include humans, dogs, cats, rabbits, hamsters and the like. 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 viewpoint of safety of clinical use. Thus, for example, when the object of administration of the prophylactic, inhibitory or therapeutic agent of the present invention is a human, a prophylactic, inhibitory or therapeutic agent comprising the above-mentioned active ingredient polypeptide b as an active ingredient is particularly preferable.

The preventive, inhibitory or therapeutic agent of the present invention for ophthalmic diseases can be used by administering to the cornea to be prevented or to the corneal disorder to be treated. As a method of administration, topical administration (eye drop administration on the cornea, subconjunctival injection administration, etc.) and the like can be mentioned. Dosage forms include contact lens types, eye ointments, injections, eye drops, and the like.

In the contact lens type, the aforementioned polypeptide of the present invention (preferably Semaphorin 3A) may be contained in a clear 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.

The dose of the aforementioned polypeptide of the present invention (preferably Semaphorin 3A) may be appropriately selected depending on the symptoms, age, body weight, administration method, and the like, and is not particularly limited.

The aforementioned polypeptide of the present invention (preferably Semaphorin 3A) has a unit dose of about 6000 to 60000U, preferably about 4000 to 40000U, more preferably about 500 to 5000U.

In one embodiment, in the case of eye drops, the eye drop dose may be about 1 to 3000ng, about 20 to 900ng, about 50 to 500ng, about 80 to 500 ng. Preferably, in one embodiment, the eye drop concentration is from about 10ng/ml to about 1000ng/ml, such as about 10ng/ml, about 100ng/ml, about 300ng/ml, about 1000 ng/ml. The above dosage forms and amounts are preferably once or several times daily, or every few days for several days to several months, depending on the degree of improvement of the symptoms.

In the case of subconjunctival injection, the amount of the aforementioned polypeptide of the invention (preferably Semaphorin 3A) is generally about 500 to 5000U, preferably about 500 to 5000U per day, and for the test animal, it may be administered once or several times separately.

In one embodiment, the amount of subconjunctival injection may be about 0.001 to 10 ng/day, about 0.05 to 5 ng/day. Preferably, in one embodiment, the concentration is about 10ng/ml to 1000ng/ml, e.g., about 10ng/ml, about 100ng/ml, about 300ng/ml, about 1000 ng/ml. Preferably, administration may be performed periodically, once or several times daily, or once or several days daily for several days to several months, depending on the degree of symptom improvement.

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

in one embodiment, the dosage form is an eye drop for treating dry eye, keratitis, conjunctivitis, conjunctival lymphoma, angiogenesis inhibition, chemical burns of the cornea, healing of the corneal epithelium, meibomian adenocarcinoma: wherein the content of the first and second substances,

the active ingredients are as follows: the aforementioned polypeptide (preferably Semaphorin 3A);

the active ingredient concentration is as follows: about 100-300 ng/ml.

In another embodiment, the dosage form is an ophthalmic solution for treating angiogenesis inhibition, chemical burns of the cornea, conjunctival lymphoid tumors, corneal epithelial healing, meibomian adenocarcinoma: wherein the content of the first and second substances,

the active ingredient concentration is as follows: about 100 ng/ml;

specification: 5 ml/bottle, containing active ingredients: 500 ng/bottle

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

In another embodiment, the dosage form is an ophthalmic solution for treating dry eye, keratitis, conjunctivitis, conjunctival lymphoma, angiogenesis inhibition, chemical burns of the cornea, healing of the corneal epithelium, meibomian adenocarcinoma: wherein the content of the first and second substances,

the concentration of active ingredients of the eye drops is as follows: about 300 ng/ml;

specification: 5 ml/vial containing 1500 ng/vial of the aforementioned polypeptide (preferably Sema 3A);

a total of 125 drops/bottle; 0.040 ml/drop.

In one embodiment, the dosage form is an ophthalmic ointment for the treatment of refractory dry eye, keratitis, conjunctivitis, conjunctival lymphoma, corneal epithelial healing, angiogenesis inhibition, corneal chemical burns, meibomian adenocarcinoma:

the active ingredient concentration is as follows: about 100-300 ng/ml;

specification: 3.5 g/mouse; contains 0.15% of the aforementioned polypeptide (preferably Sema 3A).

In one embodiment, the dosage form is a corneal soft contact film for inhibiting inflammation and pathological blood vessel and lymphatic vessel neogenesis and normalizing the function thereof, repairing corneal chemical burn scar, and maintaining corneal function normalization;

the cornea soft contact medicine film is also used for treating refractory cornea, conjunctiva and strong membrane eyelid diseases such as chronic conjunctivitis, refractory dry eye, electric ophthalmia, scleritis, chemical corneal burn, laser corneal burn, Stevens Johnson disease inflammation, predatory keratitis, meibomian adenocarcinoma and the like:

the active ingredient concentration is as follows: about 100-300 ng/ml;

contains 0.05-0.15% of the polypeptide (preferably Sema3A) per polypeptide;

specification: the diameter is 13.5mm-14.5 mm; the thickness is 0.01mm-0.09 mm.

The cornea soft contact medicine film has excellent moisture retention and water absorption substances and viscoelasticity, and helps the cornea soft contact medicine film to play quick-acting and long-acting roles.

In one embodiment, the dosage form is an injection solution for inhibiting inflammation and pathological blood vessels, lymphatic vessel neogenesis and normalizing their function, repairing corneal chemical burn scar:

the active ingredient concentration is as follows: about 100-300 ng/ml;

specification: 0.5 ml/tube; contains the polypeptide (preferably Sema3A)50 ng-150 ng/tube.

The injection can be used for reducing inflammatory cells, controlling symptoms, recovering normal tissue function, inhibiting neogenesis of diseased blood vessels/lymphatic vessels, and normalizing blood vessels/lymphatic vessels. Preferably, the injection is also used for the following inflammation-related diseases:

inflammation after ophthalmologic surgery, spinal cord edema, pre-adhesion prevention after fallopian tube surgery, prevention of postoperative peritoneal adhesion, Senear-Usher syndrome, subacute thyroiditis, thyrotoxicosis, severe hepatitis thyrotoxicosis, subacute eczema, atopic dermatitis, allergic rhinitis, induration penis, ulcerative colitis, symptomatic keratitis treatment, pollinosis, monetary eczema, symptomatic treatment of orbital pseudotumors, symptomatic treatment of blepharitis, psoriasis of arthritis, improvement of the general condition at the end of the cancer stage, bronchospasm, acute eczema, acute otitis media, acute leukemia, predisposition to hemorrhagic clotting factor disorders, scleroderma, symptomatic treatment of scleritis, guillain-barre syndrome, sudden onset of limb dermatitis, vasomotor rhinitis, erythema nodosum, periarteritis nodosa, symptomatic treatment of conjunctivitis, prevention of keloid, focal enteritis, symptomatic treatment of iridocyclitis, eosinophilic granuloma, laryngitis, laryngeal edema, erythema, erythroderma, reticulocytomatosis, dermatitis induced by home, eustachian tube stenosis, fasciolitis, eczema/dermatitis group, purpura, postotorhinolaryngological surgery, juvenile rheumatoid arthritis, amelioration of the general condition of severely debilitating diseases, childhood asthmatic bronchitis, seborrheic dermatitis, Stevens-Johnson disease, mydritis, contact dermatitis, asthmatic bronchitis, systemic lupus erythematosus, systemic vasculitis, prostate cancer, early keloid, hyperplastic pemphigus, aortic inflammation syndrome, polymyositis, multiple sclerosis, multiple myeloma, polyarteritis, Vegena granulomatosis, toxic eruptions, gouty arthritis, digital dermatitis in ring dermatitis, refractory stomatitis, refractory glossitis, recurrent metastasis of breast cancer, nephropathy, nephrotic syndrome, mucocutaneous syndrome, encephalitis, encephalomyelitis, cerebral edema, pustular psoriasis, pulmonary fibrosis, nasal folds, noninfectious chronic arthritis, dermatomyositis, cutaneous stomatitis, cutaneous reticulosis, cutaneous leukemia, diffuse interstitial pneumonia, sinusitis, Fuchs syndrome, Hebra red rash, osteoarthritis, radiation pneumonia, hodgkin's chronic eczema chronic otitis media, immune hemolytic anemia, symptomatic retinal vasculitis treatment, symptomatic choroiditis treatment, drug allergy, drug eruptions, Reiter's syndrome, pemphigus defolians, rheumatic myocarditis, rheumatoid spondylitis, limb arthritis of rheumatoid spondylitis, rheumatoid polymyalgia rheumatica, rheumatic fever, Lipschutz acute vulvar ulcer, chronic discoid lupus erythematosus, organ transplantation, genital eczema, chemical allergies, chemical addiction, external auditory eczema/dermatitis, laryngeal polyps, laryngeal nodules, otitis eczema/dermatitis, tissue transplantation, nasal vestibular eczema/dermatitis, perinasal eczema/dermatitis, drug addiction, anal eczema, otitis media with effusion, symptomatic treatment of uveitis, erosive esophagitis, malignant alopecia, non-infectious bursitis, non-infectious scapulohumeral periarthritis, non-infectious tendonitis, non-infectious peritonitis, non-infectious tendonitis, active cirrhosis, palmoplantar pustulosis, Behcet's disease, symptomatic treatment of external eye inflammation, symptomatic treatment of anterior segment inflammatory disease, symptomatic treatment of internal eye inflammation, symptomatic treatment of orbital inflammatory disease, symptomatic treatment of ocular inflammatory disease, infantile/infantile eczema, cirrhosis with persistent ascites, congestive heart failure, liver cirrhosis with cholestasis, chronic lymphocytic leukemia, lymphosarcoidosis, and malignant lymphoma.

In one embodiment, the dosage form is an oral liquid for treating airway allergies and inflammation, restoring normal mucosal function:

the active ingredients are as follows: sema 3A;

the active ingredient concentration is as follows: about 100-300 ng/ml;

the specification is (1)10ml or (2)100 ml;

the aforementioned polypeptide (preferably Sema3A) is 0.05% to 0.15% per polypeptide.

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

the active ingredients are as follows: the aforementioned polypeptide (preferably Semaphorin 3A)

The active ingredient concentration is as follows: about 100-300 ng/ml;

specification (1)5.0 ml/tube;

In one embodiment, the dosage form is a film-adhered tablet, which is used for treating wounds such as mucosal diseases, skin diseases, burns/wounds:

the active ingredient concentration is as follows: about 100-300 ng/ml;

specification: 0.05 g/piece, 0.05% -0.15% of the polypeptide (preferably Sema 3A);

wherein, the medicinal film adhering sheet comprises: firstly, oral adhesive tablets and secondly gynecological adhesive tablets; ③ mechanical trauma, chemical trauma, ulcer and the like.

Preferably, the oral adhesive tablet is prepared by the following method: the Sema3A oral adhesive tablet is prepared by direct tabletting of a mucoadhesive carbomer (CP934), sodium alginate and low-viscosity sodium carboxymethylcellulose (SCMC).

In one embodiment, the dosage form is a cream for use in treating a mucosal disorder, a skin disorder, a wound surface such as a burn/wound:

the active ingredient concentration is as follows: about 100-300 ng/ml;

specification: 5.0 g/mouse, 0.05% -0.15% of the above polypeptide (preferably Sema 3A).

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

the active ingredient concentration is as follows: about 100-300 ng/ml;

specification: 0.15 g/piece; the polypeptide (preferably Sema3A) is 0.05% to 0.15% per polypeptide.

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

the active ingredient concentration is as follows: about 100-300 ng/ml;

specification: 5.0 g/piece; the aforementioned polypeptide (preferably Sema3A) is 0.05% to 0.15% per polypeptide.

The above dressings can be divided into the following categories:

hydrogel wound dressings: hydrogel wound dressing for treating laser burns and medical colloid dressing with bacteriostasis and healing promotion functions;

liquid wound dressing;

③ the dressing for nursing the wound.

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

the active ingredient concentration is as follows: about 100-300 ng/ml;

specification: 5.0 ml/one of the aforementioned polypeptides (preferably Sema3A) is 0.05% to 0.15%.

The sprays can be divided into the following categories: a nasal spray; an oral spray; the gynecological spray comprises the following components: a biological preparation for treating gynecological inflammation.

In one embodiment, the dosage form is a film coating agent, which is used for treating wounds such as mucosal disorders, skin disorders, burns/wounds:

specification: 5.0 g/piece; contains 0.05-0.15% of the polypeptide (preferably Sema 3A)/polypeptide;

the active ingredient concentration is as follows: 100-300 ng/ml.

The prophylactic, inhibitory or therapeutic agents of the invention may be used alone or in combination with other prophylactic, inhibitory or therapeutic agents. Other prophylactic, inhibitory or therapeutic agents include antibiotics, anti-inflammatory agents, antiviral agents, cell growth promoters, wound healing agents, extracellular matrix components, vitamins, and the like.

Examples of the above-mentioned eye ointments include Semaphorin 3A/hyaluronic acid ointment, Semaphorin 3A/neomeril EE ointment, Semaphorin 3A/oils Linderon ointment, Semaphorin 3A/apocynum oleander ointment 0.1%. The Semaphorin 3A/hyaluronic acid ointment can be used as a preparation for hyaluronic acid ophthalmic surgery. The neomeril EE ointment is a combination drug of antibiotics and synthetic adrenocortical hormone drugs, and a high anti-inflammatory effect can be expected by the combined use with Semaphorin 3A. The same is true for the Linderon A ointment described above. The components of the adrenocortical hormone may be suitably reduced from those in the neocortical EE ointment and administered.

It is known to stabilize the concentration of Semaphorin in vivo by feedback action. Thus, Semaphorin acts mildly in vivo. In addition, since the administration of Semaphorin can activate the ability to divide without causing significant morphological changes in corneal endothelial cells, the prophylactic, inhibitory or therapeutic agent of the present invention is less likely to cause side effects.

In an embodiment, the present invention provides a method for preventing, inhibiting or treating corneal disease or corneal injury comprising the step of administering Semaphorin to a mammal.

In an embodiment, the present invention provides Semaphorin3A for use in the prevention, inhibition, or treatment of corneal disease or corneal injury.

In an embodiment, the present invention provides the use of Semaphorin3A in the manufacture of a medicament for the prevention, inhibition or treatment of corneal disease or corneal injury.

Drawings

In order that the present disclosure may be more fully understood, the invention will now be described in further detail by way of specific embodiments thereof, with reference to the accompanying drawings, in which:

fig. 1 is a schematic diagram of the bio-fluorescein staining for animal models according to the present invention, wherein the upper two figures are observed images of corneal injury, and the lower figure is a schematic diagram, wherein (a) is a photograph of the bio-fluorescein staining for animal models of corneal alkali burn under indoor light; (b) is a photograph under a slit-lamp blue free filter.

FIG. 2 is a photograph showing the treatment of full corneal epithelium scratching and fluorescein staining after the basic corneal burn model was created.

FIG. 3 is a photograph showing a comparison of clinical symptoms of epithelial insufficiency.

FIG. 4 is an enlarged microscopic photograph of a PBS-administered control group 14 days after treatment.

FIG. 5 shows the evaluation of corneal epithelial underdamage, and the symptoms of the treatment groups at concentrations of 100ng/ml and 1000ng/ml are significantly improved.

Fig. 6 is an evaluation of corneal angiogenesis and the results show that Sema3A inhibits neovascularization in a concentration-dependent manner.

FIG. 7 is a photograph of immunostaining for CD31 antibody and LYVE-1 antibody in corneal plates 14 days after debridement.

Fig. 8 shows that Sema3A effectively inhibited pathological angiogenesis after corneal alkali burn.

Fig. 9 shows that Sema3A effectively inhibited pathological lymphangiogenesis after corneal alkali burn.

FIG. 10 is a photograph of immunostaining of inflammatory cell infiltration of corneal tissue. (photograph of immunostaining of corneal tissue. beta. III tubulin and F4/80)

FIG. 11 is an evaluation of the number of F4/80 antibody positive cells (macrophages).

FIG. 12 is a photograph of corneal section showing nerve fiber immunostaining.

FIG. 13 is a corneal β III tubulin positive nerve fiber density assessment.

For a further understanding of the present invention, specific embodiments thereof are described in detail below with reference to the examples. It is to be understood that such description is merely for purposes of further illustrating the features and advantages of the invention, and is not intended to limit the scope of the claims.

Examples

Example 1

The purpose of this example is to demonstrate the prevention, inhibition and/or treatment effects of the polypeptide of the present invention, especially on angiogenesis/lymphangiogenesis of Semaphorin3A, and diseases associated with endothelial dysfunction of blood vessels, especially cancer, ophthalmic diseases, arteriosclerosis, cardiovascular, cerebrovascular, and renal vascular diseases, alzheimer's disease, aging diseases, scar formation, and the like.

1、Experimental methods：

A corneal alkali damage model was prepared by dropping 2ml of 0.15M NaOH, and the following examination was performed. The drug was administered 1 time every 1 day and for 7 days from the day of the development of alkaline lesions. In the experimental group, Semaphorin3A (2 μ l) was administered subconjunctivally on

days

0, 3, 6 and 9 after the operation, manufactured by LSBio corporation. Control groups were given subconjunctivally in PBS.

2、Grouping and treatment of laboratory animals：

BALB/c Male 8 weeks old

Experimental animals were randomly divided into the following groups of 8 animals each:

(1) PBS as a control group (n ═ 8)

(2) Semaphorin3A (10ng/ml 2 μ l/subconjunctival injection) dosing group (n-8);

(3) semaphorin3A (100ng/ml 2 μ l/subconjunctival injection) dosing group (n-8);

(4) semaphorin3A (1000ng/ml 2 μ l/subconjunctival injection) dosing group (n-8);

3、animal model organism staining：

A slit-lamp blue free filter (blue no filter) is used which selectively transmits only the green fluorescent color emitted by fluorescein, so an observed image of corneal damage can be obtained.

4、Fluorescein staining test: the condition of the cornea was observed with fluorescein staining. Staining, if any, means that the condition of corneal erosion and corneal epithelium is unstable.

This test is used to examine corneal ulceration (corneal wound) by using the nature of a penetrating epithelial defect and a weak adhesion zone between epithelial cells.

5、Evaluation index：

The corneal findings were recorded: the extent of corneal neovascularization and corneal epithelial defects was semi-quantitatively assessed on alkaline injury, corneal clouding, and corneal observation under a

slit lamp microscope

3, 6, 9, and 14 days after trauma as follows:

corneal opacity: five grades of 0-4 are statistically scored according to degree.

Grading of new blood vessels in cornea: the length and range of invasion into the cornea were statistically classified into five grades of 0-4.

Corneal epithelial defect range: since epithelial defects are generally elliptical, they are evaluated by the ratio of the long axis of the defect range to the corneal diameter.

The drug and control groups were given and compared using the unpaired Student's t test. P <0.01, P < 0.001; ns: there was no significant difference.

Clinical symptom scoring:

animal models were examined daily for changes in ocular clinical symptoms using slit lamp microscopy. The corneal opacity scoring system was: neovascularization (NV) and corneal epithelial defect area. Definitions the scoring system describes the degree of corneal haze (0-4) and NV (0-4) in semi-quantitative terms. On

days

2, 5, 6, 10, 14 after the corneal injury model was made, blind observers assigned a haze score for each corneal opacity:

0 ═ clear cornea;

1-minimum surface (non-corneal stroma) opacity;

2-minimum depth (corneal stroma) opacity;

3 ═ moderate corneal stroma opacity;

the corneal stroma, at 4 ═ intensity, is opaque.

NV scoring system:

0 ═ no vascular invasion of the cornea;

1-in less than two quadrants, the vessel length is less than half the corneal radius;

2-the length of the blood vessel exceeds half of the corneal radius and is less than two quadrants;

3-vessel length less than half the corneal radius in more than two quadrants,

the vessel length exceeds half of the corneal radius in more than two quadrants.

The area of corneal epithelial defect was calculated as a ratio of the defect area.

Long axis: the diameter of the cornea.

Statistical analysis

All data are expressed as mean ± SEM.

Values below the detection limit were 0 pg/ml. The comparison was performed using the unpaired Student's t test. P <0.01, P < 0.001; ns: there was no significant difference.

Tests were performed to statistically compare the clinical scores at each time point for untreated damaged corneas (controls) and Sema3A treated corneas.

6.Results of the experiment：

Epithelial under-damage model

FIG. 1(a) is a photograph of a corneal alkali burn model taken under indoor light. As shown, the area of the cornea is shown in the white broken line in fig. 1(a), and the corneal epithelium is detached after the corneal alkali burn. The black solid frame is the epithelial tissue accumulation area which is degenerated and shed after burn. FIG. 1(b) is a photograph of an alkaline corneal burn after staining with fluorescein and taking a photograph under a slit-lamp blue free filter. As shown, the cornea area is shown in the white broken line, and the arrow points to the corneal epithelial defect area where the white part fluorescein is attached.

Figure 2 shows a photograph of fluorescent staining after crawling of the full corneal epithelium. As shown in fig. 2, the area of the white broken line is the corneal area, the epithelial tissue on the surface of the cornea is completely peeled off after debridement, the whole cornea is colored by fluorescein, and the whole cornea becomes turbid like ground glass.

Comparison of clinical symptoms of epithelial deficit

Figure 3 shows a comparison of clinical symptoms of corneal epithelial deficit. Changes in corneal repair were observed on

days

3, 6, 9, and 14 after the alkaline corneal trauma, and epithelial lesions were stained with fluorescein and photographed under a microscope. Wherein: the observation of the animal model of corneal alkali burn for 2 weeks shows that the pictures of

lines

1, 3, 5 and 7 are the clinical symptom changes of corneal epithelium on

days

3, 6, 9 and 14 under indoor light; the photographs in

rows

2, 4, 6, and 8 show the clinical symptoms of corneal epithelium at

days

3, 6, 9, and 14 with slit-lamp blue free filter (blue without filter) after fluorescein staining.

According to the experimental results, the control group and the treatment group had 10ng/ml and 1000ng/ml of eye surface such as conjunctiva and cornea which still had fluorescein-stained areas 2 weeks after the burn, and the inflammation did not heal. The cornea became cloudy with inflammation and vascular invasion for more than 2 weeks, 100ng/ml had a clear effect on angiogenesis, epithelial defects and wound healing. The cornea had little fluorescein staining and tear fluid retention was adequate. The normal eye state is substantially restored.

FIG. 4 is an enlarged microscopic photograph of the PBS-administered control of FIG. 3 after 14 days of treatment. In fig. 4(a), the area of the broken white line is the area of corneal tissue, and the arrow indicates the neovasculature of cornea after alkali burn. The blood vessels are large, the corneal transmittance is influenced, and the vision of the animal model is sharply reduced. In FIG. 4(b), the area of broken white lines is shown in the area of corneal tissue, and white arrows indicate that the whole corneal degeneration after alkali burn appears white and opaque. The white solid line range is the area of corneal degenerative scar proliferation, and the black arrows indicate that the tissue is degenerative, hypertrophic, and opaque. Animal models of visual height loss or blindness.

Evaluation of epithelial deficit

Figure 5 shows the evaluation of corneal epithelial deficit. Corneal repair changes, fluorescein staining, clinical sampling, were observed on

days

2, 3, 4, 6, 10, and 14 after the creation of the alkaline trauma model (fig. 5).

The degree of corneal epithelial defects was scored as five:

longitudinal axis evaluation method (see: Den et al, effective of early system beta breast or cyclosporine A after corner early along in the interior of virtual tissue cytokine reduction)

0 ═ clear cornea;

1-minimum surface (non-corneal stroma) opacity;

2-minimum depth (corneal stroma) opacity;

3 ═ moderate corneal stroma opacity;

the corneal stroma, at 4 ═ intensity, is opaque.

The concentration-dependent clinical symptoms varied from day 6, and the symptoms were significantly improved in the 100ng/ml and 1000ng/ml treatment groups. The treatment group at a concentration of 100ng/ml had a significant effect on day 6 (. about.p < 0.01); significant difference at day 14 (. about.P <0.05)

By administration, the tissue for treating corneal burn is remarkably repaired, corneal epithelium is well healed, and the corneal transparency of the treatment group with the concentration of 100ng/ml is not different from that of a normal cornea.

Evaluation of angiogenesis

Figure 6 shows the evaluation of corneal angiogenesis. Evaluation was performed in five stages, based on the length and degree grading criteria of neovascularization, 2, 5, 6, 10, 14 days after the alkaline trauma.

The cornea was immunostained after 14 days with an anti-CD 31 antibody (vascular endothelial marker) and the vascular area was quantified using a tiled corneal patch. The neovascular length in the cornea was measured under a surgical microscope. Of these, mice were tested as mean ± s.d. with 8 mice per group (n ═ 8).

Neovascularization: its invasive corneal length and range were scored on five scales of 0 to 4.

The system method for scoring the new blood vessels and the new lymphatic vessels comprises the following steps:

0 ═ none

1-in less than two quadrants, the vessel/lymphatic vessel length is less than half the corneal radius;

2-vessel/lymphatic vessel length exceeds half of corneal radius and is less than two quadrants;

3-vessel/lymphatic length less than half of the corneal radius in more than two quadrants,

the length of the vessel/lymphatic vessel exceeds half the corneal radius, in more than two quadrants.

Calculating the area of corneal epithelial defect as a proportion of the defect area

Long axis: diameter of cornea

Looking at the 2-week results, Sema3A inhibited neovascularization in a concentration-dependent manner.

Wherein, compared to the PBS control group, 100ng/ml of Sema3A exhibited inhibition of cardiovascular formation on day 4; there was a significant difference at day 10 (. about.p <0.01), and the treatment significantly inhibited the generation and growth formation of new blood vessels in the cornea by administration.

Evaluation of immunostaining of blood vessels, lymphatic vessels, corneal plates

FIG. 7 shows immunostaining of corneal plates 14 days after debridement with CD31 antibody (vascular endothelial marker), LYVE-1 antibody (lymphatic endothelial marker).

After Sema3A was administered to the treatment groups, blood vessels and lymphatic vessels were immunostained in the treatment groups and control groups at Sema3A concentrations of 10ng/ml, 100ng/ml, and 1000 ng/ml. Quantitative evaluation was performed using the percentage of the area of the vessels and lymphatic regions.

As can be seen from FIG. 7, the PBS-treated control group had a large area of angiogenesis and lymphangiogenesis, and compared with the PBS group, the Sema 3A-concentration 100 ng/ml-treated group significantly inhibited angiogenesis and lymphangiogenesis, and normal transparency was maintained in the central portion of the cornea. Sema3A was shown to be effective in promoting epithelialization.

Evaluation of vascular/lymphangiogenesis

Fig. 8 shows the evaluation of the corneal angiogenesis region (expressed as angiogenesis region area (%)). After the Sema3A administration at 14 days after debridement, the angiogenesis area of the Sema3A treatment group with the concentration of 100ng/ml is obviously reduced, and is obviously different from that of the control group: p ═ 0.014; each group of mice had 8 mice (n-8); assay was mean ± s.d. The above results indicate that Sema3A is effective in inhibiting pathological angiogenesis after corneal alkali burn.

Fig. 9 shows the evaluation of the neogenetic region of the corneal lymphatics (expressed as the neogenetic area (%) of the lymphatics). 14 days after debridement, after the administration of the Sema3A in the treatment group, the new area of the lymph vessels in the Sema3A treatment group with the concentration of 100ng/ml is obviously reduced, and is obviously different from that in the control group: p ═ 0.02; each group of mice had 8 mice (n-8); assay was mean ± s.d. It is shown that Sema3A effectively inhibits pathological lymphangiogenesis after corneal alkali burn.

Infiltration of inflammatory cells of corneal tissue

FIG. 10 is a photograph of the corneal tissue immunostaining for β III tubulin and F4/80 (day 14 after debridement). Which shows the infiltration of inflammatory cells of corneal tissue.

Triangle white arrows indicate immunostaining-positive inflammatory cells;

a, left: immunostaining for the nerve fiber marker β III tubulin;

in the step B: when the anti-F4/80 antibody is used for mouse macrophage detection, the infiltration of F4/80 positive cells (macrophages) of the corneal tissue of a Sema3A treatment group with the concentration of 100ng/ml is obviously reduced compared with that of a control group. Sema3A was shown to inhibit the inflammatory response that occurs after alkaline trauma to the cornea.

Evaluation of the number of F4/80 antibody-positive cells (macrophages)

FIG. 11 shows the evaluation of the number of F4/80 antibody positive cells (macrophages).

On day 14 after debridement, after the administration of the Sema3A in the treatment group, the number of F4/80 antibody positive cells in the Sema3A treatment group with the concentration of 100nng/ml is obviously reduced, and compared with the control group, the number is significantly different: p < 0.0001; each group of mice was 15 (n-15) and was characterized as mean ± s.d.

Corneal section nerve fiber immunostaining

Figure 12 shows a photograph of immunostaining of corneal section nerve fibers. On day 14 post-debridement, corneal sections were immunostained with an anti- β III tubulin antibody, PGP 9.5 (neural marker). White arrows indicate that the part is nerve fibers, and the cornea of each concentration of the control group and the treatment group and corneal nerve fibers distributed in corneal tissue are immunofluorescent-stained.

As can be seen in FIG. 12, there was no disruption of the plexus when Sema3A was administered to corneal tissue at concentrations of 100ng/ml and 1000 ng/ml.

Figure 13 quantifies the effect of corneal sensory nerves (trigeminal nerves). β III tubulin positive nerve fiber density was used for evaluation.

On day 14 after debridement, the corneal nerve fiber area was quantified.

Corneal nerve fibers were quantified as the percentage of the threshold area of β -tubulin III staining positive in the representative confocal images shown in the figure.

N.s. each treatment group had no significant difference from the normal and control groups; 15 mice per group (n-15); assay was mean ± s.d.

This result demonstrates that Sema3A administered has no effect on nerve fibers in corneal tissue.

In summary, we examined the effect of Semaphorin3A on corneal epithelial defects, corneal neovascularization, and lymphangiogenesis using a mouse model of corneal alkaline injury.

Particularly in the 100ng/ml group administered Semaphorin3A, corneal epithelial defect, corneal neovascularization, corneal neolymphangiogenesis and inflammatory cell infiltration were all significantly inhibited, and there was no reduction in the cornea as compared with that of β -III tubulin positive neurofibrillary normal mice.

These results, based on our in vivo studies, indicate that Semaphorin3A is effective in promoting epithelialization, inhibiting pathological angiogenesis and inhibiting inflammation that occurs after corneal alkaline trauma.

Various aspects of the present invention have been illustrated by the above-described embodiments. It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Sequence listing

<110> Tianzhong Chunmei (TANAKA JUNMI)

<120> polypeptide for angiogenesis and lymphangiogenesis related diseases and application thereof

<130> P19H42707

<160> 4

<170> PatentIn version 3.1

<210> 2

<211> 771

<212> PRT

<213> Intelligent people

<400> 2

Met Gly Trp Leu Thr Arg Ile Val Cys Leu Phe Trp Gly Val Leu Leu

1 5 10 15

Thr Ala Arg Ala Asn Tyr Gln Asn Gly Lys Asn Asn Val Pro Arg Leu

20 25 30

Lys Leu Ser Tyr Lys Glu Met Leu Glu Ser Asn Asn Val Ile Thr Phe

35 40 45

Asn Gly Leu Ala Asn Ser Ser Ser Tyr His Thr Phe Leu Leu Asp Glu

50 55 60

Glu Arg Ser Arg Leu Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe

65 70 75 80

Asp Leu Val Asn Ile Lys Asp Phe Gln Lys Ile Val Trp Pro Val Ser

85 90 95

Tyr Thr Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys

100 105 110

Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Ala Tyr Asn Gln Thr His

115 120 125

Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Ile Cys Thr Tyr Ile

130 135 140

Glu Ile Gly His His Pro Glu Asp Asn Ile Phe Lys Leu Glu Asn Ser

145 150 155 160

His Phe Glu Asn Gly Arg Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu

165 170 175

Thr Ala Ser Leu Leu Ile Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala

180 185 190

Asp Phe Met Gly Arg Asp Phe Ala Ile Phe Arg Thr Leu Gly His His

195 200 205

His Pro Ile Arg Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro

210 215 220

Lys Phe Ile Ser Ala His Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp

225 230 235 240

Asp Lys Val Tyr Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu His

245 250 255

Ser Gly Lys Ala Thr His Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp

260 265 270

Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys

275 280 285

Ala Arg Leu Ile Cys Ser Val Pro Gly Pro Asn Gly Ile Asp Thr His

290 295 300

Phe Asp Glu Leu Gln Asp Val Phe Leu Met Asn Phe Lys Asp Pro Lys

305 310 315 320

Asn Pro Val Val Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys

325 330 335

Gly Ser Ala Val Cys Met Tyr Ser Met Ser Asp Val Arg Arg Val Phe

340 345 350

Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro

355 360 365

Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys

370 375 380

Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro Asp Asp Val Ile

385 390 395 400

Thr Phe Ala Arg Ser His Pro Ala Met Tyr Asn Pro Val Phe Pro Met

405 410 415

Asn Asn Arg Pro Ile Val Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr

420 425 430

Gln Ile Val Val Asp Arg Val Asp Ala Glu Asp Gly Gln Tyr Asp Val

435 440 445

Met Phe Ile Gly Thr Asp Val Gly Thr Val Leu Lys Val Val Ser Ile

450 455 460

Pro Lys Glu Thr Trp Tyr Asp Leu Glu Glu Val Leu Leu Glu Glu Met

465 470 475 480

Thr Val Phe Arg Glu Pro Thr Ala Ile Ser Ala Met Glu Leu Ser Thr

485 490 495

Lys Gln Gln Gln Leu Tyr Ile Gly Ser Thr Ala Gly Val Ala Gln Leu

500 505 510

Pro Leu His Arg Cys Asp Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys

515 520 525

Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ser Ala Cys Ser Arg

530 535 540

Tyr Phe Pro Thr Ala Lys Arg Arg Thr Arg Arg Gln Asp Ile Arg Asn

545 550 555 560

Gly Asp Pro Leu Thr His Cys Ser Asp Leu His His Asp Asn His His

565 570 575

Gly His Ser Pro Glu Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser

580 585 590

Thr Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp

595 600 605

Gln Phe Gln Arg Arg Asn Glu Glu Arg Lys Glu Glu Ile Arg Val Asp

610 615 620

Asp His Ile Ile Arg Thr Asp Gln Gly Leu Leu Leu Arg Ser Leu Gln

625 630 635 640

Gln Lys Asp Ser Gly Asn Tyr Leu Cys His Ala Val Glu His Gly Phe

645 650 655

Ile Gln Thr Leu Leu Lys Val Thr Leu Glu Val Ile Asp Thr Glu His

660 665 670

Leu Glu Glu Leu Leu His Lys Asp Asp Asp Gly Asp Gly Ser Lys Thr

675 680 685

Lys Glu Met Ser Asn Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg

690 695 700

Asp Phe Met Gln Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu

705 710 715 720

Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg

725 730 735

Pro Gly His Thr Pro Gly Asn Ser Asn Lys Trp Lys His Leu Gln Glu

740 745 750

Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu Phe Glu Arg Ala Pro

755 760 765

Arg Ser Val

770

<210> 4

<211> 772

<212> PRT

<213> original chicken

<400> 4

Met Gly Trp Leu Arg Gly Ile Ala Leu Leu Ser Leu Gly Val Leu Leu

1 5 10 15

Ala Gly Arg Val Asn Cys Gln His Val Lys Asn Asn Val Pro Arg Leu

20 25 30

Lys Leu Ser Tyr Lys Glu Met Leu Glu Ser Asn Asn Ile Val Asn Phe

35 40 45

Asn Gly Leu Ala Asn Ser Ser Ser Tyr His Thr Phe Leu Leu Asp Glu

50 55 60

Glu Arg Ser Arg Leu Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe

65 70 75 80

Asn Leu Val Asn Ile Lys Glu Tyr Gln Lys Ile Val Trp Pro Val Ser

85 90 95

His Ser Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Arg

100 105 110

Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Thr Tyr Asn Gln Thr His

115 120 125

Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Met Cys Thr Tyr Ile

130 135 140

Glu Val Gly Ser His Pro Glu Asp Asn Ile Phe Arg Met Glu Asp Ser

145 150 155 160

His Phe Glu Asn Gly Arg Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu

165 170 175

Thr Ala Ser Leu Leu Val Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala

180 185 190

Asp Phe Met Gly Arg Asp Phe Ala Ile Phe Arg Thr Leu Gly His His

195 200 205

His Pro Ile Arg Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro

210 215 220

Arg Phe Ile Ser Ala His Leu Ile Pro Glu Ser Asp Asn Pro Glu Asp

225 230 235 240

Asp Lys Ile Tyr Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu His

245 250 255

Thr Gly Lys Ala Thr His Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp

260 265 270

Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys

275 280 285

Ala Arg Leu Ile Cys Ser Val Pro Gly Pro Asn Gly Ile Asp Thr His

290 295 300

Phe Asp Glu Leu Gln Asp Val Phe Leu Met Asn Ser Lys Asp Pro Lys

305 310 315 320

Asn Pro Ile Val Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys

325 330 335

Gly Ser Ala Val Cys Met Tyr Ser Met Thr Asp Val Arg Arg Val Phe

340 345 350

Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro

355 360 365

Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys

370 375 380

Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro Asp Glu Val Ile

385 390 395 400

Thr Phe Ala Arg Ser His Pro Ala Met Tyr Asn Pro Val Phe Pro Ile

405 410 415

Asn Ser Arg Pro Ile Met Ile Lys Thr Asp Val Asp Tyr Gln Phe Thr

420 425 430

Gln Ile Val Val Asp Arg Val Asp Ala Glu Asp Gly Gln Tyr Asp Val

435 440 445

Met Phe Ile Gly Thr Asp Ile Gly Thr Val Leu Lys Val Val Ser Ile

450 455 460

Pro Lys Glu Thr Trp His Glu Leu Glu Glu Val Leu Leu Glu Glu Met

465 470 475 480

Thr Val Phe Arg Glu Pro Thr Val Ile Ser Ala Met Lys Ile Ser Thr

485 490 495

Lys Gln Gln Gln Leu Tyr Ile Gly Ser Ala Thr Gly Val Ser Gln Leu

500 505 510

Pro Leu His Arg Cys Asp Val Tyr Gly Lys Ala Cys Ala Glu Cys Cys

515 520 525

Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ser Ser Cys Ser Arg

530 535 540

Tyr Phe Pro Thr Ala Lys Arg Arg Thr Arg Arg Gln Asp Ile Arg Asn

545 550 555 560

Gly Asp Pro Leu Thr His Cys Ser Asp Leu Gln His His Asp Asn Pro

565 570 575

Ser Gly Gln Thr Leu Glu Glu Lys Ile Ile Tyr Gly Val Glu Asn Ser

580 585 590

Ser Thr Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala Ile Val Tyr

595 600 605

Trp Gln Phe Gln Lys Gln Asn Asp Asp His Lys Val Glu Ile Lys Val

610 615 620

Asp Asp Arg Met Ile Arg Thr Glu Gln Gly Leu Leu Leu Arg Ser Leu

625 630 635 640

Gln Arg Arg Asp Ser Gly Ile Tyr Phe Cys His Ala Val Glu His Gly

645 650 655

Phe Ile Gln Thr Leu Leu Lys Val Thr Leu Glu Val Ile Asp Thr Asp

660 665 670

His Leu Glu Glu Leu Leu His Lys Glu Glu Asp Ala Asp Ala Ser Lys

675 680 685

Thr Lys Asp Ala Thr Asn Ser Met Thr Pro Ser Gln Lys Ile Trp Tyr

690 695 700

Arg Asp Phe Met Gln Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp

705 710 715 720

Glu Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln

725 730 735

Arg Pro Ala Asn Ala Gln Val Asn Thr Asn Lys Trp Lys His Leu Gln

740 745 750

Glu Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu Phe Glu Arg Ala

755 760 765

Pro Arg Ser Val

770

Claims

1. The application of a polypeptide in preparing a medicament for preventing, inhibiting and/or treating angiogenesis diseases and lymphangiogenesis diseases,

wherein the polypeptide is selected from any one of the following (a) to (c):

More preferably, the polypeptide is a glycosylated polypeptide;

2. A method of treatment for preventing, inhibiting and/or treating angiogenesis diseases, lymphangiogenesis diseases, the method comprising administering to a subject in need thereof an effective amount of a polypeptide,

wherein the polypeptide is selected from any one of the following (a) to (c):

More preferably, the polypeptide is a glycosylated polypeptide;

3. A polypeptide for use in the prevention, inhibition and/or treatment of an angiogenic disease, a lymphangiogenic disease,

wherein the polypeptide is selected from any one of the following (a) to (c):

More preferably, the polypeptide is a glycosylated polypeptide;

4. The use of any one of claims 1-3, wherein the unit dose of the polypeptide is about 6000 to 60000U, preferably about 4000 to 40000U, or about 500 to 5000U.

5. The use of any one of claims 1-4, wherein the unit dose of said polypeptide is about 1 to 3000ng, about 20 to 900ng, about 50 to 500ng, about 80 to 500 ng.

6. The use of any of claims 1-5, wherein the concentration of the polypeptide is from about 10ng/ml to about 1000ng/ml, such as about 10ng/ml, about 100ng/ml, about 300ng/ml, about 1000ng/ml, preferably about 100 ng/ml.

7. The use of any one of claims 1-6, wherein the polypeptide is an eye drop, an eye ointment, an injection, a corneal soft contact film, an oral liquid, a mouthwash, a film adhesive tablet, a cream, a suppository, a spray.

8. The use of any one of claims 1-7, wherein the angiogenic or lymphangiogenic disease is cancer.

9. The use of any one of claims 1-8, wherein the cancer is one or more diseases selected from the group consisting of: breast cancer, colorectal cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal (renal cell) cancer, liver (adult primary) cancer, lymphoma, lymphangiomatosis/Gorham's disease, giant lymphatic malformations, melanoma (malignant melanoma), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, nasal tumors, gynecological malignancies, endometrial cancer, bladder cancer.

10. The use of any one of claims 1-9, wherein the angiogenic or lymphangiogenic disease is an ophthalmic angiogenic or lymphangiogenic disease.

11. The use of any one of claims 1-10, wherein the ophthalmic angiogenic or lymphangiogenic disease is selected from one or more of the following: neovascular glaucoma, diabetic macular disease, retinal vein occlusion, uveitis complicated by intraocular neovascularization, Behcet's disease, tuberculous uveitis, highly myopic choroidal neovascularization, malignant lymphoma, conjunctival malignant lymphoma, choroidal tumors, cytomegaloviral disorders such as cytomegaloviral retinitis, intraocular malignant tumors such as retinoblastoma, choroidal malignant melanoma, malignant lymphoma appearing in the eyeball, ocular adnexal tumors such as optic nerve tumors.

12. The use according to any one of claims 1 to 11, wherein the neovascular glaucoma comprises hypovascularity of the vascular and lymphatic vessels accompanying ocular fundus angiogenesis and secondary angiogenic glaucoma resulting from systemic diabetes.

13. The use of any one of claims 1-12, wherein the angiogenic or lymphangiogenic disease is one or more diseases selected from the group consisting of: rheumatoid Arthritis (RA), psoriasis (psoriasis), arteriosclerosis, tuberous sclerosis, alzheimer's disease such as vascular alzheimer, aging, acute myocardial infarction, coronary lesions induced by cytomegalovirus and chlamydia infections.

14. The use according to any one of claims 1 to 13, wherein the angiogenic or lymphangiogenic disease is a scar-tissue formation related disease.

15. The use of any one of claims 1 to 14, wherein the scar tissue formation related disease is one or more diseases selected from the group consisting of: accompanied by scars, ulcers, wounds, diseases caused by necrosis due to infarction, and tissue defects of various organs.

16. The use of any one of claims 1-15, wherein the angiogenic or lymphangiogenic disease is one or more diseases selected from the group consisting of: aging of aging organs, and hypofunction of vessels and lymphatic vessels caused by aging of aging organs in a deficient blood state due to hypofunction of vessels.

17. The use of any one of claims 1-16, wherein the organ-aging disorder comprises: vascular dementia and alzheimer's disease.

18. The use according to any one of claims 1 to 17, wherein the angiogenic or lymphangiogenic disease is a disease associated with cardiovascular, cerebrovascular and renal vascular disorders.

19. The use of any one of claims 1-18, wherein said polypeptide is Semaphorin 3A.

20. The use of any one of claims 1-19, wherein the polypeptide is combined with other prophylactic, inhibitory or therapeutic agents, such as Semaphorin3A in combination with hyaluronic acid, Semaphorin3A in combination with neomer EE, Semaphorin3A in combination with oxyders linkage, Semaphorin3A in combination with apocynum venetum.