Therapeutic agents for fibrosis, inflammation, and / or aging diseases

Abstract

The purpose of the present invention is to provide a therapeutic and / or preventive agent for fibrotic disease such as pulmonary fibrosis, inflammatory disease and / or geriatric disease.　The present invention pertains to a therapeutic and / or preventive agent for fibrotic disease, inflammatory disease and / or geriatric disease, which comprises extracellular vesicle granules derived from cells in tissue where the aforementioned diseases can occur or peripheral tissue, said cells being differentiated cells, and pertains to a pharmaceutical composition that contains said therapeutic and / or preventive agent.

Description

Technical Field

[0001] In one embodiment, the present invention relates to a therapeutic and / or prophylactic agent for at least one disease among fibrotic diseases, inflammatory diseases, and aging diseases, and a pharmaceutical composition containing the therapeutic and / or prophylactic agent.

Background Art

[0002] Fibrosis of tissue is an excessive formation of fibrous connective tissue that occurs during the repair or healing process of an organ or tissue. Such tissue fibrosis can occur in various organs, and fibrotic diseases such as pulmonary fibrosis and liver cirrhosis are known. The number of patients with fibrotic diseases reaches several hundred thousand in the country, but there is still no effective treatment method.

[0003] Pulmonary fibrosis is a representative fibrotic disease and can also be considered a type of inflammatory disease or aging disease. Among pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF) with an undetermined cause has a very poor prognosis with an average life expectancy of 3 to 5 years after diagnosis. The prevalence of IPF in Japan is about 20 per 100,000 people, and the frequency in the elderly over 70 years old is considered to be 10 times that. As treatment methods for pulmonary fibrosis, anti-fibrotic drugs such as pirfenidone and nintedanib, steroids, immunosuppressants, etc. are used (Non-Patent Documents 1 to 2), but the effects are limited.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In one embodiment, an object of the present invention is to provide a therapeutic and / or prophylactic agent for fibrotic diseases such as pulmonary fibrosis, inflammatory diseases, and / or aging diseases.

Means for Solving the Problems

[0006] The present inventors have found that extracellular vesicle particles derived from cells in the tissue where the above diseases can occur or the surrounding tissue thereof have excellent therapeutic and / or prophylactic effects on the above diseases, and have completed the present invention.

[0007] The present invention includes the following embodiments. (1) A therapeutic and / or prophylactic agent for at least one of fibrotic diseases, inflammatory diseases, and aging diseases, comprising extracellular vesicle particles derived from cells in the tissue where the disease can occur or the surrounding tissue thereof, wherein the cells are differentiated cells. (2) The therapeutic and / or prophylactic agent according to (1), wherein the tissue from which the cells donating the extracellular vesicle particles are derived is different from the tissue of the recipient cells. (3) The therapeutic and / or prophylactic agent according to (1), wherein the cells are epithelial cells, endothelial cells, mesothelial cells, muscle cells, or nerve cells. (4) The therapeutic and / or prophylactic agent according to (3), wherein the epithelial cells are airway epithelial cells, bronchiolar epithelial cells, or alveolar epithelial cells. (5) The therapeutic and / or prophylactic agent according to (1), wherein the tissue or the surrounding tissue thereof is selected from the group consisting of lung or airway; eye; kidney or glomerulus; liver, gallbladder or bile duct; intestinal tract; pancreas; heart; blood vessel; thyroid; brain or nerve; intra-abdominal cavity; uterus; skin; muscle; bone; and joint. (6) The therapeutic and / or prophylactic agent according to (5), wherein the tissue or the surrounding tissue thereof is lung or airway. (7) The therapeutic and / or prophylactic agent according to (4) or (6), wherein the disease is selected from the group consisting of scleroderma, pulmonary fibrosis, COPD (chronic obstructive pulmonary disease), bronchial asthma, ARDS (acute respiratory distress syndrome), pulmonary hypertension, radiation pneumonitis, pulmonary sarcoidosis, alveolar hemorrhage associated with collagen disease, interstitial pneumonia, bronchiectasis, cystic fibrosis, or bronchopulmonary dysplasia. (8) The therapeutic and / or prophylactic agent according to any one of (1) to (7), wherein the cells are derived from a healthy subject. (9) The therapeutic and / or prophylactic agent according to any one of (1) to (8), wherein the extracellular vesicle particles are exosomes. (10) A pharmaceutical composition comprising the therapeutic and / or prophylactic agent according to any one of (1) to (9) as an active ingredient. (11) The pharmaceutical composition according to (10), which is formulated for inhalation, spray administration, injection, drip infusion, oral, transdermal, nasal, topical, vaginal, transmucosal, or rectal use. This specification incorporates the disclosure of Japanese Patent Application No. 2019-074263, which is the basis of the priority of this application.

Effects of the Invention

[0008] According to the present invention, a therapeutic and / or prophylactic agent for fibrotic diseases, inflammatory diseases, and / or aging diseases can be provided.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] 1. Therapeutic and / or prophylactic agent for diseases In one embodiment, the present invention relates to a therapeutic and / or prophylactic agent for at least one of fibrotic diseases, inflammatory diseases, and aging diseases, the agent comprising extracellular vesicle particles derived from cells of a tissue in which the disease may occur or a surrounding tissue thereof.

[0011] As used herein, the term "fibrotic disease" means a disease accompanied by excessive formation of fibrous connective tissue that may occur in the process of repair or healing of an organ or tissue. A fibrotic disease may be a disease caused by or accompanied by abnormal deposition of scar tissue, and may occur in various tissues. Examples of fibrotic diseases include, but are not limited to, pulmonary fibrosis, pulmonary hypertension, liver cirrhosis, cardiomyopathy, ischemic heart disease, valvular disease, endomyocardial fibrosis, myocardial fibrosis, arteriosclerosis, renal fibrosis, nephrosclerosis, glomerulosclerosis, scleroderma, retroperitoneal fibrosis, and uterine fibrosis.

[0012] Scleroderma is a disease mainly characterized by skin sclerosis. Scleroderma includes two types: localized scleroderma and systemic scleroderma. Localized scleroderma is a disease in which only the skin is affected. Systemic scleroderma is a disease in which lesions occur not only in the skin but also in various organs throughout the body.

[0013] As used herein, the term "inflammatory disease" refers to a disease that presents a persistent or transient inflammatory state and whose symptoms can be improved by reducing the inflammation. Examples of inflammatory diseases include, but are not limited to, COPD (chronic obstructive pulmonary disease), bronchial asthma, acute exacerbation of interstitial pneumonia, ARDS (acute respiratory distress syndrome), radiation pneumonitis, pulmonary sarcoidosis, bronchiectasis, cystic fibrosis, bronchopulmonary dysplasia, viral hepatitis, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hepatocellular carcinoma, Crohn's disease, ulcerative colitis, acute pancreatitis, chronic pancreatitis, autoimmune pancreatitis, Takayasu arteritis, Graves' disease, Hashimoto's disease, diabetes, nephritis, collagen diseases (e.g., rheumatoid arthritis, SLE (systemic lupus erythematosus), vasculitis, Sjögren's syndrome, arthritis, sarcoidosis), dermatomyositis, and psoriasis.

[0014] ARDS is a severe respiratory disorder that develops as a complication of a pre-existing disease. Pre-existing diseases that can trigger ARDS include bacterial pneumonia, interstitial pneumonia, acute exacerbation of interstitial pneumonia, sepsis, viral infections, multiple trauma, burns, acute pancreatitis, inhalation of toxic gases, drug poisoning, drowning, lung contusion, radiation lung injury, blood transfusion, etc. Viral infections that can trigger ARDS include, but are not limited to, viral infections caused by influenza virus and coronaviruses such as COVID-19.

[0015] As used herein, the term "aging disease" refers to a disease that occurs due to aging and is associated with a decline in the homeostatic maintenance function in tissues and individuals. A typical phenotypic manifestation of aging is cellular senescence, and the accumulation of these senescent cells and the various mediators they secrete are involved in the induction of chronic inflammation and further structural changes and functional disorders such as fibrosis. Examples of aging diseases include, but are not limited to, Alzheimer's disease, dementia, Parkinson's disease, spinocerebellar degeneration, and multiple system atrophy.

[0016] It should be noted that the above disease classifications are not mutually exclusive, and one disease may belong to multiple classifications. For example, primary sclerosing cholangitis and hepatocellular carcinoma can be considered as a type of inflammatory disease and at the same time a type of fibrotic disease.

[0017] The therapeutic and / or prophylactic agent of the present invention contains, consists of, or consists essentially of extracellular vesicle particles derived from cells of the tissue where the above-mentioned disease can occur or the surrounding tissue thereof.

[0018] As used herein, "extracellular vesicle (EV)" refers to vesicles released from cells. Extracellular vesicle particles are known to mediate cell-to-cell communication and are involved in various biological processes such as immune responses, blood coagulation, and induction of phagocytosis. Extracellular vesicle particles are mainly classified into exosomes, microvesicles, and apoptotic bodies according to their intracellular origin.

[0019] In one aspect, the extracellular vesicle particles constituting the therapeutic and / or prophylactic agent of the present invention are exosomes or are substantially exosomes.

[0020] As used herein, "exosome" refers to extracellular vesicles with a diameter of about 20 to 200 nm released from various cells. Exosomes are known to have various functions including cell-to-cell communication, antigen presentation, and transport of proteins and nucleic acids such as mRNA and miRNA.

[0021] The method for preparing extracellular vesicle particles or exosomes is not limited, and for example, it can be prepared using a conventionally known method. For example, the cells described in this specification can be cultured in a medium, and extracellular vesicle particles or exosomes can be recovered from the culture supernatant. The culture conditions (temperature, period, etc.) can be appropriately selected. For example, the culture temperature can be about 20°C to about 40°C, about 30°C to about 40°C, about 35°C to about 39°C, about 36°C to about 38°C, or about 37°C. The culture period can be, for example, 6 h to 7 days, 12 h to 4 days, 1 day to 3 days, or about 2 days. Culturing may be performed in the presence of CO 2 and in this case, CO 2The concentration may be about 2% to about 10%, about 4% to about 6%, or about 5%. Also, the medium used for culturing can be selected according to the cell type to be used. Examples of available media include commercially available media (e.g., BEGM, DMEM, MEM, BME, RPMI 1640, Advanced RPMI 1640, F-10, F-12, DMEM-F12, α-MEM, IMDM, McCoy's 5A medium, mTeSR1 medium, or a mixture thereof) or prepared media. Various additives (e.g., serum or serum substitute, non-essential amino acids, antibiotics such as penicillin and streptomycin) can also be added to the medium. Preferably, the medium does not contain extracellular vesicle particles or exosomes derived from other components such as serum.

[0022] The method for recovering extracellular vesicle particles or exosomes from the culture solution is not limited, and known methods can be used for recovery. For example, ultracentrifugation (e.g., Thery C., Curr. Protoc. Cell Biol. (2006) Chapter 3:Unit 3.22.), polymer precipitation method, immunoprecipitation method, FACS method, ultrafiltration method, gel filtration method, HPLC method, and a method of adsorbing to a carrier such as beads using an antibody or lectin can be mentioned. Also, extracellular vesicle particles or exosomes may be recovered using a commercially available kit for isolating extracellular vesicle particles or exosomes.

[0023] Among the above recovery methods, ultracentrifugation is a standard method most commonly used for the isolation of extracellular vesicle particles or exosomes. The centrifugal force in ultracentrifugation may be, for example, 50000×g or more, 100000×g or more, or 150000×g or more, and may also be 300000×g or less, 250000×g or less, or 200000×g or less. The centrifugation time is not limited, but can be, for example, 30 minutes to 120 minutes, 60 minutes to 90 minutes, or 70 minutes to 80 minutes. Also, before centrifugation, impurities may be removed or reduced by performing filter filtration and / or centrifugation at a lower centrifugal force as necessary.

[0024] The recovery of extracellular vesicle particles or exosomes, or the confirmation of the physical properties of extracellular vesicle particles or exosomes, can be carried out according to known methods. For example, it may be visually confirmed by an electron microscope, or the particle size and the number of particles of extracellular vesicle particles or exosomes may be measured using NTA (Nano Tracking Analysis) technology. Alternatively, the presence of extracellular vesicle particles or exosomes can also be confirmed by confirming the expression of proteins and / or genes that can serve as markers of extracellular vesicle particles or exosomes.

[0025] In the present specification, the cells from which extracellular vesicle particles or exosomes can be derived are differentiated cells. As used herein, the term "differentiated cell" refers to a cell differentiated from an undifferentiated cell such as a stem cell. The "differentiated cells" in the present specification include differentiated cells present in biological tissues such as blood, skin, muscle, nerve, fat, digestive organs, respiratory organs, urinary organs, genital organs, and endocrine organs. Examples include epithelial cells, endothelial cells, mesothelial cells, muscle cells, and nerve cells.

[0026] In the present specification, the cells may be any of primary cultured cells, subcultured cells, and frozen cells. Also, in the present specification, the cells may be those suffering from the above-mentioned disease or those not suffering from the disease. In one embodiment, the cells are derived from a healthy subject. As used herein, the term "healthy subject" refers to an individual in a healthy state. However, in the present specification, healthy cells are also included in the broad sense of healthy subjects. Therefore, not only at the individual level, but also at the cell level, if it is in a healthy state like normal tissues collected from a patient suffering from the above-mentioned disease or normal parts of tissues collected from a patient suffering from the above-mentioned disease, it shall be referred to as a healthy subject.

[0027] The biological species from which the cells are derived is not limited and may be, for example, the same biological species to which the therapeutic and / or prophylactic agent or composition of the present invention is administered, and is not limited, but mammals such as primates such as humans and chimpanzees, experimental animals such as rats and mice, domestic animals such as pigs, cows, horses, sheep, and goats, and pets such as dogs and cats, and for example, humans or mice, preferably humans.

[0028] As described above, extracellular vesicle particles or exosomes can be derived from cells in the tissue where the above-mentioned disease can occur or the surrounding tissue thereof. The tissue from which the cells donating the extracellular vesicle particles are derived and the tissue of the recipient cells may be the same, in a relationship of being adjacent to each other, or different. In one embodiment, the tissue from which the cells donating the extracellular vesicle particles are derived and the tissue of the recipient cells are the same. In another embodiment, the tissue from which the cells donating the extracellular vesicle particles are derived and the tissue of the recipient cells are different.

[0029] When the tissue from which the cells donating the extracellular vesicle particles are derived and the tissue of the recipient cells are the same or in a relationship of being adjacent to each other, non-limiting combinations of the above-mentioned disease and the cells in the tissue where the disease can occur or the surrounding tissue thereof include the following: For diseases that can occur in the lung or airway (for example, pulmonary fibrosis, pulmonary hypertension, COPD, bronchial asthma, ARDS, radiation pneumonitis, pulmonary sarcoidosis, alveolar hemorrhage derived from collagen diseases (for example, SLE (systemic lupus erythematosus) or vasculitis), interstitial pneumonia associated with collagen diseases (for example, SLE, vasculitis, scleroderma, Sjögren's syndrome, rheumatoid arthritis, sarcoidosis), bronchiectasis, cystic fibrosis, bronchopulmonary dysplasia), extracellular vesicle particles derived from airway epithelial cells (for example, tracheal epithelial cells or bronchial epithelial cells) or alveolar epithelial cells (for example, type I alveolar epithelial cells or type II alveolar epithelial cells) can be used; For diseases that can occur in the eye (for example, diabetic retinopathy), extracellular vesicle particles derived from epithelial cells of the eye can be used; For diseases that can occur in the otorhinolarynx (e.g., chronic rhinosinusitis, chronic otitis media, otitis media with effusion, nasal polyps, etc.), extracellular vesicle particles derived from otorhinolaryngeal epithelial cells can be used; For diseases that can occur in the kidney or glomerulus (e.g., nephritis, renal fibrosis, nephrosclerosis, glomerulosclerosis, diabetic nephropathy, and nephritis associated with SLE and scleroderma, etc.), extracellular vesicle particles derived from kidney epithelial cells can be used; For diseases that can occur in the liver, gallbladder, or bile duct (e.g., viral hepatitis, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, liver cirrhosis, hepatocellular carcinoma), extracellular vesicle particles derived from liver epithelial cells, bile duct epithelial cells, or gallbladder epithelial cells can be used; For diseases that can occur in the intestinal tract (e.g., Crohn's disease and ulcerative colitis), extracellular vesicle particles derived from intestinal epithelial cells (e.g., small intestine or large intestine epithelial cells) can be used; For diseases that can occur in the pancreas (e.g., acute pancreatitis, chronic pancreatitis, and autoimmune pancreatitis), extracellular vesicle particles derived from pancreatic epithelial cells can be used; For diseases that can occur in the heart (e.g., cardiomyopathy, ischemic heart disease, valvular disease, endomyocardial fibrosis, and myocardial fibrosis), extracellular vesicle particles derived from heart epithelial cells or cardiomyocytes can be used; For diseases that can occur in blood vessels (e.g., arteriosclerosis, Takayasu arteritis, and vascular lesions associated with diabetes), extracellular vesicle particles derived from vascular epithelium or endothelial cells can be used; For diseases that can occur in the thyroid (e.g., Graves' disease and Hashimoto's disease), extracellular vesicle particles derived from thyroid epithelial cells can be used; For diseases that can occur in the brain or nerves (e.g., Alzheimer's disease, dementia, Parkinson's disease, spinocerebellar degeneration, multiple system atrophy, and diabetic neuropathy), extracellular vesicle particles derived from brain epithelial cells, ependymal cells, or nerve cells can be used; For diseases that can occur in the abdominal cavity (e.g., retroperitoneal fibrosis), extracellular vesicle particles derived from peritoneal epithelial cells can be used; For diseases that can occur in the uterus and ovaries (e.g., uterine fibroids, ovarian tumors), extracellular vesicle particles derived from uterine epithelial cells can be used; For diseases that can occur in the skin (e.g., scleroderma, dermatomyositis), extracellular vesicle particles derived from skin epithelial cells can be used; For diseases that can occur in bones or joints (e.g., rheumatoid arthritis), extracellular vesicle particles derived from bone cells or chondrocytes can be used; Note that scleroderma can also occur in various organs, and dermatomyositis can also occur in muscles. In this case, extracellular vesicle particles derived from cells of each tissue can be used.

[0030] When the tissue from which the cells donating the extracellular vesicle particles are derived is different from the tissue of the recipient cells, the above diseases and the combination of the tissues or the cells of the surrounding tissues where the diseases can occur are not limited. For example, for the above diseases that can occur in the lungs or airways, extracellular vesicle particles derived from cells other than the lungs, such as extracellular vesicle particles derived from kidney cells (e.g., extracellular vesicle particles derived from kidney epithelial cells), etc., can be used; For the above diseases that can occur in the eyes, extracellular vesicle particles derived from cells other than the eyes can be used; For the above diseases that can occur in the otorhinolaryngology region, extracellular vesicle particles derived from cells other than the otorhinolaryngology region can be used; For the above diseases that can occur in the kidneys or glomeruli, extracellular vesicle particles derived from cells other than the kidneys or glomeruli, such as extracellular vesicle particles derived from lung cells (e.g., extracellular vesicle particles derived from airway epithelial cells, tracheal epithelial cells, bronchial epithelial cells, or alveolar epithelial cells), etc., can be used; For the above diseases that can occur in the liver, gallbladder, or bile duct, extracellular vesicle particles other than those from the liver can be used; For the above diseases that can occur in the intestinal tract, extracellular vesicle particles derived from cells other than the intestinal tract can be used; For the above diseases that can occur in the pancreas, extracellular vesicle particles derived from cells other than the pancreas can be used; For the above diseases that can occur in the heart, extracellular vesicle particles derived from cells other than the heart can be used; For the above diseases that can occur in blood vessels, extracellular vesicle particles derived from cells other than blood vessels can be used; For the above diseases that can occur in the thyroid gland, extracellular vesicle particles derived from cells other than the thyroid gland can be used; For the above-mentioned diseases that can occur in the brain or nerves, extracellular vesicle particles derived from cells other than those in the brain or nerves can be used; For the above-mentioned diseases that can occur in the abdominal cavity, extracellular vesicle particles derived from cells other than those in the abdominal cavity can be used; For the above-mentioned diseases that can occur in the uterus, extracellular vesicle particles derived from cells other than those in the uterus can be used; For the above-mentioned diseases that can occur in the ovary, extracellular vesicle particles derived from cells other than those in the ovary can be used; For the above-mentioned diseases that can occur in the skin, extracellular vesicle particles derived from cells other than those in the skin, such as extracellular vesicle particles derived from kidney cells (for example, extracellular vesicle particles derived from kidney epithelial cells) and extracellular vesicle particles derived from lung cells (for example, extracellular vesicle particles derived from airway epithelial cells, tracheal epithelial cells, bronchial epithelial cells, or alveolar epithelial cells), etc. can be used; For diseases that can occur in bone or joints, extracellular vesicle particles derived from cells other than those in bone or joints can be used; Note that scleroderma can occur in various organs, and dermatomyositis can also occur in muscles. In this case, extracellular vesicle particles derived from cells other than the tissues affected by scleroderma can be used.

[0031] In this specification, "treatment" includes complete or partial cure, alleviation, or prevention of progression of a disease or its accompanying symptoms in a patient already having the symptoms. In this specification, "prevention" includes suppression of onset or reduction of the incidence rate in a patient having a possibility of suffering from a disease.

[0032] 2. Composition In one aspect, the present invention relates to a composition for the treatment and / or prevention of at least one disease of fibrotic diseases, inflammatory diseases, and aging diseases, such as a pharmaceutical composition, a food composition, or a supplement, containing the extracellular vesicle particles or exosomes described herein, or a therapeutic and / or prophylactic agent. The composition of the present invention may consist essentially of the extracellular vesicle particles or exosomes described herein, or a therapeutic and / or prophylactic agent.

[0033] The amount of extracellular vesicle particles or exosomes contained in the composition of the present invention (e.g., a therapeutically and / or prophylactically effective amount) can be appropriately determined by those skilled in the art considering various factors such as the sex, body weight, age of the subject, and the course of diseases and symptoms. For example, the amount of extracellular vesicle particles or exosomes contained in the composition of the present invention is not limited, but for example, per 1 kg of body weight of the subject to whom the composition is administered, it may be, for example, about 0.0001 to 100.0 mg, about 0.001 to 10.0 mg, about 0.01 to 1.0 mg, or about 0.05 to 2.0 mg.

[0034] In addition to the above extracellular vesicle particles or exosomes, or therapeutic and / or prophylactic agents, the composition of the present invention may contain other components, carriers such as pharmaceutically acceptable carriers, for example, sterile water, physiological saline, buffers, excipients, binders, disintegrants, emulsifiers, surfactants, stabilizers, lubricants, diluents, fluidity promoters, flavoring agents, coloring agents, and fragrances.

[0035] The composition of the present invention can be formulated by conventional methods. For formulation, for example, the methods described in Remington’s Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.) can be referred to.

[0036] The administration form is not particularly limited and is appropriately selected as needed. Generally, it can be administered as oral preparations such as tablets, capsules, granules, fine granules, powders, liquids, syrups, suspensions, emulsions, and elixirs, or as parenteral preparations such as injections, drip infusions, suppositories, inhalants, transdermal absorbents, transmucosal absorbents, patches, and ointments.

[0037] The administration route of the composition of the present invention is not limited, but for example, it may be administered by inhalation, spray administration, injection, drip infusion, oral, transdermal, nasal, topical, vaginal, transmucosal, or rectal routes. Also, the composition of the present invention may be formulated for inhalation, spray administration, injection, drip infusion, oral, transdermal, nasal, topical, vaginal, transmucosal, or rectal use. The administration route can be appropriately selected according to the applicable disease. For example, in the case of lung diseases, it can be administered by inhalation or spray administration.

[0038] Subjects to which the therapeutic and / or prophylactic agent or composition of the present invention can be administered include, but are not limited to, mammals such as primates such as humans and chimpanzees, experimental animals such as rats and mice, domestic animals such as pigs, cows, horses, sheep, and goats, and pet animals such as dogs and cats, for example, humans or mice, preferably humans.

[0039] Those skilled in the art can appropriately determine the dosage, dosing interval, and dosing period of the extracellular vesicle particles or exosomes, therapeutic and / or prophylactic agent, or composition described herein in consideration of various factors such as the sex, body weight, age of the subject, and the course of diseases such as diseases and symptoms. For example, the dosage of the extracellular vesicle particles or exosomes, therapeutic and / or prophylactic agent, or composition may be, for example, about 0.0001 to 100.0 mg / 1 kg body weight, about 0.001 to 10.0 mg / 1 kg body weight, about 0.01 to 1.0 mg / 1 kg body weight, or about 0.05 to 2.0 mg / 1 kg body weight. Also, the number of administrations is not limited, but may be, for example, three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, etc. Also, the dosing period is not limited, but may be, for example, one day, two days, three days, one week, two weeks, one month, six months, one year, or longer.

[0040] 3. Other aspects In one aspect, the present invention relates to a method for treating and / or preventing at least one disease of a fibrotic disease, an inflammatory disease, and an aging disease, comprising administering to a subject the extracellular vesicle particles or exosomes, therapeutic and / or prophylactic agent, or composition described herein. The administration target, dosage, number of administrations, administration route, etc. in this aspect are as described in the above "2. Composition".

[0041] In one aspect, the present invention relates to the extracellular vesicle particles or exosomes, therapeutic and / or prophylactic agent, or composition described herein for use in the treatment and / or prevention of at least one disease of a fibrotic disease, an inflammatory disease, and an aging disease.

[0042] Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited by the examples.

Examples

[0043] <Materials and Methods> The test protocol was approved by the Institutional Review Board of Tokyo Women's Medical University. Also, written consent was obtained for all uses of the materials.

[0044] (Samples: Human primary airway epithelial cells and human primary lung fibroblasts) Human primary airway epithelial cells (HBEC) and human primary lung fibroblasts were both obtained by separating and culturing from lung resection tissues in Tokyo Women's Medical University Hospital. The method of separation and culture was as previously reported (Araya J. et al., 2007, J. Clin. Invest., 117, pp. 3551~3562). Also, the human airway epithelial cell line BEAS-2B was purchased from ATCC (Manassas, USA), the human small airway epithelial cell HSAEC was purchased from Lonza, the bone marrow-derived mesenchymal stem cell BM-MSC was purchased from RIKEN, the human fetal kidney cell 293 (HEK293) was purchased from ATCC, the monocytic leukemia cell line THP1 was purchased from ATCC, and the human skin fibroblast NHDF was purchased from Lonza.

[0045] (Cell culture) The cells were cultured at 37°C, 5% CO 2It was maintained in a medium containing 10% heat-inactivated fetal bovine serum (FBS) and Antibiotic-Antimycotic (Thermo Fisher Scientific). BEAS-2B was cultured in Advanced RPMI 1640 medium (Thermo Fisher Scientific), human primary airway epithelial cells were cultured in BEGM medium (Lonza), human primary lung fibroblasts were cultured in DMEM medium (Thermo Fisher Scientific), HSAEC were cultured in SAGM medium (Lonza), BM-MSC were cultured in MessenPRO medium (Thermo Fisher Scientific), HEK293 were cultured in RPMI1640 medium (Thermo Fisher Scientific), THP1 were cultured in RPMI1640 medium (Thermo Fisher Scientific), and NHDF were cultured in DMEM medium (Thermo Fisher Scientific).

[0046] (Exosome recovery method) The human airway epithelial cell line BEAS-2B, human primary airway epithelial cells HBEC, HSAEC, BM-MSC, HEK293, and THP1 were cultured for 2 days in Advanced RPMI 1640 medium (Thermo Fisher Scientific), BEGM medium (Lonza), SAGM medium (Lonza), STEMPRO medium (Thermo Fisher Scientific), Advanced DMEM medium (Thermo Fisher Scientific), and Advanced RPMI1640 medium (Thermo Fisher Scientific), respectively, and the culture supernatants were collected. The collected culture supernatants were filtered through a 0.22 μm filter (Millipore) and then exosomes were isolated and purified using ultracentrifugation (35,000 r.p.m., 70 minutes, 4°C, SW41Ti rotor, Beckman). Note that ultracentrifugation is a technique for precipitating and isolating exosomes by ultracentrifuging the sample, and it is the standard method most commonly used for exosome isolation. The obtained exosomes were measured for particle size and number using NTA (Nano Tracking Analysis) technology (NanoSight).

[0047] (Western blotting) Protein was recovered from human primary airway epithelial cells, human primary lung fibroblasts, and human skin fibroblasts supplemented with exosomes and / or transforming growth factor-β (TGF-β) 1 using Mammalian Protein Extract Reagent (M-PER, Thermo Fisher Scientific). Sample buffer (Wako, 198-13282 or 191-13272) was added to the obtained samples, and electrophoresis was performed using SDS-PAGE. Subsequently, membrane transfer (polyvinylidene difluoride membrane, Millipore) and blocking using Blocking One (Nacalai Tesque) were carried out. After the antigen-antibody reaction, luminescence was induced using ImmunoStar LD (Wako) for quantification. As primary antibodies, alpha smooth muscle actin (α-SMA, Sigma-Aldrich, A2547), goat anti-COL1 / type I collagen (Southern Biotech, 1310-01), mouse anti-FN1 / cellular fibronectin containing extra domain A (Abcam, ab6328), mouse anti-ACTB / b-actin (Sigma-Aldrich, A5316), rabbit anti-p21 Waf1 / Cip1 (Cell Signaling Technology, 2947), rabbit anti-β-Catenin (Cell Signaling Technology, 8480), rabbit anti-non-phospho(Active)β-Catenin (Cell Signaling Technology, 19807) were used. The intensity of the obtained bands was quantified using Image J.

[0048] (Senescence associated β-galactosidase staining) TGF-β1 and / or airway epithelial cell-derived exosomes were added to primary human airway epithelial cells, and the number of stained positive cells was counted using a Senescence associated β-galactosidase staining kit (Sigma, CS0030). Staining was performed according to the manufacturer's instructions.

[0049] (Cell component fractionation in WNT / b-catenin signaling analysis) Fractionation of nuclear and cytoplasmic proteins was performed using a cell component fractionation kit (Nuclear and Cytoplasmic Extraction Reagents, Thermo Fisher Scientific, 78833) according to the manufacturer's instructions.

[0050] (Animal experiments) Animal experiments were carried out in accordance with the guidelines of the Experimental Animal Research Institute of Tokyo Medical and Dental University. Lung fibrosis model mice were used, in which 2.5 U / kg bleomycin (Nippon Kayaku Co., 4234400D4032) was dissolved in 50 μl of physiological saline and administered intratracheally to 8-12-week-old C57BL / 6J mice. On days 7 and 14 after bleomycin administration, 3 μg (50 μl) of BEAS2B-derived exosomes were administered intratracheally. The mice were sacrificed on day 17 after administration and used for the following immunohistochemical staining.

[0051] (Immunohistochemical staining) Mouse lungs were evaluated by Masson trichrome staining and hematoxylin and eosin staining as previously reported (Kobayashi K. et al., J. Immunol., 2016, 197(2), pp. 504-16). In addition, the Ashcroft score was determined from the tissues stained with Masson trichrome using the Ashcroft method to evaluate fibrosis. For tissue staining against the cell senescence marker p16, an Anti-mouse CDKN2A / p16INK4a antibody (abcam, ab54210) was used as the primary antibody. For tissue staining against the cell senescence marker p21, a rabbit anti-p21Waf1 / Cip1 (Cell Signaling Technology, 2947) was used.

[0052] The measurement of hydroxyproline content was performed using a Hydroxyproline assay kit (Chondrex, 6017).

[0053] (Statistical analysis) All experiments were repeated at least three times, and the results were expressed as mean ± SEM. For analysis, Student's t-test was used for three comparisons, and ANOVA was used for comparisons of three or more. Prism version 7 (GraphPad Software, San Diego, CA) was used, and a p-value < 0.05 was considered a statistically significant difference.

[0054] (Comparison between HBEC-derived exosomes and bone marrow-derived MSC-derived exosomes) Primary lung fibroblasts were added with TGF-β1 at 2 ng / ml and / or exosomes at 10 μg / ml (HBEC-derived exosomes or bone marrow-derived mesenchymal stem cell (BM-MSC)-derived exosomes) and cultured for 24 hours. Then the medium was changed and cultured for another 48 hours. Subsequently, Western blotting was performed as described above. BM-MSC-derived exosomes were cultured from BM-MSCs in StemPro MSC SFM (Thermo Fisher Scientific), and were recovered from the culture supernatant using ultracentrifugation in the same manner as HBEC-derived exosomes as described above.

[0055] (ARDS model mice) Lipopolysaccharide (LPS), a component of Gram-negative bacteria and used in a sepsis model, and Poly I:C, a synthetic double-stranded RNA analog that mimics viral RNA, were directly sprayed into the tracheas of mice (C57BL / 6J, 8 weeks old, male) to prepare acute respiratory distress syndrome (ARDS) model mice. Specifically, 4 hours after spraying LPS (5 μg / mg), Poly I:C (5 μg / mg) was sprayed, and 20 hours later, the mice were sacrificed, and the number of neutrophils in BALF, HE staining, and measurement of lung inflammation score were performed. When using HBEC-derived exosomes, 2×10 9 particles were aerosolized via the respiratory tract (Figure 12A).

[0056] The number of neutrophils in BALF (bronchoalveolar lavage fluid) was measured by the following method. First, the total number of cells in BALF was counted using trypan blue. Next, BALF was centrifuged in an autosmear centrifuge (CF-120, 800 rpm, 5 minutes), and the obtained supernatant was stained with Diff-Quik to measure the ratio of the number of neutrophils to the total number of cells, thereby calculating the number of neutrophils in BALF.

[0057] The lung inflammation score was measured by the following method with reference to the method described in Sun Y. Q. et al., Stem Cells, 30(12); 2692-9 (2012). In the lung sections of one mouse, five regions around the bronchi were randomly selected. For each region, the infiltration of inflammatory cells around the bronchi and blood vessels was scored on a 4-point scale from 0 to 3. The average score of the five regions was taken as the lung inflammation score.

[0058] <Results> (Analysis of prepared exosomes) Figure 1 shows the analysis results of exosomes recovered by ultracentrifugation from the culture supernatants of human primary airway epithelial cells HBEC and human airway epithelial cell line BEAS-2B (A: HBEC, B: BEAS-2B). These results indicate that exosomes were successfully recovered by ultracentrifugation. Also, the average diameter of the recovered exosomes was around about 80 nm.

[0059] (Inhibition of TGF-β1-induced myofibroblast differentiation by exosomes) In the pathogenesis of airway remodeling such as pulmonary fibrosis, COPD, and bronchial asthma, differentiation into myofibroblasts plays an important role. Therefore, the effect of exosomes on lung fibroblasts was examined.

[0060] Primary lung fibroblasts were added with 2 ng / ml of TGF-β1 and / or 10 μg / ml of exosomes (HBEC-EV) for 24 hours, the medium was changed, and the cells were further cultured for 48 hours. Subsequently, the results of confirming the expression of α-SMA, type I collagen, fibronectin, which are markers of myofibroblasts, and actin, a housekeeping protein, by Western blotting are shown in Figure 2A. Also, the results of quantifying the expression levels of each protein by dividing by the expression level of actin are shown in Figures 2B to 2D. Since the increased expression of α-SMA, type I collagen, and fibronectin induced by TGF-β1 was decreased by the addition of exosomes, it was shown that exosomes derived from HBEC inhibit TGF-β1-induced myofibroblast differentiation.

[0061] (Inhibition of TGF-β1-induced lung epithelial cell senescence by exosomes) In recent years, the involvement of lung epithelial cell senescence has become widely known in the pathological conditions of pulmonary fibrosis and COPD. Therefore, the effect of exosomes on the senescence of lung epithelial cells was examined.

[0062] Airway epithelial cells (HBEC) were cultured for 48 hours after adding 10 μg / ml of exosomes derived from airway epithelial cells (HBEC) together with 2 ng / ml of TGF-β1. As a result, the increased expression of SA-β-Gal (Senescence associated β-galactosidase) (Figure 3A) and p21 (Figure 3B), which are senescence markers, induced by TGF-β1 was suppressed. When exosomes derived from human small airway epithelial cells (HSAEC) were used, the increased expression of p21 induced by TGF-β1 was also suppressed in the same manner as exosomes derived from HBEC (Figure 3C). The inhibitory effect of exosomes derived from HBEC on the increased expression of p21 was greater than that of exosomes derived from BM-MSC (Figure 3D).

[0063] Next, the mechanism of action of exosomes in suppressing cellular senescence was investigated. It has been known that the suppression of the WNT / β-catenin signal inhibits myofibroblast differentiation in pulmonary fibrosis.

[0064] Therefore, the effect of airway epithelial cell-derived exosomes on WNT / β-catenin was examined. As a result, since exosomes suppressed TGF-β1-induced WNT / β-catenin, it was suggested that exosomes suppress TGF-β1-induced myofibroblast differentiation through the suppression of WNT / β-catenin (Figure 4, A: before fractionation, B: nucleus, C: cytoplasm).

[0065] From the above results, it was shown that airway epithelial cell-derived exosomes may have a therapeutic and / or preventive effect on pulmonary fibrosis and the like by suppressing the differentiation of myofibroblasts and / or the senescence of epithelial cells.

[0066] (Comparison with existing therapeutic drugs) An efficacy comparison was made between pirfenidone and nintedanib, which are existing therapeutic drugs for idiopathic pulmonary fibrosis. The blood concentrations of pirfenidone, 10 μg / ml and 500 μg / ml which is 50 times that concentration, and the blood concentrations of nintedanib, 100 nM and 5000 nM which is 50 times that concentration, were used as comparison targets. Primary lung fibroblasts were stimulated with each component at the described concentrations for 24 hours, the medium was changed, and after further culturing for 48 hours, analysis was performed.

[0067] The results are shown in Figure 5. Exosomes derived from airway epithelial cell lines dose-dependently suppressed type I collagen, fibronectin (FN), and α-SMA, which are TGF-β1-induced myofibroblast differentiation markers, and the inhibitory effect was equal to or greater than the blood concentrations of IPF therapeutic drugs.

[0068] (Animal experiment) C57BL / 6J mice at 8 - 12 weeks of age were intratracheally administered 2.5 U / kg bleomycin to establish a pulmonary fibrosis model mouse. By intratracheally administering exosomes derived from airway epithelial cell lines to this model mouse, the pulmonary fibrosis score observed by Masson trichrome staining was reduced (Figures 6A and B). Furthermore, by intratracheally administering exosomes derived from airway epithelial cell lines, the amount of hydroxyproline in lung tissue, which is an indicator of collagen accumulation, was reduced (Figure 7A), and the number of positive cells for p16 and p21, which are cell senescence markers, decreased (Figures 7B and C). Therefore, it was shown that exosomes derived from airway epithelial cell lines suppress fibrosis and senescence in pulmonary fibrosis mice in vivo.

[0069] (Comparison with MSC-derived exosomes) Primary lung fibroblasts were cultured for 24 hours after adding TGF-β1 and / or exosomes (exosomes derived from human bronchial epithelial cells (HBEC) or exosomes derived from bone marrow mesenchymal stem cells (BM-MSC)), and then the medium was changed and the cells were cultured for an additional 48 hours. Subsequently, the results of confirming the expression of α-SMA, a marker of myofibroblasts, and actin, a housekeeping protein, by Western blotting are shown in Figure 8. As is clear from Figure 8, the increase in α-SMA expression induced by TGF-β1 was significantly reduced by the addition of HBEC-derived exosomes compared to BM-MSC-derived exosomes. This result indicates that exosomes derived from differentiated HBEC may have a higher ability to inhibit myofibroblast differentiation in the lung than undifferentiated BM-MSC.

[0070] (Effect of exosomes derived from cells other than airway epithelial cells) The effect of exosomes derived from cells other than airway epithelial cells (HBEC) on TGF-β1-induced myofibroblast differentiation was examined by adding the exosomes to primary lung fibroblasts. Specifically, the effects of exosomes derived from human bronchial epithelial cells (HBEC), human bronchial epithelial cell line (BEAS-2B), human embryonic kidney cells 293 (HEK293), monocytic leukemia cell line (THP1), human small airway epithelial cells (HSAEC), and bone marrow mesenchymal stem cells (BM-MSC) on TGF-β1-induced myofibroblast differentiation were examined.

[0071] Regarding the increase in α-SMA expression induced by TGF-β1, BEAS-2B-derived exosomes and HEK293-derived exosomes showed an inhibitory effect equivalent to that of HBEC-derived exosomes. The effects of these exosomes were all more significant than those of BM-MSC-derived exosomes (Figures 9A and B). On the other hand, no effect was observed with THP1-derived exosomes. Regarding the increase in α-SMA and type I collagen expression induced by TGF-β1, BEAS-2B-derived exosomes and HSAEC-derived exosomes showed an inhibitory effect equivalent to that of HBEC-derived exosomes (Figure 10).

[0072] These results indicated that exosomes derived from cells other than airway epithelial cells, such as kidney cells and alveolar epithelial cells, etc., could also have a high ability to inhibit myofibroblast differentiation in the lung.

[0073] (Inhibitory effect of exosomes on TGF-β1-induced myofibroblast differentiation of dermal fibroblasts) It was examined whether exosomes derived from HBEC had an effect of suppressing fibrosis on dermal fibroblasts.

[0074] To dermal fibroblasts (NHDF), TGF-β1 at 2 ng / ml and / or exosomes at 10 μg / ml (HBEC-EV) were added and cultured for 24 hours, then the medium was changed and cultured for another 48 hours. Subsequently, the results of confirming the expression of α-SMA, type I collagen, which are markers of myofibroblasts, and actin by Western blotting are shown in Fig. 11.

[0075] Since the increased expression of α-SMA and type I collagen induced by TGF-β1 in dermal fibroblasts (NHDF) was decreased by the addition of exosomes, it was shown that exosomes derived from HBEC also suppressed TGF-β1-induced myofibroblast differentiation in dermal fibroblasts. This result indicates the possibility that exosomes derived from the lung can be a therapeutic agent for scleroderma in tissues different from the lung.

[0076] (Effect of exosomes in ARDS model) It was examined whether exosomes derived from HBEC had an effect of suppressing the inflammatory response in a mouse model of acute respiratory distress syndrome (ARDS).

[0077] Four hours after spraying lipopolysaccharide (LPS) into the trachea, poly I:C and / or exosomes (HBEC-EV) were sprayed into the trachea to prepare a mouse model of acute respiratory distress syndrome (ARDS), and the mice were dissected 20 hours later (Fig. 12A), and the inflammatory response in the lung was examined.

[0078] The increase in the number of neutrophils induced by LPS and poly I:C was significantly suppressed by HBEC-derived exosomes (Figure 12B). In addition, in pathological images evaluated by HE staining, it was shown that HBEC-derived exosomes reduced inflammatory cell infiltration around the airways and blood vessels and suppressed lung injury (Figure 12C). Furthermore, the increase in the lung inflammation score induced by LPS and poly I:C was significantly decreased by HBEC-derived exosomes (Figure 12D). These results indicate the possibility that the exosomes of the present invention can be a therapeutic agent for inflammatory diseases including ARDS.

[0079] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A therapeutic and / or prophylactic agent for pulmonary fibrosis or ARDS (acute respiratory distress syndrome), which is an exosome derived from cells of a tissue where the disease may occur or its surrounding tissue, and contains exosomes derived from airway epithelial cells, small airway epithelial cells, or alveolar epithelial cells, or renal cells, and the cells are derived from a healthy subject, said therapeutic and / or prophylactic agent.

2. A pharmaceutical composition comprising the therapeutic and / or prophylactic agent according to Claim 1 as an active ingredient.

3. The pharmaceutical composition according to Claim 2, formulated for inhalation, aerosol administration, injection, drip infusion, oral, transdermal, nasal, topical, vaginal, transmucosal, or rectal administration.

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