WO2021016727A1 - 针对纤维化、炎症、和/或老化疾病的治疗药物 - Google Patents

针对纤维化、炎症、和/或老化疾病的治疗药物 Download PDF

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WO2021016727A1
WO2021016727A1 PCT/CN2019/000152 CN2019000152W WO2021016727A1 WO 2021016727 A1 WO2021016727 A1 WO 2021016727A1 CN 2019000152 W CN2019000152 W CN 2019000152W WO 2021016727 A1 WO2021016727 A1 WO 2021016727A1
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therapeutic
diseases
preventive agent
disease
cells
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French (fr)
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落谷孝広
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上海圣特佳健康科技发展有限公司
特爧妥有限公司
落谷孝広
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Priority to JP2022506095A priority Critical patent/JP2022542953A/ja
Priority to US17/630,810 priority patent/US20220273725A1/en
Priority to CN201980096325.5A priority patent/CN114269358A/zh
Priority to PCT/CN2019/000152 priority patent/WO2021016727A1/zh
Priority to EP19940120.9A priority patent/EP4006144A4/en
Publication of WO2021016727A1 publication Critical patent/WO2021016727A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the present invention relates to a therapeutic and preventive agent for at least one of fibrotic diseases, inflammatory diseases, and aging diseases, and a method of treatment or prevention using the therapeutic or preventive agent.
  • Tissue fibrosis refers to the excessive formation of fibrous connective tissue produced during the repair or healing of organs or tissues. Such tissue fibrosis can occur in various organs, and as fibrotic diseases, for example, pulmonary fibrosis and liver cirrhosis are known. Although the number of patients with fibrotic diseases has risen to hundreds of thousands in Japan, there is still no effective treatment.
  • Pulmonary fibrosis is a representative fibrotic disease, and it can also be an inflammatory disease or an aging disease.
  • pulmonary fibrosis for idiopathic pulmonary fibrosis (IPF) whose cause cannot be determined, the average life span after diagnosis is 3 to 5 years, and the prognosis is extremely poor.
  • the incidence of IPF in Japan is about 20 per 100,000 people.
  • anti-fibrotic drugs such as pirfenidone and nintedanib, steroids, immunosuppressants, etc. can be used (Non-Patent Documents 1 to 2), but the effects are limited.
  • COPD chronic obstructive pulmonary disease
  • alveolus which is a cluster of grape-shaped pouches located deep in the branches of the bronchi, is destroyed and is in a state of emphysema, the function of uptake of oxygen and discharge of carbon dioxide will be reduced. It is believed that COPD coexists with these changes, and it cannot be recovered even through treatment.
  • IPF idiopathic pulmonary fibrosis
  • IPF interstitial pneumonia
  • dyspnea For these respiratory organs, dyspnea, chronic cough, and phlegm are most typical symptoms during work, which are wheezing when walking, going up and down stairs, etc. Some patients also have symptoms such as asthma, such as wheezing and paroxysmal dyspnea.
  • COPD management The goals of COPD management are: (1) improvement of symptoms and quality of life; (2) improvement and maintenance of exercise capacity and physical activity; (3) prevention of disease deterioration; (4) inhibition of disease progression; (5) ) Prevention and treatment of systemic and pulmonary complications; (6) Improvement of life prognosis. If you continue to smoke, the deterioration of respiratory function will be accelerated. Therefore, smoking ban is the basis of treatment. In order to avoid deterioration, it is recommended to receive influenza vaccine and pneumococcal vaccine.
  • the core of drug therapy is bronchodilators (anticholinergic drugs/ ⁇ 2 receptor agonists/theophylline drugs).
  • inhaled drugs are recommended, mainly inhaled anticholinergic drugs that dilate the bronchus for a long time, and inhaled ⁇ 2 receptor agonists.
  • inhaled steroids can be used. It has been reported that a combination of long-acting ⁇ 2 receptor agonists and inhaled steroids is also useful.
  • the core of non-drug therapy is respiratory rehabilitation (mouth breathing, abdominal breathing and other breathing training/exercise therapy/nutrition therapy, etc.).
  • home oxygen therapy can be introduced.
  • a small-sized artificial respirator and a face mask are sometimes used to assist breathing.
  • a surgical operation lung volume reduction surgery to remove an over-inflated lung is also performed.
  • Non-Patent Document 1 Azuma A, et al., Am. J. Respir. Crit. Care Med., 2005, 171, 1040 ⁇ 1047.
  • Non-Patent Document 2 Richeldi L et al., N. Engl. J. Med., 2011, 22, 365(12), 1079 ⁇ 87.
  • the problem of the present invention is to provide a therapeutic and/or preventive agent for fibrotic diseases such as pulmonary fibrosis, inflammatory diseases, and/or aging diseases.
  • the inventors of the present invention conducted repeated studies on the pathology and treatment of fibrotic diseases such as pulmonary fibrosis, inflammatory diseases, and aging diseases. As a result, they were the first to demonstrate to the world that the outer skin derived from airway epithelial cells stimulated by smoking Exosome can become a cause of the pathology of respiratory diseases such as COPD and IPF. Subsequently, further experiments focusing on exosomes were carried out, and it was discovered that exosomes derived from mesenchymal stem cells (hereinafter referred to as "MSC" (Mesenchymal Stem Cell), also called mesenchymal stem cells) exist for the above diseases. The excellent therapeutic effect and/or preventive effect. In particular, the present inventors discovered the fact that they completed the present invention by inhaling the exosomes of human MSC through the airway, thereby being able to treat these respiratory diseases and even asthma.
  • MSC mesenchymal Stem Cell
  • the present invention relates to the following inventions.
  • a therapeutic and/or preventive agent which is a therapeutic or preventive agent for at least one of fibrotic diseases, inflammatory diseases, and aging diseases, which includes MSC-derived exosomes.
  • the aforementioned MSCs are cells isolated from bone marrow, adipose tissue, umbilical cord or peripheral blood.
  • the aforementioned MSCs are cells isolated from pluripotent stem cells (including iPS cells and ES cells).
  • the aforementioned MSC are human cells.
  • the disease site of the above diseases is the lung.
  • the above diseases are selected from the group consisting of pulmonary fibrosis, COPD, bronchial asthma, ARDS (acute respiratory distress syndrome), pulmonary hypertension, radiation pneumonitis, pulmonary sarcoidosis, alveolar hemorrhage accompanied by collagen disease, or interstitial pneumonia in.
  • a therapeutic and/or preventive agent for fibrotic diseases, inflammatory diseases, and/or aging diseases can be provided.
  • Figure 1 is a graph showing the experimental method and results of the myofibroblast differentiation inhibition test performed in vitro.
  • TGF ⁇ (2ng/mL), TGF ⁇ (2ng/mL) + MSC-derived exosomes (10 ⁇ g/mL), TGF ⁇ (2ng/mL) + pirfenidone (PFD) The expression level (mRNA) of ⁇ SMA in the case of (10 ⁇ g/mL) was quantified.
  • Fig. 2 is a dosing schedule showing the evaluation of the therapeutic effect using the animal model of Example 3.
  • FIG. 3 is a graph showing the results of the animal experiment in Example 3.
  • the vertical axis represents the lung fibrosis score achieved by Masson trichrome staining.
  • the present invention relates to a therapeutic and/or preventive agent, which is a therapeutic and/or preventive agent for at least one of fibrotic diseases, inflammatory diseases, and aging diseases, which comprises MSC-derived Exosomes.
  • fibrotic disease means a disease accompanied by excessive formation of fibrous connective tissue that can be produced during the repair or healing process of organs or tissues. Fibrotic disease can be caused by abnormal scar tissue Diseases caused by deposition, or diseases associated with these diseases, fibrotic diseases can develop in a variety of tissues.
  • fibrotic diseases include: pulmonary fibrosis, high pulmonary Blood pressure, liver cirrhosis, cardiomyopathy, ischemic heart disease, valvular disease, endocardial fibrosis, myocardial fibrosis, arteriosclerosis, renal fibrosis, nephrosclerosis, glomerulosclerosis, scleroderma , Retroperitoneal fibrosis and uterine fibrosis.
  • inflammatory disease means a disease that presents a persistent or prompt inflammatory state, and the symptoms can be improved by reducing the inflammation.
  • inflammatory diseases include: COPD (chronic obstructive pulmonary disease), bronchial asthma, ARDS (acute respiratory distress syndrome), radiation pneumonia, pulmonary sarcoidosis, viral hepatitis, autoimmune Hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hepatocellular carcinoma, Crohn’s disease, ulcerative colitis, acute pancreatitis, chronic pancreatitis, autoimmune pancreatitis, aortitis , Graves’ disease, Hashimoto’s disease, diabetes, nephritis, collagen disease (for example, rheumatoid arthritis, SLE (systemic lupus erythematosus), vasculitis, Sjogren’s syndrome, arthritis, nodules) Joint disease), dermatomyo
  • aging disease means a disease caused by aging that is accompanied by a decrease in the homeostasis maintenance function of a tissue or an individual.
  • the representative manifestation of aging is cellular aging.
  • the accumulation of aging cells and the various mediators secreted are related to the initiation of chronic inflammation, and in turn are related to structural changes and dysfunctions such as fibrosis.
  • examples of aging diseases include Alzheimer's disease, dementia, Parkinson's disease, spinocerebellar degeneration, and multiple system atrophy.
  • classifications of diseases are not mutually exclusive, and one disease can belong to multiple classifications.
  • primary sclerosing cholangitis and hepatocellular carcinoma can be an inflammatory disease as well as a fibrotic disease.
  • the therapeutic and/or preventive agent of the present invention includes MSC-derived exosomes, or consists of MSC-derived exosomes, or substantially consists of MSC-derived exosomes.
  • derived from mesenchymal stem cells (MSC) means exosomes derived from MSC.
  • exosomes refer to extracellular vesicles (Extracellular Vesicles) with a diameter of about 20 to 200 nm released from various cells. It is known that exosomes can have various functions such as intercellular communication, antigen presentation, protein, mRNA, and miRNA and other nucleic acid delivery.
  • exosomes can be recovered from the culture supernatant.
  • the culture conditions can be appropriately selected.
  • the culture temperature can be set at about 20°C to about 40°C, about 30°C to about 40°C, about 35°C to about 39°C, and about 36°C to about 38°C. Or about 37°C.
  • the culture period can be, for example, 6 hours to 7 days, 12 hours to 4 days, 1 day to 3 days, or about 2 days.
  • Cultivation can be carried out in the presence of CO 2 , in which case the CO 2 concentration can be about 2% to about 10%, about 4% to about 6%, or about 5%.
  • the medium used for culture can be selected according to the type of cells used. Examples of media that can be used include commercially available media (for example, BEGM, DMEM, MEM, BME, RPMI 1640, Advanced RPMI 1640, F-10, F-12, DMEM-F12, ⁇ -MEM, IMDM, MacCOy's 5A medium, mTeSR1 medium or their mixture) or prepared medium.
  • Various additives for example, serum or serum substitutes, non-essential amino acids, antibiotics such as penicillin and streptomycin
  • the medium preferably does not contain exosomes derived from other components such as serum.
  • any known method can be used. Examples include: ultracentrifugation (for example, Thery C., Curr. Protoc. Cell Biol. (2006) Chapter 3: Unit 3.22.), polymer precipitation, immunoprecipitation, FACS, ultrafiltration, coagulation Gel filtration method, HPLC method, and method of adsorbing on a carrier such as beads using antibodies and lectins.
  • a commercially available kit for exosome isolation can also be used to recover exosomes.
  • ultracentrifugation is the most commonly used standard method for the isolation of exosomes.
  • the centrifugal force of the ultracentrifugation method can be, for example, 50,000 xg or more, 100,000 xg or more, or 1,500,000 xg or more, and can also be 300,000 xg or less, 250,000 xg or less, or 200,000 xg or less.
  • the centrifugation time is not limited, for example, it can be 30 minutes to 120 minutes, 60 minutes to 90 minutes, or 70 minutes to 80 minutes.
  • it is also possible to remove or reduce impurities through filter filtration and/or centrifugal separation with lower centrifugal force as needed.
  • Recovered exosomes or confirmation of the physical properties of exosomes can be carried out in accordance with known methods, for example, visual confirmation by electron microscope, or NTA (Nano Tracking Analysis) technology to measure the particle size and number of exosomes .
  • NTA Nano Tracking Analysis
  • the presence of exosomes can also be confirmed by confirming the expression of proteins and/or genes that can be markers of exosomes.
  • MSC that can be a source of exosomes can be obtained, for example, by isolation from bone marrow, adipose tissue, umbilical cord blood, umbilical cord, bone marrow, placenta, or peripheral blood.
  • MSC has adhesiveness to plastics and is relatively easy to proliferate on petri dishes. Therefore, this property can be used for isolation. For example, by inoculating fresh bone marrow on a petri dish and culturing for several days, MSCs in the form of fibroblasts begin to appear at the bottom of the petri dish. After culturing for 5 to 20 days, MSCs are proliferated in a manner of filling the petri dish.
  • CD34(-), CD45(-), CD105(+), CD29(+) cells can be obtained by FACS or the like from the obtained cell aliquots.
  • MSC-specific expressions on Stro-1, CD271, SSEA-4, CD146, CD49f, CD349SSEA-3, SUSD2, Stro-4, PDGFR- ⁇ , Sca-1, and ecto-5′-nucleus One or more markers of mesenchymal cells such as nucleotidase (CD73) are isolated by FACS or the like.
  • MSC can also be obtained by induction of differentiation from cells with stronger differentiation ability, such as pluripotent stem cells (including iPS cells and ES cells).
  • pluripotent stem cells including iPS cells and ES cells.
  • MSCs are induced by culturing pluripotent stem cells in the presence of GSK3 ⁇ inhibitors, TGF ⁇ inhibitors, and insulin (PLoS ONE such as Fukuta M, etc. 9(12): e112291.).
  • the MSC can also be obtained as a commercially available product (Takara Bio Inc. Japan; COSMO BIO CO., Itd., Japan, etc.).
  • the cells may also be any of primary culture cells, subculture cells, and frozen cells.
  • the type of organism that becomes the source of the cells is not limited. For example, it may be the same species as the species to which the therapeutic or preventive agent of the present invention is administered.
  • mammals such as humans and primates such as chimpanzees can be cited.
  • Animals such as experimental animals such as rats and mice; domestic animals such as pigs, cattle, horses, sheep, and goats; and pets such as dogs and cats, such as humans or mice, preferably humans.
  • treatment includes the complete or partial cure, alleviation, or prevention of progression of the disease of a patient who has already had symptoms or the accompanying symptoms.
  • prevention includes the suppression of the onset or the reduction of the morbidity in patients who are likely to suffer from the disease.
  • the amount of exosomes contained in the therapeutic or preventive agent of the present invention for example, a therapeutic and/or preventive effective amount
  • those skilled in the art can consider the progress of the subject’s sex, weight, age, and disease, etc. Various important factors such as symptoms are appropriately determined.
  • the amount of exosomes contained in the composition of the present invention is not limited, it can be, for example, about 0.0001 to 100.0 mg or about 0.001 to 10 mg per kg of body weight of the subject to which the composition is administered. , About 0.01 to 1.0 mg or about 0.05 to 2.0 mg.
  • composition of the present invention may also contain other ingredients, pharmaceutically acceptable carriers and other carriers, such as sterile water, physiological saline, buffers, excipients, Binders, disintegrants, emulsifiers, surfactants, stabilizers, lubricants, diluents, fluidity promoters, flavoring agents, coloring agents, perfumes, etc.
  • pharmaceutically acceptable carriers and other carriers such as sterile water, physiological saline, buffers, excipients, Binders, disintegrants, emulsifiers, surfactants, stabilizers, lubricants, diluents, fluidity promoters, flavoring agents, coloring agents, perfumes, etc.
  • pharmaceutically acceptable carriers and other carriers such as sterile water, physiological saline, buffers, excipients, Binders, disintegrants, emulsifiers, surfactants, stabilizers, lubricants, diluents, fluidity promoters, flavoring agents, coloring agents,
  • the administration method is not particularly limited, and can be appropriately selected as required. Generally speaking, it can be used as tablets, capsules, granules, fine granules, powders, liquids, syrups, suspensions, emulsions, elixirs, etc. It is administered orally or as parenteral preparations such as injections, drips, suppositories, inhalants, transdermal absorbers, transmucosal absorbers, sprays, patches, and ointments.
  • the administration route of the composition of the present invention is not limited. For example, it may be administered by inhalation, spray administration, injection, drip infusion, oral, transdermal, nasal, topical, vaginal or rectal administration.
  • the composition of the present invention can also be formulated for inhalation, spray administration, intravenous injection, drip infusion, oral, transdermal, intratracheal, transpulmonary, transnasal, topical, transvaginal, or transrectal administration.
  • Medicinal composition can be appropriately selected according to the applicable disease. For example, in the case of lung disease, it can be administered by inhalation or spray.
  • test subject who can administer the therapeutic and/or preventive agent or composition of the present invention is not limited, but includes mammals such as humans and primates such as chimpanzees; experimental animals such as rats and mice ; Livestock animals such as pigs, cattle, horses, sheep, and goats; and pets such as dogs and cats, such as humans or mice, preferably humans.
  • exosomes, therapeutic and/or preventive agents, or compositions described in this specification can consider the sex, weight, and age of the test subject. And various important factors such as the progress of the disease and the symptoms, etc. are appropriately determined.
  • the amount of exosomes, therapeutic and/or preventive agents, or compositions administered can be, for example, about 0.0001-100.0 mg/1 kg body weight, about 0.001-10 mg/1 kg body weight, about 0.01-1.0 mg/1 kg body weight, or about 0.05 ⁇ 2.0mg/1kg body weight.
  • the number of administrations is not limited, it can be 3 times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, and once a month. Inferior.
  • the administration period is not limited, it can be one day, two days, three days, one week, two weeks, one month, six months, one year or more.
  • the present invention relates to a method for treating or preventing at least one disease selected from fibrotic diseases, inflammatory diseases, and aging diseases, which comprises administering to a subject the exosomes described in this specification, Or therapeutic agent or preventive agent.
  • the present invention relates to an exosome described in this specification, or a therapeutic or preventive agent, which is used for the treatment of at least one disease selected from fibrotic diseases, inflammatory diseases, and aging diseases Or prevention.
  • Human MSCs are cultured in STEMPRO MSC SFM medium (THERMOFISHER). At the time point when the required number of cells is reached, they are re-inoculated into a subconfluent state, and cultured in the same medium for 48 hours, from a total of 200ML culture The culture supernatant was recovered from the liquid. The collected culture supernatant was filtered with a 0.22 micron filter (MILIPORE), and after observing large particles and fine fragments, it was centrifuged (centrifuged at 100000xG for 1 hour, BECKMAN) to collect the secreted MSC Exosomes are purified. The obtained exosomes were used to measure the particle system and the number of particles using NANOSIGHT (NANOSIGHT) as a nanoparticle tracking system.
  • NANOSIGHT NANOSIGHT
  • TGF ⁇ was added to cultured human fibroblasts to experimentally induce differentiation of the cells into myofibroblasts.
  • Human primary lung fibroblasts were obtained from lung resection tissue in the affiliated Hospital of Tokyo Jikekai Medical University. The method of isolation culture is as reported (Araya J. et aL., 2007, J. Clin. Invest., 117, pp. 3551 to 3562).
  • the cells were maintained at 37° C., 5% CO 2 , in DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS) and Antibiotic-AntimyCOtic (Thermo Fisher Scientific).
  • FBS heat-inactivated fetal bovine serum
  • Antibiotic-AntimyCOtic Thermo Fisher Scientific
  • mice were carried out in accordance with the guidelines of the Laboratory Animal Research Institute of the National Cancer Research Center of Japan. Dissolve 2.U/kg of bleomycin (Nippon Kayaku CO., 4234400D4032) in 50 ⁇ L of normal saline and administer it through the airway to 8-12 weeks old C57BL/6J mice, make and use lung fibrosis Model mice. 10 ⁇ g of exosomes secreted from MSC were administered through the airway twice, once after 1 week (day 7) after bleomycin administration (day 0), and the other time after another 1 week ( Day 14). Six days after the second administration of exosomes (day 20), the mice were euthanized, and the lung fibrosis score was measured by triple staining. The exosomes non-administered group and the bleomycin and exosomal non-administered groups were used as controls, and 10 mice were used in each group. Fig. 2 shows the administration time.
  • mice were evaluated by triple staining, haematine and eosin staining (Kobayashi K. et aL., J. ImmunoL., (2016) 197(2) , Pp: 504-16).
  • Ashcroft's method was used to determine the Ashcroft score to evaluate fibrosis.

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Abstract

一种治疗剂和预防剂，其针对肺纤维化等纤维化疾病、炎症疾病、和/或老化疾病起到了治疗效果。着眼于外泌体而针对肺纤维化等纤维化疾病、炎症疾病、以及老化疾病的病理以及治疗方法反复进行了研究，其结果是，发现了源自间充质干细胞(MSC)的外泌体存在针对上述疾病的优异的治疗效果和/或预防效果。因此，涉及一种治疗剂或预防剂以及包含该治疗和/或预防剂的医药组合物，该治疗或预防剂为纤维化疾病、炎症疾病、以及老化疾病中的至少一种疾病的治疗剂或预防剂，其包含源自间充质干细胞的外泌体。

Description

针对纤维化、炎症、和/或老化疾病的治疗药物 技术领域
本发明涉及一种纤维化疾病、炎症疾病、以及老化疾病中的至少一种疾病的治疗剂和预防剂、以及使用了该治疗剂或预防剂的治疗方法或预防方法。
背景技术
组织的纤维化是指，在器官或组织的修复或治愈过程中产生的纤维结缔组织的过剩的形成。这样的组织的纤维化能够在各种脏器中产生，作为纤维化疾病，例如已知有肺纤维化以及肝硬化等。虽然纤维化疾病的患者在日本国内已升至数十万人，但是仍不存在有效的治疗法。
肺纤维化是一种代表性的纤维化疾病，也能是一种炎症疾病或老化疾病。在肺纤维化之中，针对无法确定原因的特发性肺纤维化(IPF)，诊断后的平均寿命为3～5年，预后极差。日本的IPF的发病频率在每10万人中约为20人。作为肺纤维化的治疗法，可使用吡非尼酮、尼达尼布之类的抗纤维化药、类固醇、免疫抑制剂等(非专利文献1～2)，但是效果有限。
慢性阻塞性肺疾病(COPD：chronic obstructive pulmonary disease)是指，过去被称为慢性支气管炎、肺气肿的疾病的总称。其为长期吸入/暴露于以烟草烟雾为主的有害物质而产生的肺部的炎症性疾病，是一种以吸烟习惯为背景的发病于中老年的生活习惯病。推测存在40岁以上的人口的8.6％、约530万人的COPD患者(NICE study)，认为大多数患者处于未诊断、未治疗的状态。其在所有疾病中，占据死亡原因的第9名，对于男性而言占据第7名，根据WHO的统计，是 被视为将在2020年提升至世界死亡原因的第3名的重要的疾病。
认为COPD的最大原因是吸烟，吸烟者的15～20％会发病COPD。因吸入烟草的烟雾而在肺中的支气管产生炎症，引发咳嗽、痰多，或者支气管变细，导致空气流减少。此外，若位于支气管分支的深处的葡萄串状的小袋即肺泡(Alveolus)被破坏而处于肺气肿的状态，则氧的摄取、排出二氧化碳的功能会降低。认为COPD并存有这些变化，即使通过治疗也无法康复。
已知IPF(特发性肺纤维化)也是一种原因不明的间质性肺炎(特发性间质性肺炎)，是在特发性间质性肺炎之中发病频率最高的疾病。IPF的男性发病率高于女性发病率，且IPF的发病率随着年龄增长而增加。已知IPF也会因间质的纤维化逐渐恶化而成为肺癌的原因。
对于这些呼吸器官而言，在步行时、上下楼梯等活动身体时感到气喘的劳动时呼吸困难、慢性咳嗽、痰多为典型症状。一部分患者也有并发喘鸣、阵发性呼吸困难等如哮喘这样的症状的情况。
针对COPD的管理的目标在于：(1)症状和生活品质的改善；(2)运动能力和身体活动性的提高和维持；(3)病情恶化的预防；(4)疾病进展的抑制；(5)全身并发症和肺部并发症的预防和治疗；(6)生命预后的改善。若持续吸烟，则呼吸功能的恶化会加速，因此，禁烟是治疗的基础。为了避免病情恶化，建议接种流感疫苗、肺炎球菌疫苗。药物疗法的核心为支气管扩张药(抗胆碱能药物/β2受体激动药/茶碱类药物)。从效果、副作用的方面考虑，推荐吸入药物，主要可使用长时间扩张支气管的吸入抗胆碱能药物、吸入β2受体激动药。在气流阻塞为重症且病情反复恶化情况下，可使用吸入类固醇药。已报告长效β2受体激动药与吸入用类固醇的组合药物也是有用的。非药物疗法的核心在于呼吸康复(口呼吸、腹式呼吸等呼吸训练/运动疗法/营养疗法等)。在低氧血症有所进展的情况下，可导入家庭氧疗法。进而，在呼吸衰竭有所进展的情况下，有时也进行使 用小型人工呼吸器和面罩来帮助呼吸的换气辅助疗法。根据病例，也进行将过度膨胀的肺切除的外科手术(肺减容术)。
但是，至今为止COPD、IPF的治疗极其困难，认为是难以完全治疗(治愈)的疾病。由此，各种治疗法以抑制疾病的进展作为目标。因此，渴望开发出一种针对今后持续增加的呼吸道疾病的新治疗方法。
现有技术文献
非专利文献
非专利文献1：Azuma A，et al.，Am.J.Respir.Crit.Care Med.，2005，171，1040～1047.
非专利文献2：Richeldi L et al.，N.Engl.J.Med.，2011，22，365(12)，1079～87.
发明内容
发明所要解决的问题
在一实施方式中，本发明的问题在于，提供一种肺纤维化等纤维化疾病、炎症疾病、和/或老化疾病的治疗和/或预防剂。
用于解决问题的方案
本发明人等针对肺纤维化等纤维化疾病、炎症疾病、以及老化疾病的病理以及治疗方法反复进行了研究，其结果是向世界率先展示了：源自受到吸烟刺激的气道上皮细胞的外泌体(Exosome)能够成为COPD、IPF等呼吸道疾病的病理的一个原因。随后，进一步进行了着眼于外泌体的实验，发现了源自间充质干细胞(以下称为“MSC”(Mesenchymal Stem Cell)，也可叫做间叶系干细胞)的外泌体存在针对上述疾病的优异的治疗效果和/或预防效果。特别是，本发明人等发现 了如下事实，从而完成了本发明，即，经由气道吸入人MSC的外泌体，由此，能够治疗这些呼吸道疾病，甚至能够治疗哮喘。
因此，本发明涉及以下的发明。
(1)一种治疗和/或预防剂，其为纤维化疾病、炎症疾病、以及老化疾病中的至少一种疾病的治疗剂或预防剂，其包含源自MSC的外泌体。
(2)根据(1)所述的治疗剂或预防剂，其中，
上述MSC为从骨髓、脂肪组织、脐带或外周血离析出的细胞。
(3)根据(1)所述的治疗剂或预防剂，其中，
上述MSC为从多能干细胞(包括iPS细胞、ES细胞)离析出的细胞。
(4)根据(1)～(3)中任一项所述的治疗剂或预防剂，其中，
上述MSC为人细胞。
(5)根据(1)～(4)中任一项所述的治疗剂或预防剂，其中，
上述疾病的疾病部位为肺。
(6)根据(5)所述的治疗剂或预防剂，其中，
上述疾病选自由肺纤维化、COPD、支气管哮喘、ARDS(急性呼吸窘迫综合征)、肺性高血压症、放射性肺炎、肺结节病、伴随胶原病的肺泡出血或间质性肺炎构成的组中。
(7)根据(1)～(6)中任一项所述的治疗剂或预防剂，其为吸入剂。
(8)根据(1)～(7)中任一项所述的治疗剂或预防剂，其用于进行气管内给药或经肺给药。
发明效果
根据本发明，能够提供一种纤维化疾病、炎症疾病、和/或老化疾病的治疗和/或预防剂。
附图说明
图1为表示在体外(in vitro)进行的肌成纤维细胞分化抑制试验的实验方法以及结果的图表。在表中，从左起依次表示对添加了TGFβ(2ng/mL)、TGFβ(2ng/mL)+源自MSC的外泌体(10μg/mL)、TGFβ(2ng/mL)+吡非尼酮(PFD)(10μg/mL)的情况下的αSMA的表达量(mRNA)进行定量的结果。
图2为表示使用了实施例3的动物模型的治疗效果的评价的给药时间表。
图3为表示实施例3的动物实验的结果的图表。从左向右表示：博莱霉素(Bleomycin)以及外泌体未给药组；博莱霉素给药以及外泌体未给药组；博莱霉素给药以及外泌体给药组。纵轴表示由三重(Masson trichrome)染色实现的肺纤维化评分。
具体实施方式
1.疾病的治疗和/或预防剂
在一实施方式中，本发明涉及一种治疗和/或预防剂，其为纤维化疾病、炎症疾病、以及老化疾病中的至少一种疾病的治疗和/或预防剂，其包含源自MSC的外泌体。
在本说明书中，“纤维化疾病””意指，伴有能够在器官或组织的修复或治愈过程等中产生的纤维结缔组织的过剩的形成的疾病。纤维化疾病能够为因瘢痕组织的异常沉积而引发的疾病，或者能够为伴有这些疾病的疾病，纤维化疾病能够在多种组织中发病。虽然不作限定，但是作为纤维化疾病的例子，可列举出：肺纤维化、肺性高血压症、肝硬化、心肌病、缺血性心脏病、瓣膜病、心内膜心肌纤维化症、心肌纤维化、动脉硬化、肾纤维症、肾硬化症、肾小球硬化症、硬皮症、腹膜后纤维化症以及子宫纤维化症。
在本说明书中，“炎症疾病”意指，呈现持续性或瞬发性的炎症状态，并且能够通过减轻该炎症来改善症状的疾病。虽然不作限定，但是作为炎症疾病的例子，可列举出：COPD(慢性阻塞性肺疾病)、支气管哮喘、ARDS(急性呼吸窘迫综合征)、放射性肺炎、肺结节病、病毒性肝炎、自身免疫性肝炎、原发性胆汁性胆管炎、原发性硬化性胆管炎、肝细胞癌、克罗恩病、溃疡性结肠炎、急性胰腺炎、慢性胰腺炎、自身免疫性胰腺炎、主动脉炎、格雷夫斯病(Graves’disease)、桥本氏病、糖尿病、肾炎、胶原病(例如，类风湿性关节炎、SLE(系统性红斑狼疮)、血管炎、干燥综合征、关节炎、结节病)、皮肌炎、以及牛皮癣。
在本说明书中，“老化疾病”意指，因年龄增长引发的、伴随组织或个体的内稳态维持功能的降低而产生的疾病。老化的代表性表现形式是细胞老化，该老化的细胞的累积、所分泌的各种介质与慢性炎症的引发相关，进而与纤维化等结构变化和功能障碍相关。虽然不作限定，但是作为老化疾病的例子，可列举出：阿尔茨海默病、痴呆、帕金森病、脊髓小脑变性症以及多系统萎缩症。
需要说明的是，上述疾病的分类并不相互排斥，一种疾病能够属于多种分类。例如，原发性硬化性胆管炎以及肝细胞癌能够是一种炎症疾病，同时也能是一种纤维化疾病。
本发明的治疗和/或预防剂包含源自MSC的外泌体，或者由源自MSC的外泌体构成，或者实质上由源自MSC的外泌体构成。此处，“源自间充质干细胞(MSC)”意指，由MSC得到的外泌体。
在本说明书中，“外泌体”是指，由各种细胞释放出的直径约20～200nm的细胞外囊泡(Extracellular Vesicles)。已知外泌体能够具有以细胞间通讯、抗原递呈、蛋白质，mRNA及miRNA等核酸的输送为首的各种功能。
外泌体的制备能够使用任意的公知方法来进行。例如，在培养基中培养MSC，能够从培养上清回收外泌体。培养条件(温度以及期间等)能够适当选择，例如，培养温度能够设为约20℃～约40℃、约30℃～约40℃、约35℃～约39℃、约36℃～约38℃或约37℃。培养期间例如可为6小时～7天、12小时～4天、1天～3天、或约2天。可以在CO 2的存在下进行培养，在此情况下，CO 2浓度可为约2％～约10％、约4％～约6％、或约5％。此外，用于培养的培养基能够根据所使用的细胞种类进行选择。作为能够使用的培养基，可列举出：市售的培养基(例如，BEGM、DMEM、MEM、BME、RPMI 1640、Advanced RPMI 1640、F-10、F-12、DMEM-F12、α-MEM、IMDM、MacCOy′s 5A培养基、mTeSR1培养基或它们的混合物)或制备出的培养基。也能够在培养基中添加各种添加物(例如，血清或血清代替物、非必需氨基酸、青霉素以及链霉素等抗生素)。培养基优选不含有源自血清等其他成分的外泌体。
从培养液中回收外泌体的方法能够使用任意的公知方法。例如可列举出：超速离心法(例如，Thery C.，Curr.Protoc.Cell Biol.(2006)Chapter 3：Unit 3.22.)、聚合物沉淀法、免疫沉淀法、FACS法、超滤法、凝胶过滤法、HPLC法、以及利用抗体、凝集素而吸附于珠体等载体的方法。此外，也可使用市售的外泌体离析用试剂盒来回收外泌体。
在上述回收方法之中，超速离心法为最普遍用作外泌体的离析的标准方法。超速离心法的离心力例如可为50000xg以上、100000xg以上、或1500000xg以上，此外，可为300000xg以下、250000xg以下、或200000xg以下。虽然离心时间没有限定，但是例如能够设为30分钟～120分钟、60分钟～90分钟、或70分钟～80分钟。此外，在离心分离前，也可以根据需要，通过过滤器过滤和/或更低的离心力的离心分离来去除或减少掺杂物。
回收外泌体或外泌体的物性的确认能够按照公知的方法进行，例如，可以通过电子显微镜进行视觉确认，或者使用NTA(Nano Tracking Analysis)技术来计测 外泌体的粒径和粒数。或者，也能够通过确认能够成为外泌体的标记物的蛋白质和/或基因的表达来确认外泌体的存在。
在本说明书中，作为能够成为外泌体的来源的MSC，例如能够通过从骨髓、脂肪组织、脐带血、脐带、骨髓、胎盘或外周血进行离析来得到。MSC对于塑料有粘接性，比较容易在培养皿上增殖，由此，能够利用该性质进行离析。例如，通过将新鲜骨髓接种于培养皿上，培养数天，在培养皿的底部开始出现如成纤维细胞这样的形态的MSC。通过培养5～20天，以布满培养皿的方式使MSC增殖。可以进一步通过FACS等从所得到的细胞等分获取CD34(-)、CD45(-)、CD105(+)、CD29(+)的细胞。或者，能够利用对于MSC特异的、表达于Stro-1、CD271、SSEA-4、CD146、CD49f、CD349SSEA-3、SUSD2、Stro-4、PDGFR-α、Sca-1、以及ecto-5′-核苷酸酶(nucleotidase)(CD73)等间充质细胞的一种以上标记物，通过FACS等进行离析。
或者，MSC也能够通过从分化能力更强的细胞，例如多能干细胞(包含iPS细胞、ES细胞)进行分化诱导来得到。例如报告有：通过在GSK3β抑制剂、TGFβ抑制剂、以及胰岛素的存在下对多能干细胞进行培养，来诱导MSC(Fukuta M等PLoS ONE 9(12)：e112291.)。或者，MSC也能够作为市售品进行获取(Takara Bio Inc.日本；COSMO BIO CO.，Itd.，日本等)。
在本说明书中，细胞也可为原代培养细胞、传代培养细胞以及冷冻细胞中的任一种。成为细胞的来源的生物种类没有限定，例如可为与给药了本发明的治疗剂或预防剂的物种相同的物种，虽然没有限定，但是可列举出哺乳动物，例如：人以及黑猩猩等灵长类；大鼠以及小鼠等实验动物；猪、牛、马、羊、以及山羊等家畜动物；以及狗以及猫等宠物，例如是人或小鼠，优选是人。
在本说明书中，“治疗”包括：已经具有症状的患者的疾病或其所伴有的症状的完全或部分治愈、减轻、或进展的防止。在本说明书中，“预防”包括：具有患 病可能性的患者的发病的抑制或发病率的降低。
针对本发明的治疗剂或预防剂所含的外泌体的量(例如治疗和/或预防有效量)，本领域技术人员能够通过考虑被试验对象的性别、体重、年龄以及疾病等的进展以及症状等各种重要因素来适当确定。例如，本发明的组合物所含的外泌体的量虽然不作限定，但是例如相对于给药了组合物的被试验对象的每1kg体重，例如可为约0.0001～100.0mg、约0.001～10mg、约0.01～1.0mg或约0.05～2.0mg。
本发明的组合物除了上述外泌体或治疗和/或预防剂以外，还可以含有其他成分、药学上可接受的载体等载体，例如：灭菌水、生理盐水、缓冲剂、赋形剂、粘合剂、崩解剂、乳化剂、表面活性剂、稳定化剂、润滑剂、稀释剂、流动性促进剂、调味剂、着色剂、以及香料等。本发明的组合物能够通过常规方法进行制剂化。制剂化例如能够参照Remington’s Pharmaceutical Sciences(Merck Publishing CO.，Easton，Pa.)所述的方法。
给药方式没有特别限制，可根据需要进行适当选择，一般而言，能够作为片剂、胶囊剂、颗粒剂、细粒剂、粉剂、液剂、糖浆剂、悬浮剂、乳剂、以及酏剂等经口剂进行给药，或者作为注射剂、滴注剂、栓剂、吸入剂、经皮吸收剂、经粘膜吸收剂、喷雾剂、贴附剂、以及软膏剂等非经口剂进行给药。
本发明的组合物的给药途径没有限定，例如也可以通过吸入、喷雾给药用、注射、点滴注入、经口、经皮、经鼻、经局部、经阴道或经直肠进行给药。此外，本发明的组合物也可以为制剂化为吸入、喷雾给药、静脉注射、点滴注入、经口、经皮、气管内、经肺、经鼻、经局部、经阴道、或经直肠给药用的组合物。给药途径能够根据适用疾病来进行适当选择，例如若为肺疾病，则能够采用吸入或喷雾给药。
作为能够给药本发明的治疗和/或预防剂或组合物的被试验对象，虽然没有限定，但是可列举出哺乳动物，例如：人以及黑猩猩等灵长类；大鼠以及小鼠等实验动物；猪、牛、马、羊、以及山羊等家畜动物；以及狗以及猫等宠物，例如是人或小鼠，优选是人。
针对本说明书中记载的外泌体、治疗和/或预防剂、或组合物的给药量、给药间隔、以及给药期间，本领域技术人员能够通过考虑被试验对象的性别、体重、年龄以及疾病等的进展以及症状等各种重要因素来适当确定。例如，外泌体、治疗和/或预防剂、或组合物的给药量例如可为约0.0001～100.0mg/1kg体重、约0.001～10mg/1kg体重、约0.01～1.0mg/1kg体重或约0.05～2.0mg/1kg体重。此外，给药次数虽然不作限定，但是可为1天3次、1天2次、1天1次、2天1次、3天1次、1周1次、2周1次、1月1次等。此外，给药期间虽然不作限定，但是可为1天、2天、3天、1周、2周、1月、半年、一年或其以上。
在一方式中，本发明涉及一种选自纤维化疾病、炎症疾病、以及老化疾病中的至少一种疾病的治疗方法或预防方法，其包括向对象给药本说明书中记载的外泌体、或者治疗剂或预防剂。在另一方式中，本发明涉及一种本说明书中记载的外泌体、或者治疗剂或预防剂，其用于选自纤维化疾病、炎症疾病、以及老化疾病中的至少一种疾病的治疗或预防。
实施例
以下，使用实施例对本发明进行详细说明，但本发明的范围不限于此。需要说明的是，本申请说明书通篇中引用的文献通过参照而将整篇文献援用于本申请说明书中。
(统计学解析)
以下的实验均至少重复3次，将其结果作为平均±SEM进行表示。针对解析， 在3次的比较中使用t检验(Student′s t test)，在3次以上的比较中使用ANOVA。使用Prism version 7(GraphPad Software，San Diego，CA)，将p值＜0.05设为统计学显著性差异。
(实施例1)从间充质干细胞回收外泌体
人MSC通过STEMPRO MSC SFM培养基(THERMOFISHER社)进行培养，在达成需要的细胞数量的时间点，重新接种为亚融合(Subconfluent)状态，在同样的培养基中培养48小时，从合计200ML的培养液回收培养上清。用0.22微米的过滤器(MILIPORE社)将回收到的培养上清过滤，在观察到大粒子、细微破碎物后，通过离心法(以100000xG进行1小时离心，BECKMAN社)回收从MSC分泌出的外泌体，进行纯化。所得到的外泌体通过作为纳米粒子跟踪系统的NANOSIGHT(NANOSIGHT社)来计测粒子体系、粒数。
(实施例2)在体外(in vitro)进行的肌成纤维细胞分化抑制试验
作为在患者的肺部产生的COPD、IPF的体外(in vitro)模型，向所培养的人成纤维细胞添加TGFβ，实验性地诱导细胞向肌成纤维细胞的分化。在东京慈惠会医科大学附属医院内由肺切除组织进行分离培养而得到人原代肺成纤维细胞。分离培养的方法如已报告的那样(Araya J.et aL.，2007，J.CLin.Invest.，117，pp.3551～3562)。细胞以37℃、5％CO 2、在包含10％的热灭活胎牛血清(FBS)以及抗菌-抗真菌剂(Antibiotic-AntimyCOtic)(Thermo Fisher Scientific)的DMEM培养基中进行维持。
在从成纤维细胞的培养开始起24小时后，添加(A)TGFβ(2ng/mL)、(B)TGFβ(2ng/mL)+源自MSC的外泌体(10μg/mL)、(C)TGFβ(2ng/mL)+吡非尼酮(PFD)(10μg/mL)，在48小时后对αSMA的表达量(mRNA)进行定量。
其结果是，通过添加源自MSC的外泌体(10μg/mL)，αSMA的表达量(mRNA) 减少，纤维化的程度降低至极其优异(图1)。其效果比作为IPF的一般药物的吡非尼酮更好。
(实施例3)使用了动物模型的治疗效果的评价
动物实验按照日本国立癌症研究中心研究所的实验动物研究机构的指导原则进行实施。将2.U/kg的博莱霉素(Nippon Kayaku CO.，4234400D4032)溶解于50μL的生理盐水，经气道给药至8～12周龄的C57BL/6J小鼠，制作并使用肺纤维化模型小鼠。将从MSC分泌出的外泌体10μg经气道给药两次，一次在博莱霉素给药(第0天)的1周后(第7天)，另一次在再下1周后(第14天)。在第2次外泌体的给药的6天后(第20天)，对小鼠实施安乐死，通过三重染色来测定肺纤维化评分。以外泌体未给药组以及博莱霉素以及外泌体未给药组为对照，各组使用了10只小鼠。将给药时间示于图2。
如已报告的那样，对小鼠肺部以三重染色以及苏木精(haematine)以及伊红(eosin)染色进行评价(Kobayashi K.et aL.，J.ImmunoL.，(2016)197(2)，pp：504-16)。此外，从三重染色的组织，使用雅斯科方法(Ashcroft’s method)来决定雅斯科(Ashcroft)评分，对纤维化进行评价。
将结果示于图3。源自MSC的外泌体的给药优异地抑制了肺部的纤维化。

Claims (8)

  1. 一种治疗剂或预防剂，其为纤维化疾病、炎症疾病、以及老化疾病中的至少一种疾病的治疗剂或预防剂，其包含源自间充质干细胞的外泌体。
  2. 根据权利要求1所述的治疗剂或预防剂，其中，
    所述间叶系干细胞为从骨髓、脂肪组织、脐带或外周血离析出的细胞。
  3. 根据权利要求1所述的治疗剂或预防剂，其中，
    所述间充质干细胞为从包括iPS细胞、ES细胞的多能干细胞离析出的细胞。
  4. 根据权利要求1～权利要求3中任一项所述的治疗剂或预防剂，其中，
    所述间充质干细胞为人细胞。
  5. 根据权利要求1～权利要求4中任一项所述的治疗剂或预防剂，其中，
    所述疾病的疾病部位为肺。
  6. 根据权利要求5所述的治疗剂或预防剂，其中，
    所述疾病选自由肺纤维化、COPD、支气管哮喘、ARDS急性呼吸窘迫综合征、肺性高血压症、放射性肺炎、肺结节病、伴随胶原病的肺泡出血或间质性肺炎构成的组中。
  7. 根据权利要求1～权利要求6中任一项所述的治疗剂或预防剂，其为吸入剂。
  8. 根据权利要求1～权利要求7中任一项所述的治疗剂或预防剂，其用于进行气管内给药或经肺给药。
PCT/CN2019/000152 2019-07-31 2019-07-31 针对纤维化、炎症、和/或老化疾病的治疗药物 WO2021016727A1 (zh)

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