WO2022222987A1 - 含有干细胞胞外囊泡的药物组合物及其在呼吸道炎症治疗中的应用 - Google Patents

含有干细胞胞外囊泡的药物组合物及其在呼吸道炎症治疗中的应用 Download PDF

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WO2022222987A1
WO2022222987A1 PCT/CN2022/088118 CN2022088118W WO2022222987A1 WO 2022222987 A1 WO2022222987 A1 WO 2022222987A1 CN 2022088118 W CN2022088118 W CN 2022088118W WO 2022222987 A1 WO2022222987 A1 WO 2022222987A1
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stem cell
pharmaceutical composition
cells
extracellular vesicles
stem cells
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French (fr)
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戴成祥
雷继刚
李萍
陈应炉
李苏克
宋晓乐
沈美萍
盛一凡
任程洁
王静
李蒙
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西比曼生物科技（上海）有限公司
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    • A—HUMAN NECESSITIES
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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
    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K35/48—Reproductive organs
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Definitions

  • the invention relates to the field of stem cell therapy drugs, in particular to a pharmaceutical composition of extracellular vesicles secreted by stem cells and its application in the treatment of acute and chronic airway inflammation.
  • Lung inflammation is the body's natural response to injury. It helps the body remove pathogens, irritants and damaged cells and initiates the tissue repair process. Acute and chronic lung inflammation is common in different respiratory diseases such as asthma, cystic fibrosis, acute respiratory distress syndrome and chronic obstructive pulmonary disease (COPD). Among them, asthma and COPD are common chronic diseases in the world, affecting more than 300 million people worldwide and causing a large number of deaths each year. The morbidity and mortality of asthma and COPD are still increasing worldwide.
  • Asthma is a heterogeneous chronic airway inflammatory disease characterized by reversible airway obstruction. Its pathological features are airway hyperresponsiveness, the bronchus and its surrounding tissues are infiltrated by a large number of inflammatory cells, and the airway is remodeled and excessively narrowed.
  • COPD is a chronic airway inflammatory disease with incomplete reversible airflow limitation. Without proper treatment and control, both diseases can lead to changes in the patient's airways that can lead to irreversible alveolar damage and permanent pulmonary fibrosis, ultimately leading to loss of lung function and reduced responsiveness to therapeutic drugs.
  • the existing humanized antibody drug Omal izumab is only suitable for the treatment of severe asthma with eosinophil infiltration as the main feature, but it is not suitable for the treatment of asthma with neutrophil infiltration as the main feature.
  • the therapeutic application of this antibody drug in some children with asthma is still limited.
  • the drugs for the treatment of COPD are mainly some hormone drugs and bronchodilators. However, patients who use these drugs for a long time will also experience many side effects, such as tachycardia and hypertension.
  • the purpose of the present invention is to provide a preparation method of a pharmaceutical composition with key active substances in stem cell extracellular vesicles as main active ingredients and its application in the treatment of acute and chronic respiratory tract inflammation.
  • the first aspect of the present invention provides a preparation of stem cell extracellular vesicles, wherein the diameter of the extracellular vesicles is 30-200 nm, preferably 50-150 nm, more preferably 80-120 nm; and, the Extracellular vesicles contain bioactive factors related to anti-inflammatory or tissue damage repair, including matrix protein metalloenzyme 1 (MMP-1) and hepatocyte growth factor (HGF), where MMP-1 is present in
  • MMP-1 matrix protein metalloenzyme 1
  • HGF hepatocyte growth factor
  • the amount of MMP-1 in the formulation can be about 50 to about 5000 pg/ml, about 100 to about 4000 pg/ml, about 100 to about 3000 pg/ml, about 100 to about 2000 pg/ml, about 200 to about 3000 pg/ml, about 200 to about 4000 pg/ml, about 200 to about 2000 pg/ml, about 300 to about 2000 pg/ml, about 400 to about 5000 pg/ml, about 500 to about 5000 pg/ml/ml, about 600 to about 5000 pg/ml, about 700 to about 5000 pg/ml, about 800 to about 5000 pg/ml, about 900 to about 5000 pg/ml, about 1000 to about 5000 pg/ml, about 1000 to about 3000 pg/ml, about 1000 to about 2000 pg/ml, about 500 to about 2000 pg/ml, about 500 to about 3000 pg/
  • the amount of HGF in the formulation can be about 50 to about 8000 pg/ml, about 100 to about 7000 pg/ml, about 200 to about 6000 pg/ml, about 300 to about 8000 pg/ml, about 400 to about 8000 pg/ml, about 500 to About 8000 pg/ml, about 600 to about 8000 pg/ml, about 700 to about 8000 pg/ml, about 800 to about 8000 pg/ml, about 900 to about 8000 pg/ml, about 1000 to about 8000 pg/ml, about 1000 to about 7000 pg /ml, about 1000 to about 6000 pg/ml, about 500 to about 7000 pg/ml, about 500 to about 6000 pg/ml, about 2000 to about 8000 pg/ml, about 2000 to about 7000 pg/ml, about 2000 to about 6000 pg/ml or From about 5000 to about 6000
  • EVs extracellular vesicles
  • exosomes lipid bilayers with diameters ranging from 30–2000 nm.
  • microvesicles lipid bilayers with diameters ranging from 30–2000 nm.
  • Large extracellular vesicles can range in diameter from about 5 ⁇ m to about 12 ⁇ m.
  • the diameter of apoptotic bodies can range from about 1 ⁇ m to about 5 ⁇ m.
  • Microvesicles can range in diameter from about 100 nm to about 1 ⁇ m.
  • Exosomes can have diameters ranging from about 30 nm to about 150 nm, about 30 nm to about 100 nm, about 50 nm to about 150 nm, about 50 nm to about 100 nm, or about 50 nm to about 200 nm.
  • the stem cell extracellular vesicle preparation contains 1 ⁇ 10 8 to 5 ⁇ 10 8 stem cell extracellular vesicles/ml.
  • the stem cell extracellular vesicles are derived from the supernatant collected during the in vitro culture of human stem cells.
  • the stem cells are selected from the group consisting of human umbilical cord blood-derived stem cells, human peripheral blood-derived stem cells, human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, human adipose-derived mesenchymal stem cells, Human bone marrow-derived stem cells, or a combination thereof.
  • the stem cells are human adipose-derived mesenchymal stem cells.
  • the formulations of the invention are derived from stem or progenitor cells.
  • the methods of the invention culture stem or progenitor cells.
  • Stem cells are undifferentiated cells with the ability to self-renew and produce differentiated progeny (see Morrison et al. (1997) Cell 88:287-298). In mammals, there are two main types of stem cells: embryonic stem cells and adult stem cells, which are found in a variety of tissues.
  • the stem cells can be bone marrow-derived stem cells (BMSCs), adipose-derived stem cells (ADSCs), neural stem cells (NSCs), blood stem cells or hematopoietic stem cells.
  • BMSCs bone marrow-derived stem cells
  • ADSCs adipose-derived stem cells
  • NSCs neural stem cells
  • Stem cells can also be derived from cord blood. Stem cells can be generated by somatic cell nuclear transfer or dedifferentiation.
  • Stem cells include, but are not limited to, blood stem cells, adipose stem cells, bone marrow mesenchymal stem cells, mesenchymal stem cells, neural stem cells (NSC), skin stem cells, endothelial stem cells, liver stem cells, pancreatic stem cells, intestinal epithelial stem cells, or germinal stem cells.
  • mesenchymal stem cells are isolated from mesodermal organs, such as bone marrow, umbilical cord blood, and adipose tissue.
  • the stem cells are induced pluripotent stem cells (iPS cells or iPSCs).
  • iPSC refers to a class of pluripotent stem cells artificially generated from non-pluripotent cells, usually adult somatic or terminally differentiated cells, such as fibroblasts, hematopoietic cells, muscle cells, neurons, epidermal cells Wait.
  • the methods of the invention culture proliferating cells. In certain embodiments, the methods of the invention culture T cells (including primary T cells).
  • Cells may include autologous cells and/or biocompatible allogeneic cells or syngeneic cells harvested from the subject being treated, such as autologous cells, allogeneic cells or syngeneic cells Genetic cells (eg, mesenchymal stem cells), progenitor cells (eg, connective tissue progenitor cells) or multipotent adult progenitor cells) and/or other further differentiated cells.
  • autologous cells eg., allogeneic cells or syngeneic cells
  • Genetic cells eg, mesenchymal stem cells
  • progenitor cells eg, connective tissue progenitor cells
  • multipotent adult progenitor cells eg, multipotent adult progenitor cells
  • stem cells with differentiation potential can be used in the method for producing induced exosomes, and the stem cells with differentiation potential include but are not limited to embryonic stem cells, induced high-efficiency stem cells, cancer stem cells and tissue stem cells.
  • Tissue stem cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, breast stem cells, neural stem cells, intestinal stem cells, skin stem cells, umbilical cord blood stem cells, limbal stem cells, hair follicle stem cells, adipose tissue-derived stem cells, bone marrow stem cells, corneal stem cells, and ovary stem cell.
  • the stem cells used to generate exosomes can be selected from embryonic stem cells, induced pluripotent stem cells, cancer stem cells, mesenchymal stem cells, hematopoietic stem cells, breast stem cells, neural stem cells, intestinal stem cells, skin stem cells, umbilical cord blood stem cells, and limbal stem cells , hair follicle stem cells, adipose tissue-derived stem cells, bone marrow stem cells, corneal stem cells and ovarian stem cells.
  • the bioactive factor related to anti-inflammatory or tissue damage repair is selected from the group consisting of MMP-1, HGF, vascular endothelial growth factor (VEGF), or a combination thereof.
  • MMP-1 MMP-1
  • HGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • the bioactive factor related to anti-inflammatory or tissue damage repair is the bioactive factor produced by the human stem cells.
  • the biologically active factors related to anti-inflammatory or tissue damage repair include biologically active factors produced by paracrine of stem cells cultured in natural state, biologically active factors produced by expression of stem cells modified with exogenous genes factor, or a combination thereof.
  • the stem cell extracellular vesicle has a complete lipid bilayer membrane structure, and contains the following proteins: CD9, CD63, CD81, TSG101 and HSP70; and does not contain or substantially does not contain the protein CANX.
  • the surface of the membrane structure of the stem cell extracellular vesicle includes CD9, CD63, and CD81, and the interior of the membrane structure includes TSG101 and HSP70.
  • the membrane structure of the stem cell extracellular vesicle contains DNA and RNA with certain non-coding functions.
  • the RNA includes miRNA, tRNA, rRNA, snoRNA and snRNA.
  • the length of the RNA is 17-100 nucleotides.
  • the DNA and RNA with non-coding functions include DNA and RNA molecules with non-coding functions produced by stem cells cultured in a natural state, and DNA and RNA with non-coding functions produced by the expression of genetically engineered stem cells Molecules, non-coding functional DNA and RNA molecules, or combinations thereof, that are chemically synthesized and transduced into stem cells.
  • the RNA is selected from the following group: hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-126-5p, hsa-miR-151a-3p, hsa-miR -1246, hsa-miR-21-5p, hsa-miR-146a-5p, hsa-let-7b-3p, has-miR-1290, has-miR-23a-3p, has-let-7d-3p or its combination.
  • the cells used for producing the stem cell extracellular vesicles include cells from the following sources:
  • the cells used for producing the stem cell extracellular vesicles include primary cells and passaged cells with 1-10 passages.
  • the cells for producing the stem cell extracellular vesicles include cells that have not been genetically manipulated or cells that have been genetically manipulated.
  • the gene manipulation includes gene editing, gene introduction, gene knockdown (knock-down), gene knockout (knock-out), or a combination thereof.
  • the stem cell extracellular vesicles have the following characteristics: the extracellular vesicles are prepared by polyethylene glycol (PEG) precipitation, and the particle size of the extracellular vesicles is concentrated between 80-120 nm, and the particle size is smaller and uniform.
  • PEG polyethylene glycol
  • the polyethylene glycol (PEG) precipitation includes the steps of: using PEG of 3000-9000 PEG, using PBS to prepare a 8%-20% PEG stock solution, and sterilizing by filtration (for example, using 0.22 ⁇ m PEG). filter), added to the treated conditioned medium in a certain proportion (such as about 1:1 volume ratio) (the conditioned medium is processed by differential centrifugation and filtration to treat the culture supernatant of human-derived adipose-derived mesenchymal stem cells).
  • the second aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) the stem cell extracellular vesicle preparation described in the first aspect of the present invention, and (b) a pharmaceutically acceptable carrier .
  • the pharmaceutical composition is a cell-free and cell-debris-free pharmaceutical composition.
  • the "cell-free" means that the pharmaceutical composition does not contain live cells and dead cells.
  • the pharmaceutical composition comprises a bioactive factor related to anti-inflammatory or tissue damage repair, and the bioactive factor includes MMP1, HGF, VEGF or a combination thereof.
  • the pharmaceutically acceptable carrier is selected from the group consisting of sodium chloride, sodium phosphate, polyethylene glycol, chitosan, sodium hyaluronate, trehalose, heparin, or a combination thereof.
  • the dosage form of the pharmaceutical composition is selected from the following group: liquid dosage form, solid dosage form (eg, freeze-dried dosage form).
  • the dosage form of the pharmaceutical composition is selected from the group consisting of: aerosol inhalation, eye drops, nasal drops, and injection.
  • the pharmaceutical composition is an aerosol inhaler.
  • the pharmaceutical composition has the following characteristics: its main active ingredients are easily taken up by bronchiolar epithelial cells, alveolar epithelial cells, and inflammatory cells in the airways, thereby significantly inhibiting airway inflammation. It can be used to prevent and treat airway inflammatory diseases and injuries caused by various reasons.
  • the pharmaceutical composition is used to treat acute airway inflammation and/or chronic airway inflammation.
  • the pharmaceutical composition is used for the treatment of chronic airway inflammation, preferably for the treatment of infiltration of inflammatory cells such as eosinophils, neutrophils, monocytes and macrophages in the respiratory mucosa
  • chronic airway inflammation is the main feature, and it is better used for the treatment of bronchial asthma and chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • a third aspect of the present invention provides a method for preparing a pharmaceutical composition containing stem cell extracellular vesicles, the method comprising the steps of:
  • S1 culturing human stem cells (such as adipose-derived mesenchymal stem (progenitor) cells) to a predetermined degree of confluence (such as 75-90%);
  • human stem cells such as adipose-derived mesenchymal stem (progenitor) cells
  • the content of MMP-1 derived from extracellular vesicles is 1500-2000 pg/ml, and the content of HGF is 5500-6000 pg/ml.
  • the method further includes:
  • the method further comprises preparing the pharmaceutical composition into an aerosol inhalation preparation, an injection preparation, or a freeze-dried preparation.
  • the fourth aspect of the present invention provides a use of the stem cell extracellular vesicle preparation described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention for preparing a medicine or preparation for treating respiratory tract inflammation .
  • the airway inflammation includes acute airway inflammation and chronic airway inflammation.
  • the chronic airway inflammation is mainly characterized by the infiltration of inflammatory cells such as eosinophils, neutrophils, and monocyte-macrophages in the respiratory mucosa.
  • the chronic airway inflammation is bronchial asthma, chronic obstructive pulmonary disease (COPD), or a combination thereof.
  • COPD chronic obstructive pulmonary disease
  • the acute respiratory tract inflammation includes viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, or a combination thereof.
  • the acute respiratory tract inflammation includes a disease selected from the group consisting of acute respiratory distress syndrome (ARDS), acute lung injury (ALI), novel coronavirus (SARS-CoV-2) infectious pneumonia, or a combination thereof.
  • ARDS acute respiratory distress syndrome
  • ALI acute lung injury
  • SARS-CoV-2 novel coronavirus
  • a fifth aspect of the present invention provides a method for treating airway inflammation, comprising the step of: administering the pharmaceutical composition of the second aspect of the present invention to a subject in need.
  • the airway inflammation includes acute airway inflammation and chronic airway inflammation.
  • the chronic airway inflammation is mainly characterized by the infiltration of inflammatory cells such as eosinophils, neutrophils, and monocyte-macrophages in the respiratory mucosa.
  • the chronic airway inflammation is bronchial asthma, chronic obstructive pulmonary disease (COPD), or a combination thereof.
  • COPD chronic obstructive pulmonary disease
  • the acute respiratory tract inflammation includes viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, or a combination thereof.
  • the acute respiratory tract inflammation includes a disease selected from the group consisting of acute respiratory distress syndrome (ARDS), acute lung injury (ALI), novel coronavirus (SARS-CoV-2) infectious pneumonia, or a combination thereof.
  • ARDS acute respiratory distress syndrome
  • ALI acute lung injury
  • SARS-CoV-2 novel coronavirus
  • the required object is a human being.
  • the subject in need suffers from acute airway inflammation and/or chronic airway inflammation.
  • Figure 1 shows the identification of the biological characteristics of the main active ingredients in the pharmaceutical composition preparation of human adipose-derived mesenchymal stem cell extracellular vesicles.
  • Figure 2 shows the in vitro cellular uptake of the main active ingredients in a pharmaceutical composition formulation of human adipose-derived mesenchymal stem cell extracellular vesicles.
  • Figure 3 shows the biodistribution of DiR-labeled human adipose-derived mesenchymal progenitor exosomes in mice.
  • A the distribution in mice after 1h, 4h, 24h, 48h and 72h of exosome aerosol administration
  • B in vitro tissue of mice after 48h and 72h of exosome nebulization administration distribution of exosomes.
  • Figure 4 shows the treatment of PA-induced pneumonia in mice by nebulization of exosomes from human adipose-derived mesenchymal progenitor cells.
  • A morphological observation of mouse lung tissue HE staining;
  • B mouse lung tissue injury score;
  • C and
  • D changes in the number of total leukocytes and neutrophils in mouse bronchoalveolar lavage fluid;
  • E ) and
  • F the content of pro-inflammatory cytokines TNF- ⁇ and IL-6 in the bronchoalveolar lavage fluid of mice;
  • G the content of the anti-inflammatory cytokine IL-10 in the bronchoalveolar lavage fluid of mice.
  • Figure 5 shows the changes of chest CT in severe COVID-19 patients before and after exosome aerosol treatment of human-derived adipose-derived mesenchymal progenitor cells.
  • a and B Chest CT images of patients A and B before and after treatment showing reduced white patchy opacities in their lungs after treatment.
  • Figure 6 shows the top 20 types of miRNAs and their relative expression levels in exosomes from human adipose-derived mesenchymal progenitor cells produced in three batches.
  • Figure 7 shows a schematic diagram of the preparation of a mouse model of asthma characterized by mixed granulocytes.
  • Figure 8 shows a schematic diagram of the treatment of asthmatic mice with mixed granulocytic characteristics by a pharmaceutical composition formulation of human adipose-derived mesenchymal stem cell extracellular vesicles.
  • FIG. 9 shows a schematic diagram of a pharmaceutical composition preparation of human adipose-derived mesenchymal stem cell extracellular vesicles for treating COPD mice.
  • the medicinal preparation contains stem cell extracellular vesicles derived from human stem cells and has a specific particle size. , and contains bioactive factors related to anti-inflammatory or tissue damage repair.
  • the stem cell vesicles of the present invention are prepared by the polyethylene glycol (PEG) precipitation method. The particle size of the extracellular vesicles is concentrated between 80 and 120 nm, and the particle size is smaller and uniform.
  • the present invention has extremely excellent dispersibility and is especially suitable for direct administration to the respiratory tract of patients by means such as atomization inhalation; and the main active components in the pharmaceutical composition or preparation of stem cell extracellular vesicles of the present invention are easily absorbed by the bronchioles It is taken up by epithelium, alveolar epithelial cells, and inflammatory cells in the airway, so as to rapidly and efficiently treat or relieve chronic airway inflammation, especially diseases such as asthma and COPD. On this basis, the present invention has been completed.
  • Asthma generally refers to bronchial asthma (bronchial asthma), which is a chronic airway inflammation involving a variety of cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components.
  • bronchial asthma a chronic airway inflammation involving a variety of cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components.
  • bronchial asthma a chronic airway inflammation involving a variety of cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components.
  • a heterogeneous disease characterized by chronic inflammation associated with airway hyperresponsiveness, often with widespread and variable reversible expiratory airflow limitation leading to recurrent episodes of wheezing, shortness of breath, chest tightness, and/or coughing and other
  • COPD Chronic Obstructive Pulmonary Disease
  • COPD chronic obstructive pulmonary disease
  • EVs stem cell extracellular vesicles
  • exosomes lipid bilayers with diameters ranging from 30–2000 nm.
  • microvesicles lipid bilayers with diameters ranging from 30–2000 nm.
  • exosomes refers to a subclass of endosome-derived EVs with a diameter of 50-100 nm, which are the main source of paracrine secretions from a variety of cell types, including mesenchymal stem cells (MSCs). component.
  • MSCs mesenchymal stem cells
  • MSCs exosomes are a class of MSCs-derived EVs with a diameter in the range of 50-100 nm and a complete lipid bilayer membrane structure.
  • Exosomes are carriers of abundant cargoes, and their functions are mainly exerted by the continuous transport of microRNAs (miRNAs) and proteins.
  • miRNAs microRNAs
  • MSCs-derived exosomes more than 150 miRNAs and more than 850 unique proteins have been identified that alter various activities of target cells through different pathways.
  • MSC exosomes are involved in body development, epigenetic regulation, immune regulation (miR-155 and miR-146), tumorigenesis and tumor progression (miR-23b, miR-451, miR-223, miR-24, miR- 125b, miR-31, miR-214 and miR-122) and other physiological and pathological processes.
  • ExoCarta more than 900 proteins have been collected from MSCs exosomes.
  • MSC exosomes are loaded with some cytokines and growth factors, such as TGF ⁇ 1, interleukin-6 (IL-6), IL-10, and hepatocyte growth factor (HGF), etc. Helps in immune regulation.
  • cytokines and growth factors such as TGF ⁇ 1, interleukin-6 (IL-6), IL-10, and hepatocyte growth factor (HGF), etc.
  • VEGF Vascular endothelial growth factor
  • EMMPRIN extracellular matrix metalloproteinase inducer
  • MMP-9 have been reported in MSC exosomes, and these three proteins play an important role in stimulating angiogenesis, May be the basis for the tissue repair effect of exosomes.
  • the stem cell extracellular vesicle of the present invention and the preparation method of the pharmaceutical composition containing the same
  • the present invention provides a stem cell-derived extracellular vesicle, the diameter of the extracellular vesicle is 30-200 nm, preferably 50-150 nm, more preferably 80-120 nm; and, the extracellular vesicle is It contains bioactive factors related to anti-inflammatory or tissue damage repair, the active factors include matrix protein metalloenzyme 1 (MMP-1) and hepatocyte growth factor (HGF), wherein the content of MMP-1 is 1500-2000pg/ ml, the content of HGF is 5500-6000pg/ml.
  • MMP-1 matrix protein metalloenzyme 1
  • HGF hepatocyte growth factor
  • the described stem cells include but are not limited to human umbilical cord blood-derived stem cells, human peripheral blood-derived stem cells, human umbilical cord mesenchymal stem cells, human placental mesenchymal stem cells, human adipose-derived mesenchymal stem cells, human bone marrow-derived stem cells stem cell.
  • the stem cells are human adipose-derived mesenchymal stem cells.
  • GMP-grade human adipose-derived mesenchymal stem cells are mainly used as parent cells for producing stem cell extracellular vesicles, but they can also be stem cells derived from umbilical cord blood, umbilical cord, placenta, bone marrow and other tissues.
  • the preparation of the pharmaceutical composition of stem cell extracellular vesicles in the present invention comprises the following steps:
  • Polymers include but are not limited to polyethylene glycols with different molecular weights (PEG3000-9000) and their compositions in different ratios (1:1-1:6);
  • the stem cell extracellular vesicles obtained above can be diluted to an appropriate effective concentration with 3-10 ml of a pharmaceutically acceptable carrier or excipient.
  • the preparation of the pharmaceutical composition of stem cell extracellular vesicles is preferably allogeneic human adipose-derived mesenchymal stem cell extracellular vesicles.
  • the pharmaceutical composition or preparation of stem cell extracellular vesicles includes pharmaceutically acceptable carriers or excipients, including but not limited to sodium chloride, sodium phosphate, polyethylene glycol, chitosan, glass sodium, trehalose, heparin, etc., as well as any combination of the above substances, etc.
  • compositions or preparations of human adipose-derived mesenchymal stem cell extracellular vesicles are used for in vitro cellular uptake studies, including type II alveolar epithelial cells A549 and activated macrophages RAW247.6.
  • a HyperFlask cell factory can produce stem cell extracellular vesicles in 500ml of conditioned medium, and it is estimated that 5 ⁇ 10 10 —10 ⁇ 10 10 stem cell extracellular vesicles will be isolated in total.
  • one production operation unit in the laboratory can isolate stem cell extracellular vesicles with a total of 800-1200ml of conditioned medium from two cell factories. It is estimated that 2-5 ⁇ 10 11 stem cell extracellular vesicles can be isolated. The usage volume of 9 , one production operation unit can meet the usage volume of 100-250 patients.
  • the present invention also provides the application of the pharmaceutical composition or preparation with the key active substances in the stem cell extracellular vesicles as the main effective components for treating respiratory inflammatory diseases.
  • the pharmaceutical composition of the present invention contains (a) the stem cell extracellular vesicle preparation described in the first aspect of the present invention, and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a cell-free and cell-debris-free pharmaceutical composition.
  • the active ingredient further includes bioactive factors related to anti-inflammatory or tissue damage repair, and the bioactive factors include but are not limited to MMP1, HGF, or VEGF. These bioactive factors are mainly present in stem cell extracellular vesicles. In addition, some bioactive factors may exist outside the stem cell extracellular vesicles.
  • a particularly preferred pharmaceutical formulation is a nebulized inhalation formulation.
  • a mouse asthma model characterized by mixed granulocytes is used to verify the application of the pharmaceutical composition of the present invention in the treatment of airway inflammatory diseases. .
  • the pharmaceutical composition (1 ⁇ 10 7 -1 ⁇ 10 8 particles) of human adipose-derived mesenchymal stem cell extracellular vesicles labeled with lipophilic fluorescent dye PKH67 was used to study its distribution in the lungs of asthma model mice through respiratory inhalation It is found that the main active ingredient of the pharmaceutical composition preparation is easily taken up by the bronchiolar epithelium, the alveolar epithelial cells, and the inflammatory cells in the airway. In addition, after random grouping of mice (Table 1), the mouse asthma model characterized by mixed granulocytes was used to compare the treatment of dexamethasone, stem cells and the main active components of the preparation at different doses.
  • Table 1 The drug composition of human adipose-derived mesenchymal stem cell extracellular vesicles treats mixed granulocytic characteristics of asthmatic mice grouping
  • a nicotine (1.5-5 mg)-induced COPD mouse model is first established.
  • the mice in the control group were treated with normal saline aerosol inhalation through the airway, while the mice in the experimental group were treated with the pharmaceutical composition of human adipose-derived mesenchymal stem cell extracellular vesicles (1 ⁇ 10 5 ⁇ 1 ⁇ 10 8 particles/mouse)
  • the lung function indicators of the mice were tested and it was found that their lung function was improved.
  • the stem cell extracellular vesicles of the present invention are prepared by a polyethylene glycol (PEG) precipitation method, and the diameters of the extracellular vesicles are concentrated between 80-120 nm, and the particle diameters are smaller and uniform.
  • PEG polyethylene glycol
  • the stem cell extracellular vesicles of the present invention contain bioactive factors related to anti-inflammatory or tissue damage repair, and the content is higher than that of extracellular vesicles prepared by traditional centrifugation methods.
  • the main active ingredients in the pharmaceutical composition or preparation of stem cell extracellular vesicles of the present invention are easily taken up by bronchiolar epithelial cells, alveolar epithelial cells, and inflammatory cells in the airways.
  • the pharmaceutical composition or preparation of stem cell extracellular vesicles of the present invention can more effectively improve or treat respiratory inflammatory diseases.
  • human adipose-derived mesenchymal stem cells are used as parent cells for obtaining extracellular vesicles, and other related test materials and instruments mainly include:
  • ELISA detection kits for IL-6, IL-10, IL-13 and IL-17A centrifuge tubes, 12-well cell culture plates, centrifuges, paraffin embedding machines, paraffin microtome, frozen microtome, and plain Field microscope (40 ⁇ objective), upright fluorescence microscope (40 ⁇ objective), inverted fluorescence microscope (20 ⁇ objective), cell incubator, nebulizer, non-invasive spirometer, blood cell counter, IVIS imaging system, particle size Analyzer, Illumina sequencing platform.
  • human adipose-derived mesenchymal stem cells with GMP grade are selected as parent cells for producing stem cell extracellular vesicles, but they can also be stem cells derived from umbilical cord blood, umbilical cord, placenta, bone marrow and other tissues.
  • the main steps of preparing the stem cell extracellular vesicle preparation in the present invention are as follows:
  • the polymer precipitation method is used for separation, preferably PEG9000; 20% g/ml PEG9000 mother solution is prepared with PBS, filtered with a 0.22 ⁇ m filter, and added to the above in a volume ratio of 1:1.
  • One-step treated conditioned medium incubated at 4°C overnight, centrifuged at 4000g for 50min at 4°C, discarded the supernatant and retained the precipitate;
  • the stem cell extracellular vesicle preparation obtained above is diluted to an appropriate effective concentration with a medical carrier solution, thereby obtaining a stem cell extracellular vesicle pharmaceutical composition.
  • a control group was set to prepare stem cell extracellular vesicle preparations, and the preparation method was as follows:
  • the method of the present invention can obtain more uniformly dispersed and transparent stem cell extracellular vesicle preparations, and the preparations are rich in bioactive factors related to anti-inflammatory and tissue damage repair. , such as MMP-1, HGF (Table 2).
  • the content of MMP-1 (with anti-fibrotic effect) was at least 200 times higher than that of the control group, and the content of HGF (with certain anti-inflammatory effect) was about 2500 times higher.
  • VEGF and other factors also increased correspondingly. This shows that the stem cell extracellular vesicle preparation prepared by the present invention has excellent anti-inflammatory and anti-fibrotic properties.
  • the stem cell extracellular vesicle pharmaceutical composition (500 mL) prepared by the present invention contains 1.7 ⁇ 10 11 particles of extracellular vesicles.
  • the diameter of extracellular vesicles is 30-200 nm, preferably 50-150 nm, more preferably 80-120 nm, which is more favorable for the therapeutic effect after administration.
  • the active components of the pharmaceutical composition of human adipose-derived mesenchymal stem cell extracellular vesicles were first labeled with lipophilic fluorescent dye PKH67, and added to the type II In alveolar epithelial cells A549 and LPS-stimulated RAW264.7 macrophages, after culturing for 24h and 48h, respectively, the uptake of the active ingredients in the composition by the cells was observed under an inverted fluorescence microscope.
  • the main steps of the specific implementation are as follows:
  • Type II alveolar epithelial cells A549 and RAW264.7 macrophages were seeded in a 12-well culture plate at a density of 1 ⁇ 10 4 cells/cm 2 and 5 ⁇ 10 4 cells/cm 2 , respectively, in a cell culture incubator. (37°C, 5%CO 2 ) to culture the cells for 24h respectively;
  • RAW264.7 macrophages were stimulated with 10ng/ml LPS, and then the labeled pharmaceutical composition was added, cultured for 48 hours, and the culture dish was taken out to observe the uptake of the active ingredients in the pharmaceutical composition by cells under an inverted fluorescence microscope. .
  • the distribution of DiR-labeled extracellular vesicles in mouse lung tissue was observed by in vivo fluorescence imaging technology, and the distribution of DiR-labeled extracellular vesicles in mouse lung tissue was analyzed. residence time.
  • FIG. 3A DiR-labeled human adipose-derived mesenchymal stem cell extracellular vesicles were inhaled into the trachea of mice by aerosol needle at different time points, and their fluorescence signals were observed from different angles in mice. distribution in the body. It was found (Fig. 3A) that, in addition to the fluorescent signal of the reflux esophagus caused by tracheal administration (Fig. 3A abdomen), the fluorescent signal in this mouse was mainly concentrated in the mouse lung tissue (Fig. 3A back, right and left side).
  • the fluorescence signal in the left lung tissue of the mice was the highest, while the right lung was weaker, indicating that a large number of extracellular vesicles accumulated in the left lung lobe at this time; subsequently, the fluorescence signal in the left lung lobe was The signal decreased sharply, while the right lobe was gradually attenuated, indicating that extracellular vesicles were gradually depleted and reduced in the lung tissue. Since in vivo observation cannot accurately observe the fluorescence signal in the tissue, various types of tissue were selected for in vitro fluorescence imaging observation.
  • Each group consisted of 6 8-10-week-old C57BL/6 wild-type male mice, and the extracellular vesicle dosage of each mouse in the treatment group was calculated as 1 ⁇ 10 6 stem cell extracellular vesicles/g. 4 hours after the induction of multidrug-resistant aeruginosa, extracellular vesicles were inhaled by nebulization, once a day for 5 days. Afterwards, bronchoalveolar lavage fluid was collected for neutrophil count and determination of related inflammatory factor levels. In addition, the morphological observation of the lung tissue by HE staining and the assessment of lung injury were performed.
  • the results of total leukocyte and neutrophil counts in the bronchoalveolar lavage fluid of mice revealed that compared with the control group, the bronchoalveolar lavage fluid of mice treated with nebulization of human adipose-derived mesenchymal stem cell extracellular vesicles
  • the numbers of total leukocytes and neutrophils were significantly reduced in BALF (Fig. 4C,D); the levels of pro-inflammatory cytokines TNF- ⁇ and IL-6 in the bronchoalveolar lavage fluid were also significantly reduced (Fig. 4E,F), In contrast, the level of the anti-inflammatory cytokine IL-10 was significantly increased (Fig. 4G).
  • the daily SOFA score, the detection of relevant biochemical indicators and the observation of lung imaging, as well as the negative time of the new coronavirus in the respiratory specimens of the patients, were used to evaluate the improvement of the symptoms of the patients after treatment.
  • the inclusion criteria of this study mainly include 1) age 18-75 years old, male or female, and the person or his family members voluntarily join and sign the informed consent; 2) RT-PCR test positive or definite diagnosis of novel coronavirus pneumonia; 3 ) meet the diagnostic criteria for severe and critically ill patients.
  • Exclusion criteria mainly include 1) related virus carriers or patients with more severe allergic constitution, patients with pneumonia caused by other viruses; 2) patients with lung cancer or long-term use of immunosuppressive drugs; 3) patients undergoing hemodialysis or peritoneal dialysis, and liver function Abnormal patients; 4) Patients who are using ECMO or high-frequency oscillatory ventilation; 5) Patients who are pregnant, breastfeeding or planning to conceive within six months; 6) Patients who are judged by the investigator to be unable to participate in the study or who fail to understand and implement the program.
  • the results of this clinical trial show that the atomization of human-derived adipose-derived mesenchymal stem cell extracellular vesicles can assist in the treatment of acute lung injury induced by 2019-nCoV to a certain extent, thereby improving the symptoms of pneumonia in critically ill patients, and it is safe. .
  • Anti-inflammatory miRNAs are enriched in extracellular vesicles of human adipose-derived mesenchymal stem cells
  • microRNAs in human adipose-derived mesenchymal stem cell extracellular vesicles (haMSC-Exos) produced in three batches was investigated. Three batches of human adipose-derived mesenchymal stem cell extracellular vesicles produced under GMP conditions were selected for total microRNA extraction.
  • the Illumina TruSeq Small RNA kit was used to construct the library, including connecting the 3' end adapter and the 5' end adapter respectively, inverting the random primers into the first strand cDNA, and then performing the steps of library enrichment and gel purification. After quantifying the library, press Proportional on-machine injection detection, using Illumina platform for sequencing. Finally, the data after quality control, that is, clean data (reads), are compared with human genome data, and the expression levels of known miRNAs in each sample are counted, and TPM (Transcripts Per Million) is used to normalize the expression levels. .
  • miRNAs with anti-inflammatory and immunomodulatory functions include hsa-let-7b-5p, hsa-let-7a-5p, hsa-miR-126-5p, hsa-miR-151a- 3p, hsa-miR-1246, hsa-miR-21-5p, hsa-miR-146a-5p and hsa-let-7b-3p, etc., so they can inhibit or weaken acute lung injury or ARDS.
  • Bioinformatics prediction analysis showed that the main mechanism of anti-inflammatory effect of these miRNAs is dependent on the targeted inhibition of TLR4/MyD88/NF- ⁇ B and/or JAK2/STAT3 signaling pathway activation.
  • the pharmaceutical composition of stem cell extracellular vesicles can reach the pulmonary airway inflammation site by aerosol inhalation through the respiratory tract
  • mice in the modeling group were treated with a mixture of 10 ⁇ g chicken ovalbumin (OVA) and 0.4 mg aluminum hydroxide dissolved in 200 ⁇ l normal saline on the same day and the seventh day, and the mice were injected intraperitoneally.
  • Sensitization treatment subsequently, on the 14th, 15th, 16th and 17th days, challenge treatment was performed, and mice were treated with nebulization of 6% OVA solution dissolved in medical saline for 25 min.
  • mice were treated with aerosol inhalation on the 13th and 14th days after anesthesia. Then, 0.5h, 1h, 3h, 24h and 48h after administration, mice were subjected to IVIS fluorescence imaging and fluorescence intensity analysis of lung tissue in vitro.
  • the lung tissue was quickly placed in liquid nitrogen for freezing treatment, and after being embedded in OCT, it was frozen in liquid nitrogen again; 10 ⁇ m frozen sections were prepared by a cryostat, dried at room temperature, and the OCT embedding agent was washed away. , the nuclei were stained with DAPI and mounted with a fluorescence quencher; then, they were imaged with a fluorescence microscope to analyze the localization of PKH67-labeled extracellular vesicles in the airways of lung tissue.
  • a pharmaceutical composition formulation of stem cell extracellular vesicles reduces airway hyperresponsiveness in asthmatic mice
  • the degree of airway hyperresponsiveness depends primarily on the level of airway inflammation and the individual's immune response. It is an important feature of the presence or absence of granulocyte inflammation in asthmatic phenotypes in children and adults.
  • the asthma model in Example 7 was used to conduct a treatment study ( FIG. 8 ).
  • the specific implementation method is as follows:
  • mice 7-week-old female wild-type BALB/c mice were placed in an independent ventilation system in an SPF animal room for one week and then randomly divided into 6 groups with 10 mice in each group.
  • saline saline
  • OVA+LPS modeling group
  • the mixture of physiological saline was injected intraperitoneally into mice for sensitization treatment; then, on the 14th, 15th, 16th and 17th days, the mice were stimulated, and the mice were nebulized with medical saline and dissolved 6% OVA solution for 25min respectively.
  • mice in the drug treatment group were treated with medical saline, dexamethasone (1 mg/kg), adipose mesenchymal stem cells (1.5 ⁇ 10 5 cells/time/mouse) and their vesicle drug combination on the 13th and 14th days.
  • the biological preparations (1 ⁇ 10 5 extracellular vesicles/mouse in the low-dose group and 2 ⁇ 10 6 extracellular vesicles/mouse in the high-dose group) were administered to the mice through the respiratory tract. Twenty-four hours after the last challenge treatment (day 18), all mice were subjected to the sampling test.
  • mice were placed on a non-invasive spirometer for 10 minutes to adapt, and under the condition of stable breathing, the airway without methacholine (Mch) treatment was recorded.
  • Rat 5min airway resistance value RI value
  • concentrations of methacholine (5, 10, 25, and 50 mg/ml) inhaled by nebulization were continuously increased, and the RI values at the corresponding concentrations were detected respectively.
  • percentage curves of RI values at different Mch doses were plotted.
  • a pharmaceutical composition formulation of stem cell extracellular vesicles improves lung histopathology in asthmatic mice
  • Example 7 In order to explore the pathological characteristics of the lung tissue of asthmatic mice after the pharmaceutical composition preparation of stem cell extracellular vesicles was treated by respiratory inhalation, the asthma animal model in Example 7 was used for research, and the specific operations were as follows:
  • the ratio of dry weight to wet weight of lung tissue was used to evaluate the pulmonary edema in mice.
  • the whole lung tissue from the bronchoalveolar lavage fluid (BALF) was placed on gauze, the surface moisture was absorbed, weighed and recorded its wet weight; then placed in an 80°C incubator for 48 hours, weighed again, and recorded. Lung dry weight.
  • the pharmaceutical composition preparation of adipose-derived mesenchymal stem cell extracellular vesicles can improve pulmonary edema in mixed granulocyte-mediated asthma mice through respiratory inhalation.
  • peribronchial and perivascular inflammatory cell infiltration by H&E staining a blind review score was performed: no cellular infiltration, scored as 0; only some cells, scored as 1, and the infiltrating cells formed a ring with a width of 1 cell, scored as 2; the infiltrating cells formed a ring and the width of 2 to 4 cells was marked as 3; the infiltrated cells formed a ring and the width of more than 4 cells was marked as 4.
  • the expected result is that the pharmaceutical composition formulation of adipose-derived mesenchymal stem cell extracellular vesicles through respiratory nebulization can reduce mixed granulocyte-mediated asthma in mice lung compared with dexamethasone and adipose-derived mesenchymal stem cell treatment. Levels of inflammatory cell infiltration around bronchi and blood vessels.
  • a pharmaceutical composition formulation of stem cell extracellular vesicles reduces the levels of inflammatory factors in asthmatic mice
  • Example 7 In order to explore the level of inflammatory factors in asthmatic mice after the pharmaceutical composition preparation of stem cell extracellular vesicles is treated by respiratory nebulization inhalation, the asthma animal model in Example 7 is used to detect it, and the specific operations are as follows:
  • the pharmaceutical composition preparation of adipose-derived mesenchymal stem cell extracellular vesicles through respiratory nebulization can reduce serum IL-4 in mice with mixed granulocyte-mediated asthma , IL-5, IL-6, IL-13, IL-17A and IgE levels.
  • BALF supernatant was used to detect cytokines (IL-4, IL-5, IL-6, IL-13 and IL-17A) levels by ELISA.
  • cytokines IL-4, IL-5, IL-6, IL-13 and IL-17A
  • the pharmaceutical composition preparation of stem cell extracellular vesicles can reduce IL-4, IL-5, IL-6 in BALF of mice with mixed granulocyte-mediated asthma through respiratory nebulization , IL-13 and IL-17A levels.
  • the pelleted and resuspended suspension of bronchoalveolar lavage fluid was used to detect the total number of cells, as well as eosinophils (eosinophils), neutrophils (neutrophils), macrophages (macrophages) and lymphocytes ( lymphocytes).
  • a pharmaceutical composition formulation of stem cell extracellular vesicles improves lung function in COPD mice
  • mice were placed in a nebulizing box, and the smoke produced by burning 3 mg of nicotine was inhaled through the mouth and nose, with an interval of 30 min between every two cigarettes, for a total of 4 weeks of exposure.
  • mice in the experimental group received the inhalation treatment of the pharmaceutical composition of human adipose-derived mesenchymal stem cell extracellular vesicles (1 ⁇ 10 6 particles/mouse) every day. , aerosol inhalation once a day, continuous treatment for 5 days.
  • mice were tested for pulmonary function indicators, including maximum expiratory volume within 100ms (FEV 100 ), arterial blood oxygen partial pressure (PaO 2 ), arterial blood carbon dioxide partial pressure (PaCO 2 ) and oxygen saturation (SaO 2 ).
  • FEV 100 maximum expiratory volume within 100ms
  • PaO 2 arterial blood oxygen partial pressure
  • PaCO 2 arterial blood carbon dioxide partial pressure
  • SaO 2 oxygen saturation
  • the pharmaceutical composition preparation of human adipose-derived mesenchymal stem cell extracellular vesicles can significantly improve the maximum expiratory volume within 100ms of COPD model mice through respiratory inhalation Blood oxygen partial pressure and oxygen saturation, reduce arterial blood carbon dioxide partial pressure, thereby improving its lung function.
  • mesenchymal stem cells have the effect of promoting tissue repair and anti-inflammatory, and most of their functions are produced by interacting with target cells through the secretion of soluble factors or vesicles.
  • Mesenchymal stem cell vesicles play an important role in the treatment of many diseases, and also have great application potential in the prevention and treatment of respiratory inflammatory diseases.
  • Stem cell vesicles are important information carriers for signal communication between stem cells and target cells. Once secreted into the extracellular space, stem cell vesicles can be engulfed into target cells through interactions with target cells, and ultimately affect the phenotype of target cells to exert their unique biological functions.
  • human adipose-derived mesenchymal stem cells produced under GMP conditions are preferably used as parent cells for producing stem cell vesicles, and the pharmaceutical composition preparations for obtaining the vesicles are further produced and administered by airway inhalation It is used to treat acute airway inflammation and chronic airway inflammation such as bronchial asthma and chronic obstructive pulmonary disease, in order to provide new treatments for airway inflammation caused by various incentives.

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Abstract

提供了含干细胞囊泡的药物组合物及其在治疗急性和慢性呼吸道炎症中的用途。具体地，提供了以干细胞囊泡内含物中关键活性物质为主要有效成分的药物组合物及其制备方法，以及该药物组合物在治疗急性呼吸道炎症和慢性阻塞性肺疾病(COPD)、支气管哮喘中的应用。所述药物组合物制剂经呼吸道给药时，可有效地分布于肺部细小支气管和肺泡部位。与常规药物或干细胞治疗相比，所述药物组合物的主要活性成分极易被细小支气管上皮、肺泡上皮细胞、以及气道内的炎性细胞所摄取，进而产生显著的抑制气道炎性反应、修复气道损伤、改善气道张力等作用，从而有效预防或治疗呼吸道炎症。

Description

含有干细胞胞外囊泡的药物组合物及其在呼吸道炎症治疗中的应用 技术领域
本发明涉及干细胞治疗药物领域，具体涉及干细胞分泌的胞外囊泡的药物组合物及其在急性和慢性呼吸道炎症治疗中的应用。
背景技术
肺部炎症是身体对损伤的自然反应。它有助于机体去除病原体、刺激物和受损细胞，并启动组织修复过程。急性和慢性肺部炎症常见于不同的呼吸系统疾病，如哮喘、囊性纤维化、急性呼吸窘迫综合征和慢性阻塞性肺疾病(COPD)。其中，哮喘和COPD是世界上普遍的慢性疾病，全球范围内影响超过3亿人，可造成每年大量患者死亡。目前在全世界范围内，哮喘和COPD的发病率和死亡率仍然不断增加。
哮喘是一种以可逆性气道阻塞为特征的异质性慢性气道炎症性疾病。其病理特征表现为气道高反应性，支气管及其周围组织被大量炎症细胞浸润，且气道发生重塑而过度狭窄。COPD则属于不完全可逆性气流限制的慢性气道炎症性疾病，其病理特征主要表现为炎症细胞浸润气道组织、气道重塑和肺泡破损等。如果没有适当的治疗和控制，这两种疾病可能会导致患者气道发生变化，进而导致不可逆转的肺泡损伤和永久性肺纤维化，最终导致肺功能丧失和对治疗药物的反应性降低。
目前，尽管针对上述疾病已开发了较多的治疗药物，但仍然无法充分控制病程。治疗哮喘的常规药物主要是一些缓解和控制症状的药物。其中，缓解药物可迅速缓解支气管痉挛而缓解哮喘症状，但无法逆转疾病过程，且患者通常会产生耐受性，需要更高剂量来控制症状。而控制药物则主要是一些具有抗炎作用的皮质类固醇激素和白三烯拮抗剂等，需要长期服用，故副作用较大，也无法恢复与哮喘相关的所有变化。另外，已有的人源化抗体药物Omal izumab仅适用于以嗜酸性粒细胞浸润为主要特征的重度哮喘的治疗，但不适合以中性粒细胞浸润为主要特征的哮喘治疗。并且，该抗体药物在一些儿童哮喘患者中的治疗应用仍然存局限性。另外，治疗COPD的药物主要是一些激素类药物和支气管扩张剂等。然而，患者长期使用这些药物同样会出现许多副作用，如心动过速、高血压等。
因此，为了避免上述常规药物长期治疗所带来的弊端，控制严重患者的病程，提高患者的生活质量，迫切需要寻找一种新的治疗模式，来改进目前的治疗方法。
发明内容
本发明目的在于提供了一种以干细胞胞外囊泡中关键活性物质为主要有效成分的药物组合物的制备方法及其在急性和慢性呼吸道炎症治疗中的应用。
本发明的第一方面，提供了一种干细胞胞外囊泡制剂，所述胞外囊泡的粒径为30-200nm，较佳地50-150nm，更佳地80-120nm；并且，所述胞外囊泡中含有与抗炎或组织损伤修复相关的生物活性因子，所述生物活性因子包括基质蛋白金属酶1(MMP-1)和肝细胞生长因子(HGF)，其中MMP-1在所述制剂中的含量为1500-2000pg/ml，HGF在所述制剂中的含量为5500-6000pg/ml。
制剂中MMP-1的量可以是约50至约5000pg/ml、约100至约4000pg/ml、约100至约3000pg/ml、约100至约2000pg/ml、约200至约3000pg/ml、约200至约4000pg/ml、约200至约2000pg/ml、约300至约2000pg/ml、约400至约5000pg/ml、约500至约5000pg/ml/ml，约600至约5000pg/ml，约700至约5000pg/ml，约800至约5000pg/ml，约900至约5000pg/ml，约1000至约5000pg/ml，约1000约3000pg/ml、约1000至约2000pg/ml、约500至约2000pg/ml、约500至约3000pg/ml、或约1200至约2000pg/ml。
制剂中HGF的量可以是约50至约8000pg/ml、约100至约7000pg/ml、约200至约6000pg/ml、约300至约8000pg/ml、约400至约8000pg/ml、约500至约8000pg/ml、约600至约8000pg/ml、约700至约8000pg/ml、约800至约8000pg/ml、约900至约8000pg/ml,约1000至约8000pg/ml,约1000至约7000pg/ml,约1000至约6000pg/ml,约500至约7000pg/ml,约500至约6000pg/ml,约2000至约8000pg/ml，约2000至约7000pg/ml，约2000至约6000pg/ml，约3000至约8000pg/ml，约3000至约7000pg/ml，约3000至约6000pg/ml，约4000至约8000pg/ml、约4000至约7000pg/ml、约4000至约6000pg/ml、或约5000至约6000pg/ml。
存在三个主要类型的胞外囊泡(EVs)，即，外泌体、微囊泡和凋亡小体。所有这三个主要类型的EVs都被脂质双分子层包裹，其直径范围在30-2000nm。
大的胞外囊泡的直径范围可以从约5μm至约12μm。凋亡小体的直径范围可以从约1μm至约5μm。微囊泡的直径范围可以从约100nm至约1μm。外泌体可以具有约30nm至约150nm、约30nm至约100nm、约50nm至约150nm、约50nm至约100nm或约50nm至约200nm的直径范围。
在另一优选例中，所述干细胞胞外囊泡制剂含有1×10 8-5×10 8个干细胞胞外囊泡/ml。
在另一优选例中，所述干细胞胞外囊泡来源于人体干细胞在体外培养过程中所收集的上清液。
在另一优选例中，所述干细胞选自下组：人脐带血来源的干细胞、人外周血来源的干细胞、人脐带间充质干细胞、人胎盘间充质干细胞、人脂肪间充质干细胞、人骨髓来源的干细胞、或其组合。
在另一优选例中，所述干细胞为人脂肪间充质干细胞。
在某些实施方案中，本发明的制剂衍生自干细胞或祖细胞。在某些实施方案中，本发明的方法培养干细胞或祖细胞。干细胞是具有自我更新和产生分化后代的能力的未分化细胞(参见Morrison等人(1997)Cell 88:287-298)。在哺乳动物中，有两大类型的干细胞：胚胎干细胞和成体干细胞，它们存在于多种组织中。
干细胞可以是骨髓来源的干细胞(BMSCs)、脂肪来源的干细胞(ADSCs)、神经干细胞(NSCs)、血液干细胞或造血干细胞。干细胞也可以来自脐带血。干细胞可以通过体细胞核移植或去分化而产生。
干细胞包括但不限于血液干细胞、脂肪干细胞、骨髓间充质干细胞、间充质干细胞、神经干细胞(NSC)、皮肤干细胞、内皮干细胞、肝干细胞、胰腺干细胞、肠上皮干细胞或生殖干细胞。在某些实施方案中，间充质干细胞分离自中胚层器官，例如骨髓、脐带血和脂肪组织。
在某些实施方案中，干细胞是诱导性多能干细胞(iPS细胞或iPSC)。iPSC是指由非多能细胞人工产生的一类多能干细胞，所述非多能细胞通常是成体体细胞或终末分化细胞，例如成纤维细胞、造血细胞、肌细胞、神经元、表皮细胞等。
在某些实施方案中，本发明的方法培养增殖细胞。在某些实施方案中，本发明的方法培养T细胞(包括原代T细胞)。
细胞可以包括从被治疗的受试者收获的自体细胞和/或生物相容的同种异 基因细胞或同种同基因细胞(syngeneic cells)，例如自体细胞、同种异基因细胞或同种同基因细胞(例如，间充质干细胞)、祖细胞(例如，结缔组织祖细胞)或多能成体祖细胞)和/或其他进一步分化的细胞。
任何具有分化潜能的干细胞均可以用于产生诱导性外泌体的方法中，所述具有分化潜能的干细胞包括(但不限于)胚胎干细胞、诱导性高效干细胞、癌干细胞和组织干细胞。组织干细胞包括但不限于间充质干细胞、造血干细胞、乳腺干细胞、神经干细胞、小肠干细胞、皮肤干细胞、脐带血干细胞、角膜缘干细胞、毛囊干细胞、脂肪组织来源的干细胞、骨髓干细胞、角膜干细胞和卵巢干细胞。用于产生外泌体的干细胞可以选自胚胎干细胞、诱导性多能干细胞、癌干细胞、间充质干细胞、造血干细胞、乳腺干细胞、神经干细胞、小肠干细胞、皮肤干细胞、脐带血干细胞、角膜缘干细胞、毛囊干细胞、脂肪组织来源的干细胞、骨髓干细胞、角膜干细胞和卵巢干细胞。
在另一优选例中，所述与抗炎或组织损伤修复相关的生物活性因子选自下组：MMP-1、HGF、血管内皮细胞生长因子(VEGF)、或其组合。
在另一优选例中，所述与抗炎或组织损伤修复相关的生物活性因子是所述的人干细胞产生的生物活性因子。
在另一优选例中，所述与抗炎或组织损伤修复相关的生物活性因子包括自然状态下培养的干细胞旁分泌所产生的生物活性因子、用外源基因修饰的干细胞所表达产生的生物活性因子、或其组合。
在另一优选例中，所述干细胞胞外囊泡具有完整的脂质双分子层膜结构，并且包含以下蛋白：CD9、CD63、CD81、TSG101和HSP70；不包含或基本上不包含蛋白CANX。
在另一优选例中，所述干细胞胞外囊泡的膜结构表面包含CD9、CD63、CD81，其膜结构内部包含TSG101、HSP70。
在另一优选例中，所述干细胞胞外囊泡的膜结构内部包含还某些非编码功能的DNA和RNA。
在另一优选例中，所述RNA包括miRNA、tRNA、rRNA、snoRNA和snRNA。
在另一优选例中，所述RNA的长度为17～100个核苷酸。
在另一优选例中，所述非编码功能的DNA和RNA包括自然状态下培养的干细胞产生的非编码功能的DNA和RNA分子、经基因工程改造的干细胞表达产生的非编码功能的DNA和RNA分子、化学合成后转导进入干细胞的非编码功能的 DNA和RNA分子、或其组合。
在另一优选例中，所述RNA选自下组：hsa-let-7b-5p、hsa-let-7a-5p、hsa-miR-126-5p、hsa-miR-151a-3p、hsa-miR-1246、hsa-miR-21-5p、hsa-miR-146a-5p、hsa-let-7b-3p、has-miR-1290、has-miR-23a-3p、has-let-7d-3p或其组合。
在另一优选例中，用于生产所述干细胞胞外囊泡的细胞包括以下来源的细胞：
(a)从人体组织直接分离纯化所获得的干细胞；
(b)从人体组织直接分离纯化后，再经过GMP实验室最小程度的操作，使这些干细胞增殖后所获得的细胞；
(c)在GMP实验室经过特定基因修饰、特定基因编辑、特定基因转导、特定微小核糖核酸miRNA的导入所获得的细胞；
(d)在GMP实验室经过特殊培养条件的预处理所获得的细胞。
在另一优选例中，所述的用于生产所述干细胞胞外囊泡的细胞包括原代细胞、传代次数为1-10次的传代细胞。
在另一优选例中，所述的用于生产所述干细胞胞外囊泡的细胞包括未经基因操作的细胞、经基因操作的细胞。
在另一优选例中，所述的基因操作包括基因编辑、基因导入、基因下调(knock-down)、基因敲除(knock-out)、或其组合。
在另一优选例中，所述干细胞胞外囊泡具有以下特征：采用聚乙二醇(PEG)沉淀法制备得到，胞外囊泡粒径集中分布在80-120nm之间，粒径更小且均一。
在另一优选例中，所述聚乙二醇(PEG)沉淀包括步骤：使用PEG3000～9000的PEG，用PBS配置为8％-20％的PEG母液，经过滤除菌(如用0.22μm的过滤器过滤)后，按照一定比例(如约1:1体积比)加入到处理后的条件培养基中(所述条件培养基经差速离心和过滤处理人源脂肪间充质干细胞的培养上清液获得)，置于4℃过夜孵育；然后4℃，3,000-5000g离心30-60分钟，弃去上清，加入预冷的PBS重悬沉淀；4℃，100000-120000g超高速离心60-120分钟，弃去上清，从而得到所述干细胞胞外囊泡。
本发明的第二方面，提供了一种药物组合物，所述药物组合物含有：(a)本发明第一方面所述的干细胞胞外囊泡制剂，和(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物是无细胞和无细胞碎片的药物组合物。
在另一优选例中，所述的“无细胞”指所述的药物组合物不含有活细胞和死细胞。
在另一优选例中，所述药物组合物包含与抗炎或组织损伤修复相关的生物活性因子，所述生物活性因子包括MMP1、HGF、VEGF或其组合。
在另一优选例中，所述药学上可接受的载体选自下组：氯化钠、磷酸钠、聚乙二醇、壳聚糖、玻璃酸钠、海藻糖、肝素、或其组合。
在另一优选例中，所述的药物组合物的剂型选自下组：液态剂型、固态剂型(如冻干剂型)。
在另一优选例中，所述的药物组合物的剂型选自下组：雾化吸入剂、滴眼液、滴鼻液、注射液。
在另一优选例中，所述的药物组合物为雾化吸入剂。
在另一优选例中，所述药物组合物具有以下特征：其主要活性成分极易被细小支气管上皮、肺泡上皮细胞，以及气道内的炎性细胞所摄取，进而产生显著的抑制气道炎性反应、修复气道损伤、改善气道张力等作用，可用于预防和治疗各种原因引起的呼吸道炎症性疾病和损伤。
在另一优选例中，所述药物组合物被用于治疗急性呼吸道炎症和/或慢性呼吸道炎症。
在另一优选例中，所述药物组合物被用于治疗慢性呼吸道炎症，较佳地用于治疗以呼吸道粘膜内嗜酸性粒细胞、中性粒细胞、单核巨噬细胞等炎性细胞浸润为主要特征的慢性呼吸道炎症，更佳地用于治疗支气管哮喘、慢性阻塞性肺疾病(COPD)。
本发明的第三方面，提供了一种含干细胞胞外囊泡的药物组合物的制备方法，所述方法包括步骤：
(S1)培养人的干细胞(如脂肪间充质干(祖)细胞)，使其达到预定的汇合度(如75-90％)；
(S2)在适合产生EV的条件下，继续培养所述的细胞一段时间T1；其中，所述的T1通常为24-72小时，较佳地30-60小时；
(S3)从培养体系中去除细胞，从而分离获得含干细胞胞外囊泡的培养液，即为“条件培养液(conditioned medium)”；
(S4)将所述的条件培养液(conditioned medium)与聚乙二醇(PEG)进行混合，形成第一混合物，并放置一段时间T2，从而形成经PEG修饰的干细胞胞外囊泡；其中，所述的T2通常为6-60小时，较佳地12-48小时；
(S5)对上一步骤的第一混合物进行离心，使得所述的经PEG修饰的干细胞胞外囊泡沉淀，并弃去上清，获得经PEG修饰的干细胞胞外囊泡沉淀；
(S6)对上一步骤获得的经PEG修饰的干细胞胞外囊泡沉淀进行重悬，从而获得第一重悬混合物；
(S7)对第一重悬混合物进行离心，使所述的经PEG修饰的干细胞胞外囊泡沉淀，并弃去上清，获得经PEG修饰的干细胞胞外囊泡沉淀；
(S8)对上一步骤中获得的经PEG修饰的干细胞胞外囊泡沉淀进行重悬，从而获得可医用的干细胞胞外囊泡制剂。
在另一优选例中,在所述干细胞胞外囊泡制剂中，来源于胞外囊泡的MMP-1的含量为1500-2000pg/ml，HGF的含量为5500-6000pg/ml。
在另一优选例中，所述方法还包括：
(S9)将可医用的干细胞胞外囊泡制剂(或活性物质)与药学上可接受的载体混合，从而制得药物组合物。
在另一优选例中，所述方法还包括将所述的药物组合物制成雾化吸入制剂、注射剂、或冻干制剂。
本发明的第四方面，提供了一种本发明第一方面所述的干细胞胞外囊泡制剂或本发明第二方面所述的药物组合物的用途，用于制备治疗呼吸道炎症的药物或制剂。
在另一优选例中，所述呼吸道炎症包括急性呼吸道炎症和慢性呼吸道炎症。
在另一优选例中，所述慢性呼吸道炎症以呼吸道粘膜内嗜酸性粒细胞、中性粒细胞、单核巨噬细胞等炎性细胞浸润为主要特征。
在另一优选例中，所述慢性呼吸道炎症为支气管哮喘、慢性阻塞性肺疾病(COPD)、或其组合。
在另一优选例中，所述急性呼吸道炎症包括病毒感染性炎症、细菌感染性炎症、真菌感染性炎症、或其组合。
在另一优选例中，所述急性呼吸道炎症包括选自下组的疾病：急性呼吸窘迫综合征(ARDS)、急性肺损伤(ALI)、新型冠状病毒(SARS-CoV-2)感染性肺炎、 或其组合。
本发明的第五方面，提供了一种治疗呼吸道炎症的方法，包括步骤：给需要的对象施用本发明第二方面的药物组合物。
在另一优选例中，所述呼吸道炎症包括急性呼吸道炎症和慢性呼吸道炎症。
在另一优选例中，所述慢性呼吸道炎症以呼吸道粘膜内嗜酸性粒细胞、中性粒细胞、单核巨噬细胞等炎性细胞浸润为主要特征。
在另一优选例中，所述慢性呼吸道炎症为支气管哮喘、慢性阻塞性肺疾病(COPD)、或其组合。
在另一优选例中，所述急性呼吸道炎症包括病毒感染性炎症、细菌感染性炎症、真菌感染性炎症、或其组合。
在另一优选例中，所述急性呼吸道炎症包括选自下组的疾病：急性呼吸窘迫综合征(ARDS)、急性肺损伤(ALI)、新型冠状病毒(SARS-CoV-2)感染性肺炎、或其组合。
在另一优选例中，所述的需要的对象是人。
在另一优选例中，所述的需要的对象患有急性呼吸道炎症和/或慢性呼吸道炎症。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了人脂肪间充质干细胞胞外囊泡的药物组合物制剂中主要活性成分的生物学特征鉴定。
图2显示了人脂肪间充质干细胞胞外囊泡的药物组合物制剂中主要活性成分的体外细胞摄取。
图3显示了DiR标记的人源脂肪间充质祖细胞外泌体在小鼠体内的生物分布情况。其中，(A)在外泌体雾化给药1h、4h、24h、48h和72h后，小鼠体内分布情况；(B)在外泌体雾化给药48h和72h后，小鼠离体组织中的外泌体分布情况。
图4显示了人源脂肪间充质祖细胞外泌体雾化治疗PA诱导的小鼠肺炎。其中，(A)小鼠肺组织HE染色形态观察；(B)小鼠肺组织损伤评分；(C)和(D)小鼠肺泡灌洗液中总白细胞和中性粒细胞数量变化；(E)和(F)小鼠肺泡灌洗液中的促炎症细胞因子TNF-α和IL-6含量；(G)小鼠肺泡灌洗液中的抑炎症细胞因子IL-10含量。*P<0.05，**P<0.01，****P<0.0001。
图5显示了人源脂肪间充质祖细胞外泌体雾化治疗前后重症新冠患者胸部CT变化情况。(A和B)患者A和B在治疗前后的胸部CT成像，结果显示其治疗后的肺部白色片状阴影减弱。
图6显示了三批次生产的人源脂肪间充质祖细胞外泌体中排名前20的miRNAs种类及其相对表达量。
图7显示了混合型粒细胞特征的哮喘小鼠模型制备示意图。
图8显示了人脂肪间充质干细胞胞外囊泡的药物组合物制剂治疗混合型粒细胞特征的哮喘小鼠示意图。
图9显示了人脂肪间充质干细胞胞外囊泡的药物组合物制剂治疗COPD小鼠示意图。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地研发出了一种可有效治疗慢性呼吸道炎症的无细胞生物医药制剂，该药物制剂含有人体干细胞来源的干细胞胞外囊泡，具有特定的粒径，并且含有与抗炎或组织损伤修复相关的生物活性因子。本发明的干细胞囊泡采用聚乙二醇(PEG)沉淀法制备得到，胞外囊泡粒径集中分布在80-120nm之间，粒径更小且均一，在医用溶剂(如生理盐水等)中具有极其优异的分散性，特别适合通过雾化吸入方式等方式直接施用于患者的呼吸道；并且，本发明的干细胞胞外囊泡的药物组合物或制剂中的主要活性成分极易被细小支气管上皮、肺泡上皮细胞、以及气道内的炎性细胞所摄取，从而快速、高效地治疗或缓解慢性呼吸道炎症，尤其是哮喘和COPD等疾病。在此基础上，完成了本发明。
术语
哮喘
哮喘一般指支气管哮喘(bronchial asthma)，是由多种细胞(如嗜酸性 粒细胞、肥大细胞、T淋巴细胞、中性粒细胞、气道上皮细胞等)和细胞组分参与的气道慢性炎症为特征的异质性疾病，这种慢性炎症与气道高反应性相关，通常出现广泛而多变的可逆性呼气气流受限，导致反复发作的喘息、气促、胸闷和(或)咳嗽等症状，强度随时间变化。多在夜间和(或)清晨发作、加剧，多数患者可自行缓解或经治疗缓解。支气管哮喘如诊治不及时，随病程的延长可产生气道不可逆性缩窄和气道重塑。
慢性阻塞性肺疾病(COPD)
慢性阻塞性肺疾病(chronic obstructive pulmonary disease，COPD)是一种常见的以持续气流受限为特征的可以预防和治疗的疾病，气流受限进行性发展，与气道和肺脏对有毒颗粒或气体的慢性炎性反应增强有关。它是一种具有气流阻塞特征的慢性支气管炎和(或)肺气肿，可进一步发展为肺心病和呼吸衰竭的常见慢性疾病。
间充质干细胞胞外囊泡
干细胞胞外囊泡(extracel lular vesicles,EVs)有三个主要类型，分别为外泌体(Exosomes)、微囊泡(Microvesicles)和凋亡小体(Apoptotic bodies)。所有这三个主要类型的EVs都被脂质双分子层包裹，其直径范围在30-2000nm。
外泌体一词指的是由胞内体衍生的直径在50-100nm的EVs亚类，它们是包括间充质干细胞(mesenchymal stem cells,MSCs)在内的多种细胞类型旁分泌物的主要组成部分。
MSCs外泌体(Exosomes)是MSCs衍生的EVs中的一类直径在50-100nm范围内的、具有完整脂质双分子层膜结构的EVs。
外泌体是一种运载丰富货物的载体，其功能主要是通过不断转运微小核糖核酸(miRNAs)和蛋白来发挥的。在MSCs来源的外泌体中，已经鉴定出150多个miRNAs和850多个独特的蛋白质，通过不同的途径，改变靶细胞的各种活性。MSC外泌体参与了机体发育、表观遗传调控、免疫调节(miR-155和miR-146)、肿瘤发生和肿瘤进展(miR-23b、miR-451、miR-223、miR-24、miR-125b、miR-31、miR-214和miR-122)等生理和病理过程。根据ExoCarta的资料，已经从MSCs外泌体中收集了900多种蛋白质。一些研究表明，MSC外泌体载有一些细胞因子和生长因子,如TGFβ1、白细胞介素-6(IL-6)、IL-10和肝细胞生长因子(HGF) 等，这些因子已被证实有助于免疫调节。血管内皮生长因子(VEGF)、细胞外基质金属蛋白酶诱导剂(extracellular matrix metalloproteinase inducer,EMMPRIN)和MMP-9在MSC外泌体中均有报道，这三种蛋白在刺激血管生成中发挥重要作用，可能是外泌体组织修复作用的基础。
本发明的干细胞胞外囊泡及含有其的药物组合物的制备方法
本发明提供了一种干细胞来源的胞外囊泡，所述胞外囊泡的粒径为30-200nm，较佳地50-150nm，更佳地80-120nm；并且，所述胞外囊泡中含有与抗炎或组织损伤修复相关的生物活性因子，所述活性因子包括基质蛋白金属酶1(MMP-1)和肝细胞生长因子(HGF)，其中MMP-1的含量为1500-2000pg/ml，HGF的含量为5500-6000pg/ml。
其中，所述所述干细胞包括但不限于人脐带血来源的干细胞、人外周血来源的干细胞、人脐带间充质干细胞、人胎盘间充质干细胞、人脂肪间充质干细胞、人骨髓来源的干细胞。在本发明优选的实施方式中，所述干细胞为人脂肪间充质干细胞。
本发明所提供的以干细胞胞外囊泡中关键活性物质为主要有效成分的药物组合物或制剂的制备方法，具体技术方案如下：
在优选的实施方式中，主要采用GMP级别的人源脂肪间充质干细胞作为生产干细胞胞外囊泡的亲本细胞，但也可以是脐血、脐带、胎盘、骨髓等组织来源的干细胞。
本发明中干细胞胞外囊泡的药物组合物制备包括以下步骤：
1)收集有效条件处理的上述GMP规模化生产的人源脂肪间充质干细胞的培养上清液，差速离心和过滤处理获得条件培养基，再结合多聚物沉淀法进行囊泡分离，多聚物包括但不限于不同分子量的聚乙二醇(PEG3000～9000)及其不同比例(1:1～1:6)的组合物；
2)用PBS配置适当浓度(8％～30％g/ml)的PEG母液并过滤处理，按照1:1的体积比，与上述处理好的条件培养基混匀，低温条件下(4～8℃)孵育过夜；
3)离心(4℃,3000～5000g,45～60min)后弃上清液，加入预冷的PBS重悬沉淀，超高速离心(4℃,100000～120000g,1～2h)后弃上清液；
4)加入适量体积(3～10ml)的医用生理盐水，重悬沉淀，再经粒径分析、扫描电镜观察和免疫印迹等技术鉴定所得人源脂肪间充质干细胞胞外囊泡的 生物学特征；
5)对该干细胞胞外囊泡中主要有效成分的关键活性物质指标进行检测；
6)采用3～10ml药学上可接受的载体或辅料将上述所得干细胞胞外囊泡稀释至适当的有效浓度即可。
根据本发明实施例，所述干细胞胞外囊泡的药物组合物的制备优选异体人源脂肪间充质干细胞胞外囊泡。
根据本发明实施例，所述干细胞胞外囊泡的药物组合物或制剂包含药学上可接受的载体或辅料，包括但不限于氯化钠、磷酸钠、聚乙二醇、壳聚糖、玻璃酸钠、海藻糖、肝素等，以及上述物质的任意组合等。
此外，将优选生产的人脂肪间充质干细胞胞外囊泡的药物组合物或制剂用于体外细胞摄取研究，包括Ⅱ型肺泡上皮细胞A549和活化的巨噬细胞RAW247.6。亲脂性荧光染料PKH67标记人脂肪间充质干细胞胞外囊泡的药物组合物活性成分(1×10 7～4×10 8particles)，添加至细胞中培养24～48h后，倒置荧光显微镜下观察，结果表明该组合物活性成分能够被上述细胞摄取。
GMP条件生产干细胞胞外囊泡的工艺规模
一个HyperFlask细胞工厂可以生产500ml条件培养基的干细胞胞外囊泡，预计分离5×10 10—10×10 10干细胞胞外囊泡总量。
目前实验室产能一个生产操作单元可以分离两个细胞工厂共计800-1200ml条件培养基的干细胞胞外囊泡，预计分离2-5×10 11干细胞胞外囊泡，按照一个病人2-5×10 9的使用量，一个生产操作单元一批次生产量可以满足100-250个病人的使用量。
本发明的干细胞胞外囊泡及含有其的药物组合物的应用
本发明还提供了干细胞胞外囊泡中关键活性物质作为主要有效成分的药物组合物或制剂治疗呼吸道炎症性疾病的应用。
本发明的药物组合物含有(a)本发明第一方面所述的干细胞胞外囊泡制剂，和(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物是无细胞和无细胞碎片的药物组合物。
在本发明中，优选地，所述的活性成分还包括与抗炎或组织损伤修复相关的生物活性因子，所述生物活性因子包括但不限于MMP1、HGF、或VEGF。这些生 物活性因子主要存在于干细胞胞外囊泡中。此外，一些生物活性因子可能存在于干细胞胞外囊泡之外。
在本发明中，一种特别优选的药物制剂是雾化吸入制剂。
在优选的实施方式中，采用了混合型粒细胞表征的小鼠哮喘模型验证本发明的药物组合物在治疗呼吸道炎症疾病的应用。。
首先，采用亲脂性荧光染料PKH67标记人脂肪间充质干细胞胞外囊泡的药物组合物(1×10 7～1×10 8particles)，研究其经呼吸道吸入哮喘模型小鼠肺部的分布情况，发现该药物组合物制剂主要活性成分极易被细小支气管上皮、肺泡上皮细胞、以及气道内的炎性细胞所摄取。此外，对小鼠随机分组(表1)后，采用混合型粒细胞表征的小鼠哮喘模型，经地塞米松、干细胞及该制剂不同剂量的主要活性成分对比治疗后，结果发现该制剂的主要活性成分在低剂量(1×10 5～1×10 8particles/mouse)和高剂量(2×10 6～2×10 9particles/mouse)条件下治疗小鼠哮喘时，均能够比常规药物地塞米松(1～2mg/kg)及干细胞(1×10 4～1.5×10 5cells/time/mouse)治疗更有效地降低其气道高反应性和炎症因子水平，从而有效地改善肺组织病理学特征。
表1 人脂肪间充质干细胞胞外囊泡的药物组合物治疗混合型粒细胞特征哮喘小鼠分组
Figure PCTCN2022088118-appb-000001
此外，在优选的实施方式中，首先建立了尼古丁(1.5～5mg)诱导的COPD小鼠模型。对照组小鼠给予生理盐水雾化经气道吸入治疗，而试验组小鼠经人脂肪间充质干细胞胞外囊泡的药物组合物制剂(1×10 5～1×10 8 particles/mouse)的经气道雾化吸入治疗，每天在雾化箱内雾化吸入一次，连续治疗5天。对小鼠进行肺功能指标进行检测发现其肺功能得到改善。
本发明的主要优点包括：
(1)本发明的干细胞胞外囊泡采用聚乙二醇(PEG)沉淀法制备得到，胞外囊泡粒径集中分布在80-120nm之间，粒径更小且均一。
(2)本发明的干细胞胞外囊泡含有与抗炎或组织损伤修复相关的生物活性因子,且含量比传统离心方法制备的胞外囊泡更高。
(3)本发明的干细胞胞外囊泡的药物组合物或制剂中的主要活性成分极易被细小支气管上皮、肺泡上皮细胞、以及气道内的炎性细胞所摄取。
(4)与常规药物或干细胞治疗相比，本发明的干细胞胞外囊泡的药物组合物或制剂可更有效地改善或治疗呼吸道炎症性疾病。
下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实验材料和仪器
本发明优选的实施方式中采用人脂肪间充质干细胞作为获取胞外囊泡的亲本细胞，其它涉及的试验材料及仪器，主要包括：
野生型BALB/c小鼠(7周龄，雌性，SPF级)、野生型C57BL/6小鼠(8-10周龄，雄性，SPF级)、麻醉剂(50mg/kg氯胺酮和30mg/kg西拉嗪)、Ⅱ型肺泡上皮细胞A549、RAW264.7巨噬细胞、耐多药铜绿菌(PA)、鸡卵清蛋白(Ovalbumin,OVA,Sigma-Aldrich)、氢氧化铝、香烟、脂多糖(LPS)、地塞米松(dexamethasone,Dex)、乙酰甲胆碱(methacholine)、医用生理盐水、不同分子量的PEG、无菌磷酸盐缓冲液(PBS)、1ml无菌注射器及20G针头、PKH67染料、DiR染料、石蜡块、H&E染色液、PAS染液、10％中性甲醛固定液、OCT包埋剂、液氮、Illumina TruSeq Small RNA试剂盒、小鼠TNF-α、IgE、IL-4、IL-5、IL-6、IL-10、IL-13及IL-17A的ELISA检测试剂盒、离心管、12孔细 胞培养板、离心机、石蜡包埋机、石蜡切片机、冰冻切片机、普通明场显微镜(40×物镜)、正置荧光显微镜(40×物镜)、倒置荧光显微镜(20×物镜)、细胞培养箱、雾化箱、无创检测肺功能仪、血细胞计数器、IVIS成像系统、粒径分析仪、Illumina测序平台。
数据统计分析方法
所有试验数据均采用GraphPad Prism 6.0软件进行统计分析，且均表示成平均值±标准误(Mean±SEM)。两组数据之间的比较时，采用Mann-Whitney U test进行数据比较分析；多组数据之间的比较时，采用One-way ANOVA与Tukey进行数据比较分析。P值小于0.05时被认为具有显著性差异。
实施例1
干细胞胞外囊泡的药物组合物的制备
在本发明所有的实施例中，选用具备GMP级别的人源脂肪间充质干细胞作为生产干细胞胞外囊泡的亲本细胞，但也可以是脐血、脐带、胎盘、骨髓等组织来源的干细胞。
本发明中干细胞胞外囊泡制剂制备的主要步骤如下：
(1)收集有效条件处理的上述GMP规模化生产的人源脂肪间充质干细胞的培养上清液，差速离心和过滤去除细胞碎片而获得含胞外囊泡的条件培养基；
(2)在本步骤中，用多聚物沉淀法进行分离，优选PEG9000；用PBS配置20％g/ml的PEG9000母液，用0.22μm的过滤器过滤后，按照1:1体积比加入到上一步骤经处理的条件培养基中，置于4℃过夜孵育后，再经4℃，4000g离心50min，弃上清液并保留沉淀物；
(3)加入预冷的PBS重悬沉淀，4℃，120000g超高速离心1h，去上清液，加入5ml医用载体溶液重悬沉淀，得到无PEG沉淀的样品,并以此作为试验组样品；
(4)试验组样品再经过粒径分析、形态的扫描电镜观察，以及CD9、CD63、CD81和TSG101等标志蛋白的免疫印迹鉴定，确认获得的人源脂肪间充质细干胞胞外囊泡(图1)；
(5)检测该囊泡中主要有效成分的关键活性物质，体外验证其药效潜能；
(6)采用医用载体溶液将上述所得干细胞胞外囊泡制剂稀释至适当的有效 浓度，从而获得干细胞胞外囊泡药物组合物。
同时设置对照组制备干细胞胞外囊泡制剂，制备方法如下：
(1)收集有效条件处理的上述GMP规模化生产的人源脂肪间充质干细胞的培养上清液，差速离心和过滤去除细胞碎片而获得含胞外囊泡的条件培养基；
(2)将上一步骤经处理的含胞外囊泡的条件培养基，4℃，120000g超高速离心1h，去上清液，加入5ml医用载体溶液重悬沉淀，获得对照组样品。
结果显示，与对照组相比，本发明的方法可获得更多的粒子分散均匀且透明的干细胞胞外囊泡制剂，且该药物制剂中富含与抗炎和组织损伤修复相关的生物活性因子，例如MMP-1、HGF(表2)。
表2.干细胞胞外囊泡药物制剂中富含与抗炎和组织损伤修复相关的生物活性因子
Figure PCTCN2022088118-appb-000002
其中，MMP-1(具有抗纤维化作用)的含量比对照组高出至少200倍，且HGF(具有一定的抗炎作用)的含量高出约2500倍。此外，VEGF等因子也有相应幅度的增加。这表明本发明制备的干细胞胞外囊泡制剂具有优异的抗炎症和抗纤维化性能。
本发明制备的干细胞胞外囊泡药物组合物(500mL)中含有1.7×10 11粒子数量的胞外囊泡。胞外囊泡粒径为30-200nm，较佳地50-150nm，更佳地80-120nm，更有利于给药后的治疗效果。
另外，试验组中干细胞胞外囊泡药物组合物未检出PEG成分(低于检测下限)。
实施例2
干细胞胞外囊泡的药物组合物的体外细胞摄取
为了检测干细胞胞外囊泡的药物组合物主要活性成分被细胞摄取情况，首先采用亲脂性荧光染料PKH67标记人脂肪间充质干细胞胞外囊泡的药物组合物活性成分，并分别添加至Ⅱ型肺泡上皮细胞A549和LPS刺激的RAW264.7巨噬细胞中，分别培养24h和48h后，倒置荧光显微镜下观察该组合物中活性成分被细胞摄取的情况，具体实施的主要步骤如下：
(1)采用亲脂性荧光染料PKH67标记本发明优选生产出来的人脂肪间充质干细胞囊泡外囊泡的药物组合物中的主要活性成分(2×10 7胞外囊泡)；
(2)将Ⅱ型肺泡上皮细胞A549和RAW264.7巨噬细胞分别按1×10 4细胞/cm 2和5×10 4细胞/cm 2密度接种于12孔培养板中，在细胞培养箱内(37℃,5％CO 2)分别培养细胞24h；
(3)将标记好的上述药物组合物加入Ⅱ型肺泡上皮细胞A549培养板中，培养24h，倒置荧光显微镜下观察该药物组合物中活性成分被细胞摄取的情况；
(4)RAW264.7巨噬细胞经10ng/ml LPS刺激后再加入标记好的上述药物组合物，培养48h，取出培养皿在倒置荧光显微镜下观察该药物组合物中活性成分被细胞摄取的情况。
结果显示，人脂肪间充质干细胞胞外囊泡的药物组合物活性成分能够被Ⅱ型肺泡上皮细胞A549吞入并显著地积累于细胞内；同时，该干细胞胞外囊泡的药物组合物中活性成分也能够被活化的巨噬细胞所摄取(图2)。
实施例3
雾化吸入荧光标记的EV在小鼠体内生物分布研究
在本实施例中，研究人源脂肪间充质干细胞胞外囊泡经雾化吸入后，在小鼠肺组织中的分布情况及其持续时间。采用荧光标记胞外囊泡示踪法，对其进行探究。
使用DiR标记的人源脂肪间充质干细胞胞外囊泡(3×10 9干细胞胞外囊泡/g)，经雾化处理30分钟并吸入小鼠(N＝3)肺部后，分别选取雾化吸入后的1h、4h、24h、48h和72h时间点，采用活体荧光成像技术对DiR标记的胞外囊泡在小鼠肺组织中的分布情况进行观察，并分析其在小鼠肺部的滞留时间。
如图3A所示，DiR标记的人源脂肪间充质干细胞胞外囊泡经雾化针于小鼠气管内雾化给药吸入后的不同时间点，从不同角度观测其荧光信号在小鼠体内的分布情况。结果发现(图3A)，除了气管给药造成反流食道的荧光信号外(图3A腹部)，该小鼠体内的荧光信号主要集中在小鼠肺组织中(图3A背部、右侧及左侧)，尤其是在给药后的4h，小鼠左侧肺组织中荧光信号最高，而右侧肺较弱，表明此时左肺叶聚集了大量的胞外囊泡；随后，左肺叶中的荧光信号急剧降低，而右侧肺叶则逐渐减弱，表明胞外囊泡在肺组织中逐渐被消耗而减少。由于体内观察无法精确观测到组织中的荧光信号，取其各类组织进行离体荧光 成像观察。如图3B所示，与对照组相比，在胞外囊泡气管内雾化给药后的48h和72h后，心、肝、脾、肾和气管中均未发现任何荧光信号，而仅在肺组织出现荧光信号，且48h的荧光信号较强，而72h的则较弱。
综上所述，该结果表明胞外囊泡经气管内雾化给药后，其主要分布于肺组织中，而其它组织中几乎未见分布。
实施例4
雾化吸入EV治疗感染性肺损伤动物模型有效性研究
在本实施例中，研究人源脂肪间充质干细胞胞外囊泡经雾化吸入后，治疗耐多药铜绿菌(PA)诱导的小鼠肺炎模型的有效性。分别设置PBS诱导小鼠的空白对照组，PA诱导小鼠肺炎组，人源脂肪间充质干细胞胞外囊泡雾化吸入治疗PA诱导小鼠肺炎组，以及小鼠成纤维细胞胞外囊泡治疗小鼠肺炎组。每组均为6只8-10周龄的C57BL/6野生型雄性小鼠，且治疗组中的每只小鼠胞外囊泡用量均按1×10 6干细胞胞外囊泡/g计算。耐多药铜绿菌诱导4小时后，进行胞外囊泡雾化吸入，每日雾化1次，持续5天。之后，收集肺泡灌洗液，用于中性粒细胞计数，以及相关炎症因子水平测定。另外，对肺组织进行HE染色的形态学观察及其肺损伤评估。
结果如图4所示，肺组织HE染色的形态学观察表明，与对照组相比，经人源脂肪间充质干细胞胞外囊泡雾化治疗的PA所致肺炎小鼠的肺损伤明显好转(图4A)，且肺组织评分结果也显示，该小鼠肺损伤评分显著降低，而小鼠成纤维细胞胞外囊泡治疗后的小鼠肺损伤评分未见显著降低(图4B)。此外，对小鼠肺泡灌洗液中总白细胞和中性粒细胞计数结果揭示，与对照组相比，经人源脂肪间充质干细胞胞外囊泡雾化治疗后的小鼠肺泡灌洗液中的总白细胞和中性粒细胞的数量均显著降低(图4C,D)；其肺泡灌洗液中的促炎症细胞因子TNF-α和IL-6水平也显著降低(图4E,F)，而抑炎症细胞因子IL-10的水平显著升高(图4G)。
综上所述，这些结果表明人源脂肪间充质干细胞胞外囊泡雾化治疗能够显著抑制PA诱导的小鼠肺损伤，降低其炎症反应水平，从而有效治疗该模型肺炎小鼠。
实施例5
雾化吸入EV治疗新冠病毒肺炎急性肺损伤临床试验
在本实施例中，初步探索异体脂肪间充质干细胞胞外囊泡经雾化吸入治疗新型冠状病毒肺炎(COVID-19)重型和危重型患者的安全性和有效性。
拟定纳入30例重型和危重型NCP受试者，分为低、中、高三个剂量组爬坡。所有受试者除ICU常规治疗外，联合给予雾化吸入异体脂肪MSCs-Exo治疗，每次2-8×10 8干细胞胞外囊泡/3ml，每日1次，连续5天。记录并分析28天内的不良反应发生率和临床改善时间，以及患者脱机例数，ICU护理天数，幸存者机械通气持续时间和死亡率等。此外，通过每日SOFA评分，相关生化指标的检测和肺部影像学观察，以及患者呼吸道标本中新冠病毒转阴时间，对患者经治疗后的症状改善进行评判。本研究入组标准主要包括1)年龄18-75岁，男女不限，且本人或家属自愿加入并签署知情同意书；2)RT-PCR检测呈阳性或明确诊断为新型冠状病毒肺炎患者；3)符合重型和危重型患者的诊断标准。排除标准主要包括1)相关病毒携带者或较严重过敏体质患者，其他病毒引起的肺炎患者；2)肺癌或长期使用免疫抑制药物的患者；3)进行血液透析或腹膜透析的患者，以及肝功能异常患者；4)正在使用ECMO或高频振荡通气的患者；5)孕期、哺乳期或半年内计划孕育的患者；6)研究者评判不能参与研究的或未能理解和执行该方案的患者。
在已经完成的人源脂肪间充质干细胞胞外囊泡(haMSC-Exos)经雾化吸入治疗重症新型冠状病毒肺炎(COVID-19)的探索性临床研究中，除了验证在重症呼吸系统疾病患者中的安全性之外，还观察到hMSC-Exos雾化治疗后，新冠患者胸部影像学有显著改善(图5A,B)，受试者肺部渗出较前改善。同时，出现呼吸支持方式的降级。此外，有2例在首次治疗后分别于第4-5天脱离呼吸机吸氧治疗。首次治疗后7-14天内，所有受试者临床症状均改善，临床症状改善率为100％。达到出院标准，并办理出院。首次治疗后14-28天内，有6例受试者临床症状持续改善，改善率为85.7％。
综上所述，该临床试验结果表明人源脂肪间充质干细胞胞外囊泡雾化可在一定程度上辅助治疗新冠病毒诱发的急性肺损伤，从而改善重症患者的肺炎症状，且具安全性。
实施例6
人源脂肪间充质干细胞胞外囊泡中富含抗炎相关的miRNAs
在本实施例中，研究了三批次生产的人源脂肪间充质干细胞胞外囊泡(haMSC-Exos)中microRNAs(miRNAs)表达情况。分别选取GMP条件下生产的三批次人源脂肪间充质干细胞胞外囊泡产品进行总的microRNA提取。采用Illumina TruSeq Small RNA试剂盒构建文库，包括分别连接3’端接头和5’端接头，随机引物反转成第一条链cDNA，然后进行文库富集及胶纯化等步骤，对文库定量后按比例上机进样检测，采用Illumina平台进行测序。最后，将质控后的数据，即clean data(reads)与人类基因组数据进行比对，对各样本中已知miRNAs进行表达量统计，并利用TPM(Transcripts Per Million)进行表达量的均一化处理。
结果发现，本次测序共鉴定到400个已知miRNAs；其中，表达量前20的miRNAs统计及其相对表达水平如图6所示。在这20个已知的miRNAs中，具备抗炎和免疫调节功能的miRNAs包括hsa-let-7b-5p、hsa-let-7a-5p、hsa-miR-126-5p、hsa-miR-151a-3p、hsa-miR-1246、hsa-miR-21-5p、hsa-miR-146a-5p和hsa-let-7b-3p等，故而能够对急性肺损伤或ARDS发挥抑制或削弱作用。生物信息学预测分析表明，这些miRNAs发挥抗炎作用的主要作用机制是依赖于靶向抑制TLR4/MyD88/NF-κB和/或JAK2/STAT3信号通路活化。
综上所述，这些结果表明人源脂肪间充质干细胞胞外囊泡可依赖于自身含有的miRNAs而产生主要生物学功能，进而在炎症性疾病或损伤中发挥药效作用。
实施例7
干细胞胞外囊泡的药物组合物经呼吸道雾化吸入可到达肺部气道炎症部位
为了验证干细胞胞外囊泡的药物组合物经呼吸道雾化吸入后，可以更有效地分布于肺部气道炎症部位，对其在肺组织中的生物分布进行了研究。
首先，建立以嗜酸性粒细胞和中性粒细胞混合为主要特征的哮喘小鼠模型(图7)。将7周龄的雌性野生型BALB/c小鼠置于SPF级动物房内的独立通风系统中适应性饲养一周。然后，对造模组(OVA+LPS)小鼠处理时，在当天和第7天采用10μg鸡卵清蛋白(OVA)和0.4mg氢氧化铝溶于200μl生理盐水的混合物，腹腔注射小鼠进行致敏性处理；随后，在第14、15、16和17天进行 激发处理，采用医用生理盐水溶解的6％OVA溶液雾化处理小鼠25min。
将试验设置两组，分别为医用生理盐水的对照(100μl)组和标记亲脂性荧光染料PKH67的人脂肪间充质干细胞胞外囊泡(100μl,1×10 7胞外囊泡)经呼吸道雾化吸入治疗组，且每组各3只小鼠。两组小鼠经麻醉处理后，分别在第13和14天时雾化吸入处理。然后，选择给药后的0.5h、1h、3h、24h和48h对小鼠进行肺组织离体的IVIS荧光成像和荧光强度分析。之后，将肺组织迅速置于液氮中冷冻处理，经OCT包埋后，再次液氮冷冻处理；冰冻切片机制备10μm的冰冻切片，置于室温下晾干后，洗去其OCT包埋剂，进行DAPI染色细胞核，使用荧光淬灭剂对其封片；然后，采用荧光显微镜对其进行成像并分析PKH67标记的胞外囊泡在肺组织气道中定位情况。
结果表明，标记的胞外囊泡被定位于小鼠肺组织炎症部位的支气管及血管周围。
实施例8
干细胞胞外囊泡的药物组合物制剂可降低哮喘小鼠气道高反应性
气道高反应性的程度主要取决于气道炎症水平和个体免疫反应。它是儿童和成人哮喘表型中是否存在粒细胞炎症的一个重要特征。为了探究干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入可改善粒细胞介导的哮喘，采用实施例7中的哮喘模型对其进行治疗研究(图8)。具体实施方法如下：
将7周龄的雌性野生型BALB/c小鼠置于SPF级动物房内的独立通风系统中适应性饲养一周后，随机分成6组，每组10只小鼠。对生理盐水组(saline)和造模组(OVA+LPS)小鼠处理时，在当天和第七天分别采用医用生理盐水或10μg鸡卵清蛋白(OVA)和0.4mg氢氧化铝溶于200μl生理盐水的混合物，腹腔注射小鼠进行致敏性处理；随后，在第14、15、16和17天进行激发处理，分别采用医用生理盐水及其溶解的6％OVA溶液雾化处理小鼠25min，在第15天仅给一次1μg LPS气管内给药处理。其中，药物治疗组小鼠在第13和14天采用医用生理盐水、地塞米松(1mg/kg)、脂肪间充质干细胞(1.5×10 5细胞/次/小鼠)及其囊泡药物组合物制剂(低剂量组1×10 5胞外囊泡/小鼠、高剂量组2×10 6胞外囊泡/小鼠)分别经呼吸道给药治疗小鼠。在最后一次激发处理24h后(第18天)，对所有小鼠进行取材试验。
为了检测各组小鼠气道高反应性，将小鼠安置在无创检测肺功能仪上适应 10min后，在其稳定呼吸的条件下，记录未经乙酰甲胆碱(methacholine,Mch)处理的小鼠5min气道阻力值(RI值)。然后，不断增加雾化吸入的乙酰甲胆碱浓度(5、10、25和50mg/ml)，分别检测其相应浓度下的RI值。最后，绘制不同Mch剂量下RI值的百分比曲线。
结果表明，与地塞米松和脂肪间充质干细胞治疗相比，脂肪间充质干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入可降低混合型粒细胞介导的哮喘小鼠气道高反应性。
实施例9
干细胞胞外囊泡的药物组合物制剂可改善哮喘小鼠肺组织病理学特征
为了探究干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入治疗后的哮喘小鼠肺组织病理特征情况，采用实施例7中的哮喘动物模型进行研究，具体操作如下：
(1)采用肺组织干重与湿重比值评估小鼠肺水肿情况。将取完肺泡灌洗液(BALF)的全肺组织，置于纱布上，吸干其表面水分，称量并记录其湿重；然后置于80℃恒温箱内处理48h，再次称量，记录肺干重。与地塞米松和脂肪间充质干细胞治疗相比，脂肪间充质干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入可改善混合型粒细胞介导的哮喘小鼠肺水肿。
(2)吸取5ml PBS经右心室注射，对小鼠肺组织进行灌流，同时采用10％中性甲醛溶液进行固定，用于石蜡包埋和4μm切片制备。然后，将切片进行H&E染色，40×物镜下随机取视野进行拍照。根据H&E染色的支气管周围和血管周围炎症细胞浸润情况进行盲审评分：无细胞浸润，记为0；只有一些细胞，记为1，浸润细胞形成环状且为1个细胞的宽度，记为2；浸润细胞形成环状且为2～4个细胞的宽度，记为3；浸润细胞形成环状且为多于4个细胞的宽度，记为4。预期结果是，与地塞米松和脂肪间充质干细胞治疗相比，脂肪间充质干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入可降低混合型粒细胞介导的哮喘小鼠肺支气管和血管周围炎症细胞浸润水平。
(3)将切片进行PAS染色，40×物镜下随机取视野进行拍照。根据染色显示分泌粘液的杯状细胞(goblet cell)数量进行盲审评分，以评估支气管中粘液分泌情况。无杯状细胞，记为0；少于25％杯状细胞，记为1，25％～50％杯状细胞，记为2；50％～75％杯状细胞，记为3；多于75％杯状细胞，记为4。
结果表明，与地塞米松和脂肪间充质干细胞治疗相比，脂肪间充质干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入可显著降低混合型粒细胞介导的哮喘小鼠肺支气管杯状细胞粘液分泌水平。
实施例10
干细胞胞外囊泡的药物组合物制剂可降低哮喘小鼠炎症因子水平
为了探究干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入治疗后的哮喘小鼠炎症因子水平，采用实施例7中的哮喘动物模型对其进行检测，具体操作如下：
(1)收集终点小鼠心脏血，室温下凝固30min后，室温下离心(1800g,10min)，获取血清，冻存置-80℃冰箱，备用于相关细胞因子(IL-4、IL-5、IL-6、IL-13和IL-17A)及IgE的ELISA检测。与地塞米松和脂肪间充质干细胞治疗相比，脂肪间充质干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入可降低混合型粒细胞介导的哮喘小鼠血清中IL-4、IL-5、IL-6、IL-13、IL-17A和IgE水平。
(2)收集终点小鼠肺泡灌洗液(BALF)：切开暴露并充分游离小鼠气管，置入20G的灌洗针头，接1ml装有无菌生理盐水的针筒，反复灌洗3次后尽可能收集灌洗液，然后离心(4℃,500g,20min)，取上清液。采用500μl红细胞裂解液重悬沉淀，以裂解残留的红细胞，离心(4℃,600g,5min)后，再用500μl无菌PBS重悬沉淀。
(3)BALF上清液用于ELISA检测细胞因子(IL-4、IL-5、IL-6、IL-13和IL-17A)水平。与地塞米松和干细胞治疗相比，干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入可降低混合型粒细胞介导的哮喘小鼠BALF中IL-4、IL-5、IL-6、IL-13和IL-17A水平。
(4)将肺泡灌洗液的沉淀重悬液用于血细胞计数器检测总的细胞数，以及嗜酸性粒细胞(eosinophil)、中性粒细胞(neutrophil)、巨噬细胞(macrophage)及淋巴细胞(lymphocyte)的数量。
结果表明，与地塞米松和脂肪间充质干细胞治疗相比，脂肪间充质干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入可显著降低混合型粒细胞介导的哮喘小鼠BALF中炎症细胞流入水平。
实施例11
干细胞胞外囊泡的药物组合物制剂可改善COPD小鼠肺功能
为了探究干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入治疗后的COPD小鼠肺功能，首先建立了尼古丁诱导的COPD小鼠疾病模型，然后对其进行治疗，检测其肺功能相关指标(图9)，主要操作如下：
(1)将7周龄的雌性野生型BALB/c小鼠置于SPF级动物房内的独立通风系统中适应性饲养一周。
(2)将BALB/c小鼠置于雾化箱内，经口鼻吸入3mg尼古丁燃烧产生的烟雾，每两支香烟中间间隔30min，共暴露4周。
(3)然后将其随机分为对照组与试验组，每组8只。将生理盐水经气道雾化吸入对照组小鼠，而试验组小鼠每天接受人脂肪间充质干细胞胞外囊泡的药物组合物制剂(1×10 6particles/mouse)的雾化吸入治疗，每天雾化吸入一次，连续治疗5天。
(4)治疗5天完成后，对小鼠进行肺功能指标进行检测，包括100ms内最大呼气量(FEV 100)、动脉血氧分压(PaO 2)、动脉血二氧化碳分压(PaCO 2)和氧饱和度(SaO 2)。
结果表明，与对照组小鼠的治疗相比，人脂肪间充质干细胞胞外囊泡的药物组合物制剂经呼吸道雾化吸入可显著改善COPD模型小鼠100ms内的最大呼气量，提高动脉血氧分压和氧饱和度，降低动脉血二氧化碳分压，从而改善其肺功能。
讨论
大量研究表明，间充质干细胞具有促进组织修复和抗炎作用，其大部分功能是通过分泌的可溶性因子或囊泡，与靶细胞发生相互作用而产生的。间充质干细胞囊泡在诸多疾病的治疗中发挥重要作用，在预防和治疗呼吸道炎症性疾病中同样具有巨大的应用潜力。干细胞囊泡是干细胞与靶细胞之间进行信号交流的重要信息载体。一旦被分泌到细胞外空间，干细胞囊泡就可以通过与靶细胞的相互作用而被吞入靶细胞，并最终影响靶细胞的表型，从而发挥其特有的生物学功能。
因此，在本发明中，优选GMP条件下生产的人脂肪间充质干细胞作为生产干细胞囊泡的亲本细胞，并进一步生产获得该囊泡的药物组合物制剂，通过气 道雾化吸入的给药途径，用于治疗急性呼吸道炎症以及支气管哮喘和慢性阻塞性肺疾病等慢性呼吸道炎症，以期为各类诱因而导致的呼吸道炎症提供新的治疗方法。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种干细胞胞外囊泡制剂，其特征在于，所述胞外囊泡的粒径为30-200nm，较佳地50-150nm，更佳地80-120nm；并且，所述胞外囊泡中含有与抗炎或组织损伤修复相关的生物活性因子，所述生物活性因子包括基质蛋白金属酶1(MMP-1)和肝细胞生长因子(HGF)，其中MMP-1在所述制剂中的含量为1500-2000pg/ml，HGF在所述制剂中的含量为5500-6000pg/ml。
  2. 如权利要求1所述的制剂，其特征在于，所述干细胞胞外囊泡来源于人体干细胞在体外培养过程中所收集的上清液。
  3. 如权利要求1所述的制剂，其特征在于，所述干细胞选自下组：人脐带血来源的干细胞、人外周血来源的干细胞、人脐带间充质干细胞、人胎盘间充质干细胞、人脂肪间充质干细胞、人骨髓来源的干细胞、或其组合。
  4. 如权利要求1所述的制剂，其特征在于，所述干细胞胞外囊泡具有以下特征：采用聚乙二醇(PEG)沉淀法制备得到，胞外囊泡粒径集中分布在80-120nm之间，粒径更小且均一。
  5. 如权利要求4所述的制剂，其特征在于，所述聚乙二醇(PEG)沉淀包括步骤：使用PEG3000～9000的PEG，用PBS配置为8％-20％的PEG母液，经过滤除菌(如用0.22μm的过滤器过滤)后，按照一定比例(如约1:1体积比)加入到处理后的条件培养基中(所述条件培养基经差速离心和过滤处理人源脂肪间充质干细胞的培养上清液获得)，置于4℃过夜孵育；然后4℃，3,000-5000g离心30-60分钟，弃去上清，加入预冷的PBS重悬沉淀；4℃，100000-120000g超高速离心60-120分钟，弃去上清，从而得到所述干细胞胞外囊泡。
  6. 一种药物组合物，其特征在于，所述药物组合物含有：(a)如权利要求1所述的干细胞胞外囊泡制剂，和(b)药学上可接受的载体。
  7. 如权利要求6所述的药物组合物，其特征在于，所述的药物组合物的剂型选自下组：雾化吸入剂、滴眼液、滴鼻液、注射液。
  8. 一种含干细胞胞外囊泡的药物组合物的制备方法，所述方法包括步骤：
    (S1)培养人的干细胞(如脂肪间充质干(祖)细胞)，使其达到预定的汇合度(如75-90％)；
    (S2)在适合产生EV的条件下，继续培养所述的细胞一段时间T1；其中，所述的T1通常为24-72小时，较佳地30-60小时；
    (S3)从培养体系中去除细胞，从而分离获得含干细胞胞外囊泡的培养液，即 为“条件培养液(conditioned medium)”；
    (S4)将所述的条件培养液(conditioned medium)与聚乙二醇(PEG)进行混合，形成第一混合物，并放置一段时间T2，从而形成经PEG修饰的干细胞胞外囊泡；其中，所述的T2通常为6-60小时，较佳地12-48小时；
    (S5)对上一步骤的第一混合物进行离心，使得所述的经PEG修饰的干细胞胞外囊泡沉淀，并弃去上清，获得经PEG修饰的干细胞胞外囊泡沉淀；
    (S6)对上一步骤获得的经PEG修饰的干细胞胞外囊泡沉淀进行重悬，从而获得第一重悬混合物；
    (S7)对第一重悬混合物进行离心，使所述的经PEG修饰的干细胞胞外囊泡沉淀，并弃去上清，获得经PEG修饰的干细胞胞外囊泡沉淀；
    (S8)对上一步骤中获得的经PEG修饰的干细胞胞外囊泡沉淀进行重悬，从而获得可医用的干细胞胞外囊泡制剂；
    并且，所述方法还包括：
    (S9)将可医用的干细胞胞外囊泡制剂(或活性物质)与药学上可接受的载体混合，从而制得药物组合物。
  9. 一种如权利要求1-5任一项所述的干细胞胞外囊泡制剂或如权利要求6所述的药物组合物的用途，用于制备治疗呼吸道炎症的药物或制剂。
  10. 如权利要求9所述的用途，其特征在于，所述呼吸道炎症包括急性呼吸道炎症和慢性呼吸道炎症。
PCT/CN2022/088118 2021-04-21 2022-04-21 含有干细胞胞外囊泡的药物组合物及其在呼吸道炎症治疗中的应用 WO2022222987A1 (zh)

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