WO2021180237A1 - 含人体细胞衍生的细胞膜外囊泡的雾化吸入制剂、制法及其应用 - Google Patents

含人体细胞衍生的细胞膜外囊泡的雾化吸入制剂、制法及其应用 Download PDF

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WO2021180237A1
WO2021180237A1 PCT/CN2021/080869 CN2021080869W WO2021180237A1 WO 2021180237 A1 WO2021180237 A1 WO 2021180237A1 CN 2021080869 W CN2021080869 W CN 2021080869W WO 2021180237 A1 WO2021180237 A1 WO 2021180237A1
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cells
extracellular vesicles
pharmaceutical composition
peg
human
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French (fr)
Chinese (zh)
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戴成祥
李萍
王静
李苏克
雷继刚
陈应炉
宋晓乐
刘必佐
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Cellular Biomedicine Group Wuxi Ltd
Cellular Biomedicine Group Shanghai Ltd
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Cellular Biomedicine Group Wuxi Ltd
Cellular Biomedicine Group Shanghai Ltd
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Priority to EP21768139.4A priority Critical patent/EP4134105A4/en
Priority to US17/910,444 priority patent/US20230120324A1/en
Priority to JP2022554328A priority patent/JP2023516458A/ja
Priority to AU2021234601A priority patent/AU2021234601A1/en
Publication of WO2021180237A1 publication Critical patent/WO2021180237A1/zh
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    • 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
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A61P39/00—General protective or antinoxious agents
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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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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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present invention relates to the field of biomedicine, in particular to an atomized inhalation preparation containing extracellular vesicles derived from human cells, a preparation method and application thereof.
  • ARDS Acute respiratory distress syndrome
  • ARDS is most common in pneumonia, sepsis, aspiration of gastric contents, or severe trauma, accounting for about 10% of patients in intensive care units worldwide. Although some progress has been made in the past few decades, the mortality rate is still as high as 30-40% in most studies. In the United States, there are about 200,000 ARDS cases each year, and the hospital mortality rate is as high as 38.5%, and in the past few decades, this situation has not improved significantly.
  • Acute lung injury (ALI) and ARDS have the same pathological changes, the most common is diffuse alveolar injury.
  • the pathological basis of ALI/ARDS is the injury of alveolar epithelium and alveolar capillary endothelium caused by a variety of inflammatory cells (macrophages, neutrophils, lymphocytes, etc.) mediated local lung inflammation and uncontrolled inflammation.
  • the main pathological feature is the formation of protein-rich pulmonary edema and hyaline membrane in alveolar exudate caused by increased pulmonary microvascular permeability, which may be accompanied by pulmonary interstitial fibrosis.
  • the pathophysiological changes are dominated by decreased lung compliance, increased intrapulmonary shunt, and unbalanced ventilation blood flow ratio.
  • the clinical manifestations are respiratory frequency, respiratory distress, and refractory hypoxemia. Chest X-ray shows diffuse infiltrates of both lungs, and multiple organ failure is often complicated in the later stage.
  • ARDS ARDS based on clinical diagnostic criteria agreed by experts.
  • the focus of patient management is to implement lung protection ventilation strategies. No specific drug therapy has yet been determined.
  • the long-term prognosis of patients surviving ARDS is increasingly regarded as an important research goal, because many ARDS patients survive with sequelae such as organ function and/or cognitive psychology.
  • Future research directions include promoting the early recognition of ARDS, adding prognostic and/or predictive functions in clinical research to identify subgroups that may have therapeutic effects, and continuing efforts to understand the basic mechanisms of lung injury.
  • SARS-CoV-2 coronavirus
  • ARDS acute respiratory distress syndrome
  • SARS-CoV-2 and other coronaviruses there are currently no specific vaccines and antiviral treatments. These infectious diseases have seriously affected human life and health, and the development of effective therapeutic drugs is imminent. It is of great social significance to develop low-toxic and high-efficiency drugs for diseases such as SARS-CoV-2 and other coronaviruses such as pneumonia and ARDS to meet the clinical needs of patients at home and abroad.
  • the purpose of the present invention is to provide a high-efficiency, low-toxicity, safe and mass-produced medicine for treating diseases such as ARDS.
  • a pharmaceutical composition in the first aspect of the present invention, contains (a) an active substance derived from human somatic cells, and the active substance is an extracellular capsule produced by human somatic cells Vesicles; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a cell-free pharmaceutical composition.
  • the "cell-free" means that the pharmaceutical composition does not contain live cells and dead cells.
  • the somatic cells are selected from the group consisting of stem cells, progenitor cells, immune cells, or combinations thereof derived from human tissue, bone marrow and/or blood.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of a liquid dosage form and a solid dosage form (such as a lyophilized dosage form).
  • the dosage form of the pharmaceutical composition is selected from the group consisting of aerosol inhalation, eye drops, and nasal drops.
  • the pharmaceutical composition has the following characteristics:
  • (P1) Better storage stability than living cell preparations; preferably, the storage includes storage at room temperature and/or low temperature (such as -196°C to 25°C, preferably -100°C to 0°C);
  • the "storage stability better than living cell preparations” means that the stability of the pharmaceutical composition is better than the stability of living cell preparations under the same conditions;
  • (P2) High dispersibility; preferably, when the pharmaceutical composition is an aqueous liquid preparation (such as a solution prepared with physiological saline), it is placed at 0-25°C for 6-24 hours, and it appears colorless and transparent. And there is no visible flocs and sediments.
  • an aqueous liquid preparation such as a solution prepared with physiological saline
  • the pharmaceutical composition is applied to a site other than the skin.
  • the pharmaceutical composition is applied to a site selected from the group consisting of eyeball conjunctiva, eyelid conjunctiva, retina, oral cavity, nasal cavity, upper respiratory tract, lower respiratory tract, gastrointestinal tract, lung, or combination.
  • the pharmaceutical composition is administered to cells selected from the group consisting of vascular endothelial cells, alveolar type 1 epithelial cells, alveolar type 2 epithelial cells, monocytes macrophages, neutrophils, Dendritic cells, antigen presenting cells, T lymphocytes, fibroblasts, central nerve cells, peripheral nerve cells and peripheral nerve fibers, or combinations thereof.
  • the stem (progenitor) cells are selected from the group consisting of mesenchymal stem cells derived from human adipose tissue, alveolar epithelial progenitor cells derived from human lung tissue, and mesenchymal stem cells derived from human umbilical cord Wharton’s glue.
  • Stem cells mesenchymal stem cells derived from human endometrial tissue, stem cells derived from human placental tissue, epithelial cells derived from human placental amniotic membrane, mononuclear cells in human blood, hematopoietic stem cells in human bone marrow tissue, and human bone marrow tissue
  • Mesenchymal stem cells derived from human periosteum keratinocytes derived from human skin tissue, dendritic cells derived from human blood tissue, CD4-positive T lymphocytes derived from human blood tissue, and platelets derived from human blood tissue , Or a combination thereof.
  • the extracellular vesicles include nanovesicles surrounded by a lipid bilayer membrane structure.
  • the diameter of the nanovesicles is between 30-1000 nanometers.
  • the nanovesicles contain different types of microribonucleic acid (miRNA), small ribonucleic acid (sRNA), non-coding DNA fragments, and transfer ribonucleic acid (t-RNA) with specific functions. ), soluble cytokines, growth factors, and other proteins.
  • miRNA microribonucleic acid
  • sRNA small ribonucleic acid
  • t-RNA transfer ribonucleic acid
  • soluble cytokines soluble cytokines
  • growth factors and other proteins.
  • the soluble active cytokine is selected from the following group: soluble free lipoprotein molecules, glycoprotein molecules, biosignal molecules, tissue growth factor ⁇ (TGF ⁇ ), hepatocyte growth factor (HGF) , Vascular Endothelial Cell Growth Factor (VEGF), Epidermal Growth Factor (KGF), Interleukin-10 (IL-10), Interleukin-1 ⁇ Receptor Antagonist (IL-1 ⁇ A), or a combination thereof.
  • TGF ⁇ tissue growth factor ⁇
  • HGF hepatocyte growth factor
  • VEGF Vascular Endothelial Cell Growth Factor
  • KGF Epidermal Growth Factor
  • IL-10 Interleukin-10
  • IL-1 ⁇ A Interleukin-1 ⁇ Receptor Antagonist
  • the soluble active cytokine is a soluble active cytokine produced by the human somatic cells.
  • the soluble active cytokine includes the active cytokine produced by paracrine of human somatic cells cultured in the natural state, and the active cytokine produced by the expression of human somatic cells modified with exogenous genes. , Or a combination thereof.
  • the extracellular vesicles specifically express the following proteins: CD9, CD63, CD81 and TSG101; they do not express or substantially do not express the protein CANX.
  • the diameter of the extracellular vesicles is preferably 50-500 nm.
  • the diameter is an average diameter.
  • the cells used to produce the extracellular vesicles include cells from the following sources:
  • the cells used to produce the extracellular vesicles include primary cells and passage cells with 1-10 passages.
  • the cells used to produce the extracellular vesicles include cells that have not been genetically manipulated, and cells that have been genetically manipulated.
  • the gene manipulation includes gene editing, gene introduction, gene knock-down, gene knock-out, or a combination thereof.
  • the cells used to produce the extracellular vesicles are pretreated cells.
  • the somatic cells are adipose-derived mesenchymal stem (progenitor) cells, placental amniotic membrane-derived mesenchymal stem cells, or a combination thereof.
  • the fat-derived mesenchymal stem (progenitor) cells are obtained through the following methods: for those that have undergone ethical review and record, signed informed consent, and undergo strict laboratory inspections to meet the requirements of fatty stem ( Progenitor) cell products are built into the standard male or female healthy volunteers, after abdominal wall skin puncture liposuction, or abdominal wall surgery to cut fat, stored in the cell preservation solution, transported to a qualified GMP laboratory at low temperature, and separated , Purification, proliferation, and the obtained adipose-derived mesenchymal progenitor cells, that is, working bank cells (intermediate products).
  • the mesenchymal stem (progenitor) cells derived from human adipose tissue are produced in a GMP production laboratory in accordance with the intermediate product production process, and meet the negative standards of various pathogenic tests.
  • Specific surface markers such as CD73-positive, CD90-positive, CD105-positive cell percentage is about 98%, and the percentage of CD34-positive/CD45-positive and HLA-DR-positive cells is less than 2%, which meets the standards for antibiotic residue and serum and serum substitute residue detection standards , P1-P6 generation adipose mesenchymal stem (progenitor) cells.
  • the adipose-derived mesenchymal stem (progenitor) cells are P3-P4 generation adipose-derived mesenchymal stem (progenitor) cells.
  • the somatic cells are stored at -196°C to -80°C (preferably, -196°C to -135°C) for 0-36 months (preferably 0-135°C).
  • Mesenchymal stem (progenitor) cells derived from adipose tissue at 24 months or 0.5-24 months).
  • the production process of the extracellular vesicles and soluble active cytokines derived from human somatic cells includes the steps:
  • the culture conditions are 37 ⁇ 1°C, 5 ⁇ 0.5% CO 2 , to the degree of cell fusion Reach 80 ⁇ 10%, and then use a specific pretreatment medium for culture, the culture condition is 37 ⁇ 1°C, 5 ⁇ 0.5% CO 2 , and culture for 36-72 hours;
  • the differential centrifugation combined with polyethylene glycol PEG precipitation includes the steps of: using PEG3000-PEG9000 PEG, configuring 8%-30% PEG with PBS, and sterilizing through filtration (such as After filtering with a 0.22 ⁇ m filter), add it to the treated conditioned medium according to a certain ratio (for example, about 1:1 volume ratio), and incubate overnight at 4°C. Centrifuge at 3,000-5000g for 30-60 minutes at 4°C, and discard the supernatant.
  • the extracellular vesicles obtained by the production process have the following biomarker characteristics: CD9 positive (ie CD9 + ), CD63 positive, CD81 positive, TSG101 positive, and CANX negative.
  • the production process has the following characteristics: it has a clinical grade cell product production scale under GMP laboratory conditions.
  • the clinical-grade production scale means that one production operation unit can separate 800-1200ml conditioned medium obtained from two cell factories, and 2-5 ⁇ 10 11 extracellular vesicles can be separated. Calculated based on the total dose of 2-5 ⁇ 10 9 extracellular vesicles used locally for a patient, one production operation unit and one batch production can meet the local consumption of 100-250 patients. Or, with 100 production operation units, a batch of production can reach a total of 10,000-25,000 people, and it will take 5-6 days.
  • the feature of the production process is: the separated extracellular vesicles, soluble active cytokines and PEG of a specific molecular weight are cross-linked to form hydrophilic particles with a specific particle size (for example, less than 3 microns). ,
  • a specific particle size for example, less than 3 microns.
  • the extracellular vesicles, soluble active cytokines, and polyethylene glycol PEG cross-linked hydrophilic particles are dispersed in isotonic sodium chloride solution, low molecular hyaluronic acid solution, artificial tears , Gel solution, so as to make atomized inhalation liquid, eye drops, nasal drops, and external gel preparations.
  • the pharmaceutical composition is applied to mucosal cells, and the mucosal cells can take up extracellular vesicles.
  • the pharmaceutical composition of the present invention is an aerosol inhaler.
  • the pharmaceutical composition of the present invention (especially aerosol inhaler) is used to treat infectious lung injury, preferably for viral lung injury, and more preferably for coronavirus (such as SARS).
  • coronavirus such as SARS
  • the pharmaceutical composition is an aerosol prepared from adipose tissue-derived progenitor cells and placental amniotic membrane-derived mesenchymal stem cells derived extramembranous vesicles and soluble cytokines and isotonic sodium chloride solution Inhale liquid.
  • the pharmaceutical composition is used to treat diseases caused by viruses.
  • the virus is selected from the group consisting of influenza virus, SARS coronavirus, SARS coronavirus 2, MERS coronavirus infection.
  • the pharmaceutical composition (such as aerosolized inhalation liquid) is used for the treatment of viral acute lung injury, through the aerosolized inhalation treatment, so as to prevent the release of acute lung injury inflammatory factors and reduce the height of the alveolar.
  • the infiltration of protein liquid and the damage of alveolar epithelial cells significantly increase the rescue success rate of patients with acute lung injury, and improve the lung function and quality of life of surviving patients.
  • a method for preparing extracellular vesicles which includes the steps:
  • T1 is usually 24-72 hours, preferably 30-60 hours;
  • S4 Mix the conditioned medium and polyethylene glycol (PEG) to form a first mixture, and leave it for a period of T2 to form PEG-modified extracellular vesicles; wherein, The T2 mentioned is usually 6-60 hours, preferably 12-48 hours;
  • the method further includes:
  • the medicinal extracellular vesicle preparation (or active substance) is mixed with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition.
  • the method further includes preparing the pharmaceutical composition into an aerosol inhalation preparation, an injection, or a lyophilized preparation.
  • an extracellular vesicle preparation is provided, and the extracellular vesicle preparation is prepared by the method described in the second aspect of the present invention.
  • the extracellular vesicle preparation includes nanovesicles surrounded by a lipid bilayer membrane structure.
  • the diameter of the nanovesicles is between 30-1000 nanometers.
  • the nanovesicles contain different types of microribonucleic acid (miRNA), small ribonucleic acid (sRNA), non-coding DNA fragments, and transfer ribonucleic acid (t-RNA) with specific functions. ), soluble cytokines, growth factors, and other proteins.
  • miRNA microribonucleic acid
  • sRNA small ribonucleic acid
  • t-RNA transfer ribonucleic acid
  • soluble cytokines soluble cytokines
  • growth factors and other proteins.
  • the soluble active cytokine is selected from the following group: soluble free lipoprotein molecules, glycoprotein molecules, biosignal molecules, tissue growth factor ⁇ (TGF ⁇ ), hepatocyte growth factor (HGF) , Vascular endothelial cell growth factor (VEGF), epidermal cell growth factor (KGF), interleukin-10 (IL-10), interleukin-1 ⁇ receptor antagonist (IL-1 ⁇ A), or a combination thereof.
  • TGF ⁇ tissue growth factor ⁇
  • HGF hepatocyte growth factor
  • VEGF Vascular endothelial cell growth factor
  • KGF epidermal cell growth factor
  • IL-10 interleukin-10
  • IL-1 ⁇ A interleukin-1 ⁇ receptor antagonist
  • the soluble active cytokine is a soluble active cytokine produced by the human somatic cells.
  • the soluble active cytokine includes the active cytokine produced by paracrine of human somatic cells cultured in the natural state, and the active cytokine produced by the expression of human somatic cells modified with exogenous genes. , Or a combination thereof.
  • the extracellular vesicles specifically express the following proteins: CD9, CD63, CD81 and TSG101; they do not express or substantially do not express the protein CANX.
  • the fourth aspect of the present invention there is provided a use of the pharmaceutical composition according to the first aspect of the present invention or the extracellular vesicle preparation according to the third aspect of the present invention, which are used for the preparation of prevention and/or treatment of inflammation, Or injury drugs.
  • the inflammation is selected from the group consisting of viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, autoimmune inflammation, or a combination thereof;
  • the injury is selected from the following group: ischemic injury, hypoxic injury, chemical injury, physical injury, or a combination thereof.
  • the drug is used to treat diseases caused by viruses.
  • the virus is selected from the group consisting of influenza virus, SARS coronavirus, SARS coronavirus 2, MERS coronavirus infection.
  • a method for preventing and/or treating inflammation or injury including the steps of: administering the pharmaceutical composition according to the first aspect of the present invention or the third aspect to a subject in need The above-mentioned extracellular vesicle preparation.
  • the inflammation is selected from the group consisting of viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, autoimmune reactive inflammation, or a combination thereof.
  • the injury is selected from the group consisting of ischemic injury, hypoxic injury, chemical injury, physical injury, or a combination thereof.
  • the drug is used to treat acute respiratory distress syndrome.
  • the medicine is an atomized inhalation liquid.
  • the drug is used to treat viral acute lung injury.
  • the drug is treated by aerosol inhalation, so as to prevent the release of inflammatory factors in acute lung injury, reduce the infiltration of high-protein liquid in the alveoli and the damage of alveolar epithelial cells.
  • the subject is a human.
  • Figure 1 shows the production of extracellular vesicles collected by Western blotting and NTA detection by PEG precipitation and ultracentrifugation.
  • Figure (1A) M is Marker; 1 to 4 are respectively 8% PEG 6000, 12% PEG 6000, 16% PEG 6000, 20% PEG 6000 isolated CD63 expression level of extracellular vesicles (the same amount of protein Loading);
  • Figure (1D) NTA detects the size of the extracellular vesicles separated by PEG6000 and ultracentrifugation; *p ⁇ 0.05.
  • Figure 2 shows the uptake of extracellular vesicles by fibroblasts. Among them, blue: nucleus stained by Hochest; green: extracellular vesicles labeled with PKH-67; ruler: 100 ⁇ m.
  • Figure 3 shows the number of extracellular vesicles stored at 4°C, -20°C and -80°C for 9 weeks.
  • Figure 4 shows the expression of CD81, a specific marker of extracellular vesicles stored at 4°C, -20°C and -80°C for 9 weeks.
  • Figure 5 shows the state of extracellular vesicles under the transmission electron microscope after half an hour of re-fusion at room temperature.
  • Figure 5A shows the state of extracellular vesicles stored at -80°C for 1 week under transmission electron microscopy
  • Figure 5B shows the state of extracellular vesicles under transmission electron microscopy after refusion at room temperature for half an hour.
  • Figure 6 shows the particle size concentration and size of extracellular vesicles after half an hour of re-fusion at room temperature.
  • Figure 7 shows that extracellular vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage activation.
  • A Pro-inflammatory macrophage morphological observation
  • B Pro-inflammatory macrophage morphological statistical analysis.
  • Control is the control group
  • LPS is the macrophage treatment group with lipopolysaccharide alone
  • LPS+Dex is the lipopolysaccharide and dexamethasone treatment group
  • LPS+haMPCs-Exo is the lipopolysaccharide and adipose mesenchymal progenitor cell extramembrane vesicle treatment group
  • the red arrow indicates activated macrophages (pro-inflammatory macrophages); *P ⁇ 0.05, the scale is 50 ⁇ m.
  • Figure 8 shows that extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage pro-inflammatory factor gene expression.
  • (A), (B) and (C) respectively show real-time fluorescent quantitative PCR to detect the gene expression levels of macrophage inflammatory factors TNF- ⁇ , IL-1 ⁇ and IL-6.
  • Control is the control group
  • LPS is the macrophage treatment group with lipopolysaccharide alone
  • LPS+Dex is the lipopolysaccharide and dexamethasone treatment group
  • LPS+haMPCs-Exo is the lipopolysaccharide and adipose mesenchymal progenitor cell extramembrane vesicle treatment group ;***P ⁇ 0.001.
  • Figure 9 shows that extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage pro-inflammatory factor release.
  • LPS induces macrophages and is processed by human adipose-derived mesenchymal progenitor cells with extra-membrane vesicles.
  • B ELISA detects the levels of inflammatory factors IL-6 and TNF- ⁇ released by macrophages.
  • Control is the control group
  • LPS is the macrophage treatment group with lipopolysaccharide alone
  • LPS+Dex is the lipopolysaccharide and dexamethasone treatment group
  • LPS+haMPCs-Exo is the lipopolysaccharide and adipose mesenchymal progenitor cell extramembrane vesicle treatment group ; **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figure 10 is a schematic diagram of extracellular vesicles, soluble cytokines and PEG cross-linked to form hydrophilic particles.
  • FIG 11 shows a schematic process route (from step S4 to S8) of the present invention.
  • the biomedical preparation contains extracellular vesicles derived from human cells. Specific particle size and modified with specific PEG, so it can not only be stored stably for a long time, but also has excellent dispersibility in medical solvents (such as physiological saline, etc.). It is especially suitable for direct application to patients through aerosol inhalation.
  • the present invention has been completed on this basis.
  • Coronavirus belongs to the Nidovirales (Nidovirales) Coronaviridae (Coronaviridae), which is an enveloped positive-stranded RNA virus, and its subfamily includes four genera of ⁇ , ⁇ , ⁇ and ⁇ .
  • HCoV-229E and HCoV-NL63 belong to the ⁇ genus coronavirus
  • HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all ⁇ genus coronavirus Virus.
  • the new coronavirus (SARS-CoV-2) that broke out at the end of 2019 has about 80% similarity with SARS-CoV and 40% similarity with MERS-CoV, and it also belongs to the beta coronavirus.
  • EVs extracellular vesicles
  • exosomes lipid bilayers with diameters ranging from 30-2000nm.
  • microvesicles lipid bilayers with diameters ranging from 30-2000nm.
  • exosomes refers to a subclass of EVs with a diameter of 50-100 nm derived from endosomes. They are the main paracrine secretions of various cell types including mesenchymal stem cells (MSCs). component.
  • MSCs mesenchymal stem cells
  • MSCs exosomes are a type of MSCs-derived EVs with a diameter in the range of 50-100nm and a complete lipid bilayer membrane structure.
  • Exosomes are a kind of carriers that carry abundant goods, and their function is mainly through the continuous transfer of microribonucleic acid (miRNAs) and proteins.
  • miRNAs microribonucleic acid
  • proteins proteins that can change the various activities of target cells through different ways.
  • 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.
  • MSC exosomes contain some cytokines and growth factors, such as TGF ⁇ 1, interleukin-6 (IL-6), IL-10 and hepatocyte growth factor (HGF), etc. These factors have been confirmed to have Helps immune regulation.
  • cytokines and growth factors such as TGF ⁇ 1, interleukin-6 (IL-6), IL-10 and hepatocyte growth factor (HGF), etc. These factors have been confirmed to have Helps immune regulation.
  • VEGF vascular endothelial growth factor
  • EMMPRIN extracellular matrix metalloproteinase inducer
  • MMP-9 have been reported in MSC exosomes. These three proteins play an important role in stimulating angiogenesis. It may be the basis of the repair function of exosomes.
  • NIR near-infrared
  • EVs labeled with superparamagnetic iron oxide nanoparticles have high-resolution and sensitive magnetic resonance analysis capabilities, which provide a basis for accurate detection of deep organs.
  • DiI-labeled MSC exosomes can be shown to reach the brain, liver, lung and spleen.
  • DID-labeled EVs specifically accumulate in the kidneys of AKI mice, indicating that exosomes seem to be able to home to the injury site.
  • EVs Compared with intravenous injection, nasal administration can better accumulate brain exosomes at the injury site.
  • the biodistribution of EVs through systemic administration is a dynamic process: within about 30 minutes after administration, EVs rapidly distributes in the liver, spleen and lungs, and then is processed by the liver and kidneys to enter the elimination stage, from 1 to 1 after administration. Clear EVs within 6 hours.
  • exosomes play a key role in regulating tumor-specific T cell activation by carrying and presenting functional MHC peptide complexes.
  • Exosomes released from bone marrow-derived MSCs can effectively improve the chronic transplantation of mice by inhibiting the activation and infiltration of CD4-positive T cells, reducing the production of pro-inflammatory cytokines, promoting the production of IL-10-expressing Tregs, and inhibiting Th17 cells.
  • Host-resistant disease (cGVHD) Host-resistant disease
  • EVs derived from human pluripotent stromal cells can inhibit autoimmunity in type 1 diabetes (T1D) and experimental autoimmune uveoretinitis (EAU) models. EVs can inhibit the activation of antigen-presenting cells (APC), inhibit the development of Th1 and Th17 cells, and increase the expression of the immunosuppressive factor IL-10.
  • T1D type 1 diabetes
  • EAU experimental autoimmune uveoretinitis
  • Human bone marrow MSCs exosomes can promote the proliferation of regulatory T cell subsets and up-regulate the expression of cytokines IL-10 and TGF- ⁇ 1 in peripheral blood mononuclear cells (PBMCs) to enhance the immunosuppressive ability of asthma patients.
  • PBMCs peripheral blood mononuclear cells
  • miR-181c exerts an anti-inflammatory effect in the inflammation model of burn rats by down-regulating the TLR4 signaling pathway.
  • a single intrarenal injection of porcine autologous fat MSCs-EVs can reduce the levels of multiple pro-inflammatory cytokines (TNF- ⁇ , IL-6, IL-1- ⁇ ) in the renal vein of a pig model of renal artery stenosis, and inhibit inflammation Increased levels of factor IL-10, accompanied by the migration of macrophages from pro-inflammatory cells to repair macrophages, once again proved the immunomodulatory potential of EVs.
  • TNF- ⁇ , IL-6, IL-1- ⁇ pro-inflammatory cytokines
  • Adipose MSCs exosomes can significantly reduce the increase in serum alanine aminotransferase and aspartate aminotransferase levels in the C57BL/6 mouse hepatitis model induced by Concanavalin A (Con A), reduce liver inflammation, and reduce the hepatitis model
  • the levels of pro-inflammatory cytokines (TNF- ⁇ , IFN- ⁇ , IL-6, IL-18, IL-1 ⁇ ) in mouse serum inhibited the activation of inflammasome in the liver of model mice.
  • Sepsis is a systemic inflammatory response caused by the body's fight against microbial infections. Despite the application of advanced antibiotics, the mortality rate of sepsis in the intensive care unit is still high, so researchers have targeted MSCs for treatment of such systemic inflammatory diseases.
  • MSC-EVs in animal models of sepsis caused by cecal ligation has been extensively studied. In a rat model of sepsis, intravenous administration of adipose MSCs-EVs alleviated systemic inflammation, organ damage and subsequent lethality.
  • the efficacy of EVs produced from MSCs pretreated with IL-1 ⁇ to treat sepsis was significantly higher than that of EVs derived from MSCs without pretreatment.
  • the study also shows that EVs obtained from induced MSCs can effectively polarize macrophages and differentiate them into M2 type, which is the anti-inflammatory phenotype of macrophages.
  • M2 type which is the anti-inflammatory phenotype of macrophages.
  • miR-146a the content of miR-146a in MSCs and EVs pretreated with IL-1 ⁇ is significantly increased.
  • Transfer of miR-146a packaged in EVs into macrophages can polarize it to M2 type [Casado JG, et al. Frontiers in Veterinary Science. 2017; 4:39]
  • AT2R-MSCs AT2R-MSCs
  • LPS lipopolysaccharide
  • MSCs exosomes alleviate lung ischemia/reperfusion injury in mice by transporting anti-apoptotic miR-21-5p.
  • the miR-21-5p in exosomes reduces apoptosis induced by oxidative stress by targeting PTEN and PDCD4 in lung tissue.
  • exosomal miR-146a enhanced the therapeutic effect of IL-1 ⁇ pretreated MSC. It was further found that IL-1 ⁇ stimulated MSCs, which could up-regulate the expression of MSCs miR-146a, the latter Can be packaged in exosomes. Then, this exosome miR-146a was transferred to macrophages, resulting in M2 polarization, which eventually led to an increase in the survival rate of septic mice. Therefore, the use of specific miRNA overexpression to modify the exosomes of MSCs is a promising new direction for the development of ARDS treatment.
  • ARDS is an acute systemic inflammatory response caused by direct or indirect lung injury caused by factors such as smoking, drowning, aspiration, sepsis, trauma, ischemia, and exposure to toxins. Severe inflammation causes changes in vascular permeability, leading to acute pulmonary edema.
  • the pathological process of ARDS has three main stages: exudative phase, proliferative phase and fibrotic phase.
  • the inflammatory cascade caused by lung injury and the dysfunction of the alveolar-capillary barrier leads to an increase in the permeability of alveolar epithelium and pulmonary capillary endothelial cells, which are the characteristics of the exudative phase.
  • the pathological manifestations of lung tissue are diffuse alveolar injury with exudation, microvascular injury with secondary pulmonary edema, necrosis of alveolar type 1 (AT1) epithelial cells, aggregation of inflammatory cells, and release of active mediators.
  • Alveolar inflammation is mainly caused by polymorphonuclear neutrophils, monocytes and macrophages.
  • Other pro-inflammatory mechanisms are also involved, such as lung cells, inflammatory cells and fibroblasts that release large amounts of pro-inflammatory cytokines.
  • a transparent membrane is formed in the alveoli, accompanied by the infiltration of inflammatory cells, including T lymphocytes, neutrophils and macrophages.
  • inflammatory cells including T lymphocytes, neutrophils and macrophages.
  • extracellular matrix is deposited in the alveoli, accompanied by continuous chronic inflammation.
  • the inflammatory cascade plays a key role in the process of apoptosis, proliferation, migration and other processes closely related to ARDS. Continuous damage and damage that cannot be repaired in time are the main pathological manifestations of ARDS fiber proliferation.
  • fibroblast proliferation there are fibroblast proliferation, AT2 cell proliferation and lung tissue repair.
  • the repair mechanism of damaged alveolar epithelium is not fully understood. It includes the proliferation of AT2 cells. AT2 cells migrate along the basement membrane to form a new epithelial barrier, and interact with extracellular matrix and other cells including alveolar macrophages. Effect, leading to significant changes in lung structure and function in some cases. Computed tomography (CT) of the lungs can detect dense fibrosis and honeycomb structures during the fibrous proliferation phase of ARDS.
  • CT Computed tomography
  • Severe diseases associated with ARDS can cause a high risk of ventilator-acquired pneumonia, acute myocardial infarction, and acute pulmonary embolism.
  • the invention provides an extracellular vesicle derived from mesenchymal stem cells (including adipose-derived mesenchymal stem progenitor cells, umbilical cord mesenchymal stem cells and placental amnion mesenchymal stem cells).
  • mesenchymal stem cells including adipose-derived mesenchymal stem progenitor cells, umbilical cord mesenchymal stem cells and placental amnion mesenchymal stem cells.
  • the extracellular vesicles prepared by the specific optimized process of the present invention have a specific particle size distribution and are cross-linked with PEG to form hydrophilic particles, which are not only extremely suitable for application and action in the form of aerosols In the lungs (especially the lower respiratory tract), it can exist stably in aqueous solution for a long time, and has excellent dispersibility (better than the cells themselves).
  • FIG. 10 For ease of understanding, the applicant provides a schematic diagram 10. As shown in Figure 10, representative extracellular vesicles, soluble cytokines and PEG are cross-linked to form hydrophilic particles.
  • the extracellular vesicles obtained by the separation method and process described in the present invention have a particle size in the range of 50-150 nm.
  • the extracellular vesicles, soluble cytokines and PEG are cross-linked to form hydrophilic particles with a particle size of less than 3 microns (such as about 1-3 microns or 0.5-3 microns), and the stability and dispersibility in aqueous solutions are better than the cells themselves.
  • soluble cytokines and PEG cross-linked to form hydrophilic particles respectively formulated with physiological saline, artificial tears, hyaluronic acid and other solutions for atomized inhalation, eye drops, nasal drops and external use , Is conducive to local mucosal cells to absorb and utilize these extracellular vesicles and soluble active cytokines. Its biological distribution in the body is consistent with that of simple extramembranous vesicles.
  • mesenchymal stem cells are preferably used to prepare cells of extracellular vesicles.
  • Representative examples of mesenchymal stem cells include (but are not limited to): adipose-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental amniotic mesenchymal stem cells, or combinations thereof.
  • the present invention also provides a pharmaceutical composition containing (a) an active substance derived from human somatic cells, and the active substance is an extracellular vesicle produced by human somatic cells; and (b) ) A pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a cell-free pharmaceutical composition.
  • the active ingredients also include soluble active cytokines, and these soluble active cytokines are mainly present in vesicles outside the cell membrane.
  • these soluble active cytokines may exist outside the extracellular vesicles.
  • Some soluble active cytokines can also be modified by PEG.
  • a particularly preferred pharmaceutical preparation is an aerosol inhalation preparation.
  • the invention also provides a method for preparing extracellular vesicles.
  • a typical method is to use mesenchymal stem cells (including adipose mesenchymal stem (progenitor) cells, umbilical cord mesenchymal stem cells and placental amniotic mesenchymal stem cells) as raw materials to prepare the extracellular vesicles of the present invention.
  • mesenchymal stem cells including adipose mesenchymal stem (progenitor) cells, umbilical cord mesenchymal stem cells and placental amniotic mesenchymal stem cells
  • a typical preparation method includes the following steps:
  • S1 Culture cells (such as adipose mesenchymal stem (progenitor) cells) to reach a predetermined confluence (such as 75-90%);
  • T1 is usually 24-72 hours, preferably 30-60 hours;
  • S4 Mix the conditioned medium and polyethylene glycol (PEG) to form a first mixture, and leave it for a period of T2 to form PEG-modified extracellular vesicles; wherein, The T2 is usually 6-60 hours, preferably 12-48 hours;
  • FIG. 11 A schematic process route from step S4 to S8 is shown in FIG. 11.
  • a HyperFlask cell factory can produce 500ml conditioned medium of extracellular vesicles, and it is expected to separate 5 ⁇ 10 10-10 ⁇ 10 10 total extracellular vesicles.
  • one production operation unit in the laboratory can separate two cell factories with a total of 800-1200ml extracellular vesicles of conditioned medium. It is expected to separate 2-5 ⁇ 10 11 extracellular vesicles, according to a patient’s 2-5 ⁇ 109 Usage, one production operation unit can meet the usage of 100-250 patients in one batch.
  • a HyperFlask cell factory can produce 500-600ml conditioned medium extracellular vesicles, and it is expected to separate 2-5 ⁇ 10 11 total extracellular vesicles.
  • one production operation unit can separate 800-1200ml extracellular vesicles of conditioned medium produced by two cell factories, and it is expected that 2-5 ⁇ 10 11 extracellular vesicles can be separated and purified. According to a local dosage of 2-5 ⁇ 10 9 for a patient, one production operation unit can meet the usage of 100-250 patients in one batch.
  • hydrophilic particles of the pharmaceutical composition of the present invention that are uniformly dispersed in an aqueous solution and smaller than 3 microns can be administered by aerosol inhalation and can enter the respiratory tract, especially the lower respiratory tract, with unusually high efficiency.
  • Adipose-derived mesenchymal stem (progenitor) cells For male or female healthy volunteers who have undergone ethical review and record filing, signed informed consent, and passed strict laboratory inspections to meet the criteria for building adipose tissue stem cell products into the group, they will undergo abdominal wall puncture and aspiration. Fat, or abdominal wall surgery to cut fat, place it in the cell preservation solution and transport it to a qualified GMP laboratory at low temperature. After separation, purification, and proliferation, the adipose mesenchymal progenitor cells obtained are the working bank cells (middle product).
  • the ⁇ MEM containing 5% EliteGro was centrifuged at 120,000g for 6h at 4°C.
  • the medium is changed and the pretreated medium is used for culturing at 37°C, 5% CO 2 , and culturing for 48 hours.
  • the culture supernatant was collected, centrifuged at 3,000g for 15 minutes, then the supernatant was taken out, and centrifuged at 10,000g for 30 minutes at 4°C. Take the supernatant, which is the culture medium containing extracellular vesicles, which is referred to as "conditioned medium” hereinafter.
  • extracellular vesicles were prepared by PEG precipitation method and ultracentrifugation method, respectively.
  • Example 1 Take the conditioned medium (Example 1), ultracentrifuge at 120,000g for 70 minutes at 4°C, resuspend it in pre-cooled PBS, and resuspend the pellet with an appropriate amount of isotonic sodium chloride solution after ultracentrifugation at 120,000g for 70 minutes to obtain Extracellular vesicles.
  • Example 1 Prepared with PBS at a concentration of 8%, 12%, 16%, or 20% PEG6000, filtered with a 0.22 ⁇ m filter, and added to the treated conditioned medium (Example 1) at a volume ratio of 1:1, and placed After overnight incubation at 4°C.
  • extracellular vesicles obtained by using 12% PEG 6000 to separate 200ml medium and the extracellular vesicles obtained from 350ml medium by ultracentrifugation were loaded according to the same volume and the same amount of protein for CANX, TSG101 and CD81 identification And NTA detection.
  • NTA results showed that the concentration of extracellular vesicles isolated by PEG 6000 was 1.35 ⁇ 10 8 extracellular vesicles/ml medium, and the concentration of extracellular vesicles isolated by ultracentrifugation was 6.8 ⁇ 10 7 extracellular vesicles/ ml medium.
  • the size of the extracellular vesicles separated by PEG 6000 was 126.6 ⁇ 2.09nm, and the size of the extracellular vesicles separated by ultracentrifugation was 137.8 ⁇ 3.8nm.
  • Figure 2 shows that the extracellular vesicles isolated by PEG 6000 do not affect the uptake of extracellular vesicles by fibroblasts.
  • the separated and prepared extracellular vesicles were prepared into aqueous solutions with physiological saline and placed at 4°C, -20°C and -80°C, respectively, and stored for 9 weeks. After 9 weeks, the extracellular vesicles placed at 3 temperatures were tested for NTA and related performance tests.
  • test samples include:
  • Sample 1 Freeze-dried powder of basic medium (negative control), stored at 4°C for 4 weeks;
  • Sample 2 Freeze-dried powder of stem cell culture solution (positive control), stored at 4°C for 4 weeks;
  • Sample 3 Freeze-dried powder of extracellular vesicles, stored at room temperature for 4 weeks;
  • Sample 4 Freeze-dried powder of extracellular vesicles, stored at 4°C for 4 weeks;
  • Sample 5 Freeze-dried powder of extracellular vesicles, stored at -20°C for 4 weeks;
  • Extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage activation
  • LPS lipopolysaccharide
  • control without any drug treatment
  • 0.1 ⁇ g/ml LPS alone treatment group the 0.1 ⁇ g/ml LPS and 5 ⁇ g/ml dexamethasone (Dex) co-treatment group
  • Extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage pro-inflammatory factor gene expression levels
  • real-time fluorescent quantitative PCR was used to detect the expression of its main inflammatory factor genes level.
  • the specific operation method is basically the same as that described in Example 1, but the drug treatment time is 24 hours.
  • the total RNA extraction method is carried out using the Biyuntian RNA extraction kit instructions, while the real-time fluorescent quantitative PCR detection uses the TaqMan probe method for simultaneous detection of target genes and internal reference genes, and relative quantitative analysis according to the 2- ⁇ CT method.
  • Extramembrane vesicles of human adipose-derived mesenchymal progenitor cells inhibit LPS-induced macrophage release of pro-inflammatory factors
  • the level of inflammatory factors secreted by them was detected by ELISA.
  • the specific operation method is the same as that described in Example 2, but the drug treatment time is 24 hours. See Figure 9A for the experimental scheme.
  • the ELISA method to detect macrophage inflammatory factors was performed in accordance with the instructions of the Biyuntian TNF- ⁇ and IL-6 kit, and then statistical analysis was performed.
  • TNF- ⁇ and IL-6 levels were also extremely significantly reduced, reaching the levels after dexamethasone treatment (Figure 9B).
  • extramembrane vesicles have a more excellent inhibitory effect on IL-6, suggesting that they have better application prospects in the intervention treatment of cytokine storm suppression of new coronavirus pneumonia.
  • the above data indicate that the extramembrane vesicles of human adipose-derived mesenchymal progenitor cells can significantly inhibit the release of pro-inflammatory factors induced by LPS in macrophages, thereby inhibiting their pro-inflammatory response.
  • the exosomes were inhaled by aerosol, once a day for 5 days. After that, blood samples were collected for peripheral blood white blood cell and neutrophil counts and bacterial load measurement. Collect alveolar lavage fluid for neutrophil count, bacterial load measurement, and related pro-inflammatory factor level determination. In addition, the bacterial load of the lung tissue was measured, and the morphological observation of the lung tissue was performed by HE staining.
  • the enrollment criteria of this study mainly include 1) 18-75 years old, male or female, and I or my family members voluntarily join and sign the informed consent; 2) RT-PCR test positive or a clear diagnosis of new coronavirus pneumonia patients; 3 ) Meet the diagnostic criteria for severe and critically ill patients.
  • Exclusion criteria mainly include 1) related virus carriers or patients with severe allergies, 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 planning to give birth during pregnancy, lactation or within six months; 6) the investigator judges patients who cannot participate in the study or who fail to understand and implement the program.
  • some alternative cell therapies include embryonic stem cells (ESCs) treatment, induced pluripotent stem cells (iPSCs) treatment in vitro, and Mesenchymal stem cells (Mesenchymal stem cells).
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • Mesenchymal stem cells Mesenchymal stem cells
  • stromal/stem cells MSCs
  • lung epithelial progenitor cells Pulmonary epithelial progenitor cells, EpPCs
  • Endothelial progenitor cells Endothelial progenitor cells, EnPCs
  • MSCs are the most studied stem cell therapy. MSCs are a kind of pluripotent stem cells that can self-renew and proliferate. They can suppress the immune response in vitro and have the potential to differentiate into alveolar type 2 cells (AT2 cells). MSCs-mediated inflammation suppression and MSC-related lung repair and regeneration effects are likely to be the main treatment for lung diseases such as ARDS, pneumonia, asthma, chronic obstructive pulmonary disease (COPD) and pulmonary interstitial fibrosis (IPF). reason.
  • ARDS ARDS
  • COPD chronic obstructive pulmonary disease
  • IPF pulmonary interstitial fibrosis
  • ARDS is a serious clinical syndrome caused by the destruction of the alveolar epithelial barrier, accompanied by interstitial edema and inflammatory cell infiltration, leading to progressive acute respiratory failure.
  • synthetic corticosteroids, surfactants, inhaled nitric oxide, antioxidants, protease inhibitors, and various other anti-inflammatory treatments, such as simvastatin and ibuprofen many drugs have been used to treat ARDS, but No drug can significantly reduce the mortality of ARDS, and the mortality of ARDS is still maintained at 34%-44%.
  • ARDS is usually a complication of severe sepsis, especially after infection with Gram-negative bacteria. MSCs treatment can prevent the occurrence of ARDS in an animal model of sepsis induced by E. coli-derived lipopolysaccharide (LPS)[Curley G F,et al.Critical Care Med.2017,45(2):e202--e212.][Lee JW, et al. Stem Cell 2011, 29(6):913–919.].
  • LPS E. coli-derived lipopolysaccharide
  • MSCs In the ARDS mouse model induced by lipopolysaccharide, intravenous injection of MSCs can significantly improve alveolar injury and inflammation.
  • IL-10 interleukin-10
  • MSCs reduce the infiltration of neutrophils in the lungs and reduce the production of inflammatory factor TNF- ⁇ by immune cells infiltrating the lungs[Gupta N, et al. Journal of Immunology 2007,179,(3):1855–1863.][Mei SHJ,etal.PLoS Medicine,2007,4(9):e269.].
  • MSCs promote the regeneration of AT 2 cells, prevent endothelial cell apoptosis, and promote ARDS damage Repair of the lung epithelial barrier[Lee JW,et al. Proceedings of the National Academy of Sciences of the United States of America2009,106(38):16357-16362.][Hu S,et al.Stem Cell Research&Therapy2016,7( 1):66.].
  • KGF keratinocyte growth factor
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • MSCs extracellular vesicles
  • EVs extracellular vesicles
  • MSCs produce antibacterial proteins, which can directly inhibit the growth of bacteria in the inflamed lung.
  • Intratracheal injection of MSCs can significantly reduce lung injury and inflammation, and promote bacterial clearance in a lipocalcin-2-dependent manner, thereby increasing the survival rate of experimental animals with bacterial pneumonia.
  • the activation of TLR-4 in MSCs induced by LPS enhances the secretion of lipocalcin-2, and lipocalcin-2 can bind to the siderophore of bacteria, reduce iron absorption, and inhibit bacterial growth. Consistent with these findings are the recently reported findings: In an experimental model of bacterial pneumonia, Gupta et al. [Gupta N, et al. Thorax 2012, 67(6): 533–539] found mutations in TLR-4 in MSCs Significantly weakened their therapeutic effects. Therefore, it indicates a potential new cell-based treatment that may eliminate antibiotic-resistant Gram-negative strains in the lungs.
  • the research of the present invention shows that the mesenchymal stem cell has a significant advantage of extra-membrane vesicles.
  • Stem cells have great potential in the treatment of many diseases.
  • the therapeutic effect of stem cells is largely dependent on the paracrine cytokines and extracellular vesicles (EVs) or exosomes mediated.
  • EVs extracellular vesicles
  • EVs is a nano-sized membrane structure vesicle, which has the mediating function of cell-to-cell communication.
  • EVs derived from MSCs include cytokines, growth factors, signal lipids, mRNA, miRNA/siRNA and other substances.
  • MSC-EVs may represent a new cell-free treatment method. Compared with existing MSCs therapies, it has obvious advantages: 1) No risk of tumor formation in the body; 2) Lower 3) EVs can be derived from endogenous MSCs, or they can be obtained from the supernatant (conditioned medium) in the process of culturing allogeneic human MSCs through separation and purification, which can meet clinical-level large-scale production and purification.
  • the secondary yield is high, which is convenient for batch quality control and testing; 4)
  • the seed stem cells of specific proliferation generations that produce EVs can be modified in vitro to enrich more specific EVs for different therapeutic purposes; 5)
  • Single suspension state The diameter of MSC (18-40 ⁇ m) is 3-7 times the diameter of red blood cell (6 ⁇ m), and it is easy to deposit and adhere to form micro clots. After intravenous injection, there is a risk of clogging the capillaries.
  • EVs are nano-membrane structured vesicles in the diameter range of 50-500nm, which have good dispersibility in aqueous solution and are easy to be made into injection preparations; 6) EVs have good membrane structure stability and are more resistant to cryopreservation and freeze-thaw processes.
  • MSC-derived EVs Due to the membrane structure characteristics and nanoparticle characteristics of EVs, they can be actively swallowed and taken up by target tissue cells, thereby obtaining the biological information and/or active drugs carried by EVs in order to exert the direct signal of EVs to cells Conduction and targeted drug delivery functions. 8) EVs can be loaded with certain active drugs and become a natural drug delivery system. 9) Multiple research groups have reported the therapeutic potential of MSC-derived EVs. MSCs-EVs may be used as biomarkers and therapeutic agents for ARDS in the future. MSCs-EVs also seem to play a key role in the recovery process of ARDS.
  • MSC-derived microvesicles have therapeutic advantages through airway or intravenous administration.
  • Research by Zhu et al. showed that in the lung injury induced by E. coli endotoxin, intratracheal infusion of MVs derived from MSCs can reduce the extracellular water content of lung tissue, reduce pulmonary edema, and reduce the permeability of alveolar membranes to protein.
  • MSC-MVs also reduced the influx of neutrophils and reduced the level of macrophage inflammatory protein-2 in the alveolar lavage fluid (BAL).
  • MSCs regulate macrophages in ALI through EVs-mediated mitochondrial transfer.
  • MSC-MVs contain a large amount of Angiopoietin-1, and the immunomodulatory properties of MSCs on macrophages are partly mediated by the transfer of Angiopoietin-1 mRNA to macrophages.
  • HGF hepatocyte growth factor
  • MSCs exosomes reduce endothelial cell apoptosis, increase IL-10 production and reduce IL-6 production, and partially improve the LPS-induced acute lung injury model in mice. Pulmonary microvascular permeability.
  • MSCs-MVs can restore the integrity of the alveolar membrane tight junctions, and can also reduce the permeability enhancement effect of IL-1 ⁇ , TNF- ⁇ and IFN- ⁇ on human lung microvascular endothelial cells.
  • Anti-CD44 and Angiopoietin-1 siRNA pretreatment can eliminate this therapeutic effect of MSCs-MVs, indicating that the transfer of CD-44 and Angiopoietin-1 mRNA is involved in the repair and treatment mechanism of MSCs-MVs.
  • Pretreatment of MSCs can be used to enrich the expression of MVs subgroups, thereby increasing their therapeutic potential.
  • Invention Item 1 A pharmaceutical preparation containing an active substance derived from human cells.
  • the composition of the preparation has good stability and high dispersion, and is easily absorbed by the body except the skin. It is suitable for preventing and treating various causes Inflammation and injury.
  • Invention Item 2 The human cell-derived active substance of Invention Item 1, which includes, but is not limited to, extracellular vesicles and soluble active cytokines produced by stem cells, progenitor cells and immune cells derived from human tissues, bone marrow and blood .
  • Invention Item 3 The pharmaceutical preparation according to Invention Item 1, which includes, but is not limited to, aerosol inhalation liquid, eye drops, and nasal drops.
  • Invention Item 4 The pharmaceutical preparation described in Invention Item 1 has good stability, including but not limited to storage under normal temperature conditions, storage below -20°C, storage under low temperature conditions below -70°C, and low temperature conditions below -135°C When stored and stored at -196°C, the stability of the active substance is significantly better than the storage time of the preparation containing living cells under the same conditions.
  • Invention Item 5 The pharmaceutical preparation of Invention Item 1 has a high degree of dispersion, wherein it is colorless and transparent after standing for 6-24 hours under the condition of 0-25°C, and there is no visible flocs and sediments.
  • Invention Item 6 The pharmaceutical preparation described in Invention Item 1 is easy to use on other parts of the human body except skin, where these parts include, but are not limited to, eyeball conjunctiva, eyelid conjunctiva, retina, oral cavity, nasal cavity, upper respiratory tract, lower respiratory tract, stomach Intestinal tract, vascular endothelial cells, alveolar type 1 epithelial cells, alveolar type 2 epithelial cells, monocytes, macrophages, neutrophils, dendritic cells, antigen presenting cells, T lymphocytes, fibroblasts, central nervous system Cells, peripheral nerve cells and their peripheral nerve fibers.
  • eyeball conjunctiva eyelid conjunctiva
  • retina The pharmaceutical preparation described in Invention Item 1 is easy to use on other parts of the human body except skin, where these parts include, but are not limited to, eyeball conjunctiva, eyelid conjunctiva, retina, oral cavity, nasal cavity, upper respiratory tract, lower respiratory tract, stomach Intestinal tract, vascular
  • Invention Item 7 The pharmaceutical preparation described in Invention Item 1 is suitable for the prevention and treatment of inflammation and injury caused by various reasons, including but not limited to viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, and self Immunoreactive inflammation, ischemic injury, chemical injury, physical injury.
  • Invention Item 8 The human tissue, bone marrow, and blood-derived stem cells, progenitor cells, and immune cells described in Invention Item 2, including but not limited to mesenchymal stem cells derived from human adipose tissue, and alveolar epithelial progenitors derived from human lung tissue Cells, mesenchymal stem cells derived from human umbilical cord Wharton’s glue, mesenchymal stem cells derived from human endometrial tissue, stem cells derived from human placental tissue, epithelial cells derived from human placental amniotic membrane, mononuclear cells in human blood, Hematopoietic stem cells in human bone marrow tissue, mesenchymal stem cells in human bone marrow tissue, mesenchymal stem cells derived from human periosteum, keratinocytes derived from human skin tissue, dendritic cells derived from human blood tissue, and those derived from human blood tissue CD4-positive T lymphocytes, platelets
  • Invention Item 9 The human cell-derived extracellular vesicles according to Invention Item 2, which include, but are not limited to, nanovesicles surrounded by a lipid bilayer membrane structure, with a diameter between 30-1000 nanometers, It contains a wealth of different types of microribonucleic acid (miRNA), small ribonucleic acid (sRNA), non-coding DNA fragments, transport ribonucleic acid t-RNA, soluble cytokines, growth factors, and other proteins with specific functions.
  • miRNA microribonucleic acid
  • sRNA small ribonucleic acid
  • non-coding DNA fragments transport ribonucleic acid t-RNA
  • soluble cytokines growth factors, and other proteins with specific functions.
  • Invention Item 10 The soluble active cytokine derived from human cells according to Invention Item 2, which includes, but is not limited to, soluble free lipoprotein molecules, glycoprotein molecules, biosignal molecules, tissue growth factor B (TGF ⁇ ), Stem cell growth factor (HGF) vascular endothelial cell growth factor (VEGF), epidermal cell growth factor (KGF), interleukin-10 (IL-10), interleukin-1 receptor antagonist (IL-1 ⁇ A).
  • TGF ⁇ tissue growth factor B
  • HGF Stem cell growth factor
  • VEGF vascular endothelial cell growth factor
  • KGF epidermal cell growth factor
  • IL-10 interleukin-10
  • IL-1 ⁇ A interleukin-1 receptor antagonist
  • Invention Item 11 The soluble active cytokine derived from human cells described in Invention Item 2 and Invention Item 10, including but not limited to paracrine produced by human cells cultured in a natural state, and modifying human cells with specific genes Overexpressed.
  • Invention Item 12 The extracellular vesicles of Invention Item 2 and Invention Item 9, wherein the membrane protein markers CD9, CD63, CD81 and TSG101 are specifically expressed, but CANX is not expressed.
  • the diameter of extracellular vesicles is preferably 50-500 nm.
  • Invention Item 13 The human tissue, bone marrow, and blood-derived stem cells, progenitor cells, and immune cells described in Invention Item 2 and Invention Item 8, wherein these cells can be obtained by direct isolation and purification from human tissue, or these cells can be obtained from humans After the tissue is directly separated and purified, it undergoes minimal manipulation in the GMP laboratory to make these cells proliferate, or these cells undergo specific gene modification, specific gene editing, specific gene transduction, specific microribonucleic acid miRNA in the GMP laboratory The result of introduction, or the pretreatment of these cells under special culture conditions in a GMP laboratory.
  • Invention Item 14 The human tissue, bone marrow, and blood-derived stem cells, progenitor cells, and immune cells according to Invention Item 2 and Invention Item 8, wherein these cells are preferably adipose-derived mesenchymal stem cells or placental amniotic membrane-derived mesenchyme Cytoplasmic stem cells.
  • Inventive item 15 Inventive item 2, Inventive item 8, Inventive item 13, Inventive item 14, the stem cells, progenitor cells and immune cells derived from human tissues, bone marrow and blood described in Invention Item 14, more preferably fat-derived mesenchymal stem ( Progenitor) cells, mesenchymal stem cells derived from placental amniotic membrane.
  • Progenitor fat-derived mesenchymal stem
  • Invention Item 16 The adipose-derived stem (progenitor) cells described in Invention Item 15 are further preferably adipose-derived stem (progenitor) cells, wherein those that have undergone ethical review, have signed an informed consent, and have undergone strict laboratory inspections are in line with the establishment of adipose-derived stem cell products.
  • Standard male or female healthy volunteers have undergone abdominal wall puncture liposuction or abdominal wall surgery to cut fat, preserved in a patent-protected cell preservation solution, and transported to a qualified GMP laboratory at low temperature. After patent-protected separation, purification, Working bank cells (intermediate products) of proliferated adipose-derived mesenchymal progenitor cell products.
  • Invention Item 17 The further preferred human adipose tissue-derived mesenchymal progenitor cells described in Invention Item 15 and Invention Item 16, wherein the specific human adipose tissue-derived mesenchymal stem (progenitor) cells are in GMP
  • the production laboratory meets the negative standards of various pathogenic tests, and meets the specific surface markers such as CD73 positive, CD90 positive, CD105 positive cell percentage is about 98%, CD34 positive, CD45 positive and HLA -P1-P6 generation adipose-derived mesenchymal stem (progenitor) cells that have a percentage of DR-positive cells less than 2% and meet the criteria for antibiotic residues and serum and serum substitute residual detection standards, more preferably P3-P4 generation adipose-derived mesenchymal stem (progenitor) cells Stem (progenitor) cells.
  • Invention Item 18 The further preferred adipose tissue-derived mesenchymal stem (progenitor) cells described in Invention Item 15-17 are stored under deep low temperature conditions of -196°C to -80°C for 0-36 months, and more preferably -196°C to -135°C, stored for 0-24 months.
  • Invention Item 19 The human cell-derived extracellular vesicles and soluble active cytokines described in Invention Item 2 and Invention Items 9-12, wherein the production process is:
  • Invention Item 20 The differential centrifugation combined with polyethylene glycol PEG precipitation described in the production process (2) of Invention Item 19, wherein PEG3000-PEG9000 is used, 8%-30% PEG is configured with PBS, and After filtering with a 0.22 ⁇ m filter, add it to the treated conditioned medium at a volume ratio of 1:1, incubate overnight at 4°C, centrifuge at 3,000-5000g for 45-60 minutes at 4°C, and discard the supernatant.
  • Invention Item 21 The extracellular vesicles obtained by the production process of Invention Item 19 and Invention Item 20 are characterized by the biomarkers CD9 positive, CD63 positive, CD81 positive, TSG101 positive and CANX negative.
  • Invention Item 22 The production process described in Invention Items 19-21, in which, under GMP laboratory conditions, it can be scaled up to the clinical-level production scale of cell products, and its outstanding advantages are: 1 production operation unit can separate 2 cell factories The 800-1200ml conditioned medium obtained can isolate 2-5 ⁇ 10 11 extracellular vesicles. Calculated according to the total dose of 2-5 ⁇ 10 9 extracellular vesicles used locally for a patient, one production operation unit and one batch of production can meet the local consumption of 100-250 patients. With 100 production operation units, a batch of production can reach a total of 10,000-25,000 people, and it will take 5-6 days.
  • Invention Item 23 The production process according to Invention Item 19-22, wherein the separated extracellular vesicles, soluble active cytokines and PEG of specific molecular weight are cross-linked to form hydrophilic particles less than 3 microns at -196°C
  • the stability under freezing conditions at -20°C and the dispersibility in an aqueous solution at room temperature are better than those of the prior art.
  • Invention Item 24 The extracellular vesicles, soluble active cytokines, and polyethylene glycol PEG cross-linked hydrophilic particles according to Invention Item 23, including but not limited to those in isotonic sodium chloride solution, low molecular transparent From the acid solution, artificial tears, and gel solutions, it is prepared into aerosolized inhalation liquid, eye drops, and nasal drops for external use.
  • the uptake of the extracellular vesicles obtained by the process of the present invention by local mucosal cells is not affected by PEG.
  • Invention Item 25 The pharmaceutical preparations containing human extracellular membrane vesicles and soluble active cytokines described in Invention Item 1 and Invention Items 23-24 are preferably prepared as aerosol inhalants, which are suitable for the treatment of infectious lung injury.
  • Invention Item 26 The pharmaceutical preparations of Invention Item 1 and Invention Item 25, wherein adipose tissue-derived stem (progenitor) cells and placental amniotic membrane-derived mesenchymal stem cell-derived extramembrane vesicles and soluble cytokines are preferred and isotonic
  • the aerosol inhalation liquid prepared by sodium chloride aqueous solution is suitable for viruses, including but not limited to influenza virus, SARS-coronavirus, SARS-coronavirus 2, MERS-coronavirus infection, and aerosol inhalation treatment of acute lung injury caused by infection, in order to prevent Acute lung injury releases inflammatory factors, reduces the infiltration of high protein fluid in the alveoli and alveolar epithelial cell damage, significantly improves the rescue success rate of patients with acute lung injury, and improves the lung function and quality of life of surviving patients.
  • viruses including but not limited to influenza virus, SARS-coronavirus, SARS-coronavirus 2, MERS-cor

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