Therapeutic or prophylactic agent for disease

Abstract

This therapeutic or prophylactic agent is for at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and geriatric diseases, and contains extracellular vesicles derived from basal cells as an active ingredient.

Description

Treatment or prevention of disease

[0001] The present invention relates to a therapeutic or preventive agent for at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases.

[0002] The inventors have previously found that extracellular vesicles (EVs) derived from human bronchial epithelial cells (HBECs) are effective as a therapeutic agent for pulmonary fibrosis (see Patent Document 1).

[0003] International Publication No. 2020 / 209304

[0004] However, it is difficult to stably maintain the properties of bronchial epithelial cells, resulting in decreased cell proliferation and reduced therapeutic efficacy. For practical application, the production of EVs through mass culture is important, and their therapeutic efficacy must be investigated. Therefore, the present invention aims to provide a therapeutic or preventive agent with sustained efficacy.

[0005] The present invention includes the following aspects: [1] A therapeutic or preventive agent for at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases, the therapeutic or preventive agent comprising basal cell-derived extracellular vesicles as an active ingredient. [2] The therapeutic or preventive agent according to [1], wherein the basal cells are human bronchial basal cells (HBBCs). [3] The therapeutic or preventive agent according to [1], which is administered via the airway. [4] A therapeutic or preventive composition for at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases, the composition comprising the therapeutic or preventive agent according to any one of [1] to [3] and a pharmaceutically acceptable excipient. [5] A drug delivery system to the lungs comprising human bronchial basal cell-derived extracellular vesicles and a drug.

[0006] According to the present invention, a therapeutic or prophylactic agent whose effect is maintained can be provided.

[0007] Photographs showing the results of immunocytochemical staining of HBEC (P1), HBEC (P6), and HBBC (P25). Graphs showing the cell proliferation activity of HBEC and HBBC. Photographs showing the results of SA-β-gal staining of HBEC (P1), HBEC (P7), and HBBC (P25). Figures showing the results of nanoparticle tracking analysis showing the particle size of purified HBEC-EVs and HBBC-EVs using ultracentrifugation. The vertical axis of the graph represents the number of EV particles (×10 8) / mL, and the horizontal axis represents the particle size (nm) of EVs. This graph shows the results of comparing the amount of EVs secreted (particles / mL) from primary cultured HBECs and HBBCs. This graph shows the results of Western blotting to examine the expression of various myofibroblast markers after stimulation of primary cultured HBECs and HBBCs with TGF-β1 and / or EVs. This graph shows the results of Western blotting to examine the expression of senescent cell markers after stimulation of primary cultured HBECs and HBBCs with TGF-β1 and / or EVs. This graph shows the results of Western blotting to evaluate the effect of HBBC-EVs on NF-κB p65 in THP-1 cells treated with LPS (lipopolysaccharide). This graph shows the results of qRT-PCR to confirm the expression of NF-κB pathway-related inflammatory markers after the addition of HBBC-EVs to LPS-treated THP-1 cells. This figure shows the results of examining the expression of miRNAs (miR-16, miR-26a, miR-26b, miR-141, miR-148a, and miR-200a) in EVs derived from HBECs and HBBCs. This figure shows the experimental scheme for evaluating the anti-fibrotic effect of HBBC-derived EVs. (A) HE staining and Masson trichrome staining results for untreated mice (control), a pulmonary fibrosis model mouse created by bleomycin administration (BLM control), and a pulmonary fibrosis model mouse administered with HBBC-derived EVs (BLM+EVs). (B) Lung fibrosis scoring (Ashcroft score) results for untreated mice (control), a pulmonary fibrosis model mouse created by bleomycin administration (BLM control), and a pulmonary fibrosis model mouse administered with HBBC-derived EVs (BLM+EVs). (A) Imaging results of DiR-labeled EVs derived from each cell type administered intratracheally to 8-12 week-old C57BL / 6J mice. (B) Graph showing the quantification results of (A). Graph showing the results of comparing 2D culture of HBEC and HBBC. Graph showing the results of comparing the amount of EV secretion from HBEC (P4) and HBBC (P28). Photographs of HBEC and HBBC cells attached to microcarrier beads after 3D culture.Photographs showing the results of immunocytostaining of HBEC (P1), HBEC (P6), and HBBC (P25). Photographs showing the results of immunocytostaining of HBEC (P1), HBEC (P6), and HBBC (P25). (A) A table showing a conventional freeze-drying protocol. (B) A table showing the freeze-drying protocol of the present embodiment. Graphs showing the particle number retention rate of samples obtained by rehydrating freeze-dried products obtained by each freeze-drying protocol. "FD65h" indicates a rehydrated sample of freeze-dried product obtained using a conventional freeze-drying protocol with a drying time of 65 hours and 0.1% poloxamer. "FD30h" indicates a rehydrated sample of freeze-dried product obtained using the freeze-drying protocol of this embodiment with a drying time of 30 hours and 0.1% poloxamer. "Poloxamer 0.02% + FD30h" indicates a rehydrated sample of freeze-dried product obtained using the freeze-drying protocol of this embodiment with a drying time of 30 hours and 0.02% poloxamer. This graph shows the particle size distribution of a rehydrated sample (after FD) and a sample before lyophilization (before FD) of a freeze-dried product obtained using the freeze-drying protocol of this embodiment with a drying time of 30 hours and 0.1% poloxamer. This graph shows the particle size distribution of a rehydrated sample (after FD) and a sample before lyophilization (before FD) of a freeze-dried product obtained using the freeze-drying protocol of this embodiment with a drying time of 30 hours and 0.02% poloxamer. Graphs showing the EV elution positions measured by multi-angle light scattering (MALS) for rehydrated samples (after FD) and samples before lyophilization (before FD) of lyophilized products using the lyophilization protocol of this embodiment with a drying time of 30 hours. The left graph shows the graph for 0.1% poloxamer, and the right graph shows the graph for 0.02% poloxamer. Graphs showing the CD9 marker protein retention rate for samples rehydrated with lyophilized products using each lyophilization protocol."FD65h" indicates a rehydrated sample of freeze-dried product using a conventional freeze-drying protocol with a drying time of 65 hours and 0.1% poloxamer. "FD30h" indicates a rehydrated sample of freeze-dried product using the freeze-drying protocol of this embodiment with a drying time of 30 hours and 0.1% poloxamer. "Poloxamer 0.02% + FD30h" indicates a rehydrated sample of freeze-dried product using the freeze-drying protocol of this embodiment with a drying time of 30 hours and 0.02% poloxamer. This graph shows the particle count retention rate of samples freeze-dried using the freeze-drying protocol of this embodiment with a drying time of 30 hours and 0.1% poloxamer, stored at room temperature for 3 weeks and rehydrated. "Immediately after FD" indicates a sample rehydrated immediately after freeze-drying, and "After 3W" indicates a sample rehydrated after lyophilization and storage at room temperature for 3 weeks. 1 is a graph showing the particle size distribution of samples freeze-dried using the freeze-drying protocol of this embodiment, a drying time of 30 hours and 0.1% poloxamer, stored at room temperature for 3 weeks and then rehydrated. "Before FD" indicates the sample before freeze-drying, "immediately after FD" indicates the sample rehydrated immediately after freeze-drying, and "after 3W" indicates the sample rehydrated after freeze-drying and then stored at room temperature for 3 weeks. This graph shows the CD9 marker protein retention rate of samples freeze-dried using the freeze-drying protocol of this embodiment, a drying time of 30 hours and 0.1% poloxamer, stored at room temperature for 3 weeks and then rehydrated. "Immediately after FD" indicates the sample rehydrated immediately after freeze-drying, and "after 3W" indicates the sample rehydrated after freeze-drying and then stored at room temperature for 3 weeks. This figure shows the results of adding a sample (0.1% poloxamer + FD30h) freeze-dried using the freeze-drying protocol of this embodiment, a drying time of 30 hours, and 0.1% poloxamer to MRC-5 treated with TGF-β1, and evaluating the expression of collagen and α-SMA (anti-fibrotic effect). This figure also shows a graph of the particle counts of samples freeze-dried using the freeze-drying protocol of this embodiment, a drying time of 30 hours, and 0.1% poloxamer, after long-term storage at room temperature and rehydration. "0M": before freeze-drying; "1M": stored for 1 month; "3M": stored for 3 months; "6M": stored for 6 months; "12M": stored for 12 months.This figure shows the results of examining the anti-fibrotic effect of samples that were freeze-dried using the freeze-drying protocol of this embodiment, with a drying time of 30 hours and 0.1% poloxamer, and then rehydrated after storage at room temperature for 12 months (12M). This figure shows the evaluation scheme for the effect of an EV preparation rehydrated from a freeze-dried product as an inhalant. This figure shows a graph showing the particle size distribution in "pre-nebulizer" and "post-nebulizer" samples. This figure shows the results of examining the anti-fibrotic effect of "pre-nebulizer" and "post-nebulizer" samples. This figure shows the results of flow cytometry analysis of P0 cells isolated from the airways labeled with fluorescent anti-integrin αvβ8 antibody. This figure shows phase-contrast images of αvβ8+ cells (HBBCs) and αvβ8- cells. This figure shows the results of evaluating collagen and α-SMA expression in primary lung fibroblasts treated with TGF-β1 (2 ng / ml) by adding αvβ8+ cell-EVs or αvβ8- cell-EVs (10 μg / ml). This figure shows an explanatory diagram of the air-liquid interface culture method. This figure shows the results of RNA expression analysis. This figure shows the results of immunocytochemical staining (ICC) / immunofluorescence staining (IF) analysis of HBEC or HBBC after air-liquid interface culture.

[0008] [Therapeutic or Preventive Drug] In one embodiment, the present invention provides a therapeutic or preventive drug for at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases, the therapeutic or preventive drug comprising basal cell-derived extracellular vesicles (hereinafter also referred to as "EVs") as an active ingredient.

[0009] In this embodiment, the term "fibrotic disease" refers to a disease accompanied by excessive formation of fibrous connective tissue, which may occur during the repair or healing process of an organ or tissue. Fibrotic diseases are diseases caused by or accompanied by abnormal deposition of scar tissue, and may occur in a variety of tissues. Examples of fibrotic diseases include pulmonary fibrosis, pulmonary hypertension, liver cirrhosis, cardiomyopathy, ischemic heart disease, valvular disease, endomyocardial fibrosis, myocardial fibrosis, arteriosclerosis, renal fibrosis, nephrosclerosis, glomerulosclerosis, scleroderma, retroperitoneal fibrosis, and uterine fibrosis.

[0010] In this embodiment, "scleroderma" refers to a disease whose main symptom is skin hardening. Scleroderma includes two types: localized scleroderma and systemic sclerosis. Localized scleroderma is a disease that affects only the skin. Systemic scleroderma causes lesions not only in the skin but also in various organs throughout the body.

[0011] In this embodiment, the term "inflammatory disease" refers to a disease that presents with a persistent or transient inflammatory state and whose symptoms can be improved by reducing the inflammation. Examples of inflammatory diseases include COPD (chronic obstructive pulmonary disease), bronchial asthma, acute exacerbation of interstitial pneumonia, ARDS (acute respiratory distress syndrome), radiation pneumonitis, pulmonary sarcoidosis, bronchiectasis, cystic fibrosis, bronchopulmonary dysplasia, viral hepatitis, autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, hepatocellular carcinoma, Crohn's disease, ulcerative colitis, acute pancreatitis, chronic pancreatitis, autoimmune pancreatitis, aortitis, Graves' disease, Hashimoto's disease, diabetes, nephritis, collagen diseases (e.g., rheumatoid arthritis, SLE (systemic lupus erythematosus), vasculitis, Sjögren's syndrome, arthritis, and sarcoidosis), dermatomyositis, and psoriasis.

[0012] In this embodiment, "ARDS" refers to a severe respiratory disorder triggered by a preceding disease. Examples of preceding diseases that can trigger ARDS include bacterial pneumonia, interstitial pneumonia, acute exacerbation of interstitial pneumonia, sepsis, viral infection, multiple trauma, burns, acute pancreatitis, inhalation of toxic gases, drug poisoning, drowning, pulmonary contusion, radiation pneumonitis, and blood transfusion. Examples of viral infections that can trigger ARDS include influenza virus and coronavirus infections such as COVID-19.

[0013] In this embodiment, "aging diseases" refers to diseases that occur due to a decline in homeostatic functions in tissues or individuals caused by aging. A typical phenotype of aging is cellular senescence, and the accumulation of these senescent cells and the various mediators they secrete are involved in the induction of chronic inflammation, as well as structural changes such as fibrosis and functional impairment. Examples of aging diseases include Alzheimer's disease, dementia, Parkinson's disease, spinocerebellar degeneration, and multiple system atrophy.

[0014] In this embodiment, the basal cells are progenitor cells of large airways (large airways) and are not differentiated cells. Basal cells are not particularly limited, and include epidermal basal cells and bronchial basal cells, with bronchial basal cells being preferred.

[0015] The species from which the basal cells are derived is not limited, but is preferably a mammal. Examples of mammals include humans, chimpanzees, monkeys, rats, mice, pigs, cows, horses, sheep, goats, dogs, and cats, with humans and mice being preferred, and humans being more preferred. Healthy humans are particularly preferred.

[0016] Human bronchial basal cells may express SRY-related HMG-box (SOX) family proteins, preferably SOX2+, and more preferably SOX2+ / SOX9-.

[0017] The basal cells are preferably immortalized cells. The immortalization method is not particularly limited, and examples include transformation using Epstein-Barr virus or cancer-related genes. Examples of cancer-related genes include the SV40 T antigen gene, HPV E6E7 gene, v-abl gene, myc gene, hTERT gene, CDK4 gene, CyclinD1 gene, or combinations of these genes. Examples of immortalization methods include introducing the CDK4R24C gene and the hTERT gene into bronchial basal cells. CDK4R24C is a mutant cyclin-dependent kinase 4. It is preferable to introduce the CyclinD1 gene in addition to the CDK4R24C gene and the hTERT gene. Another example is a combination of the HPV E6E7 gene and the hTERT gene.

[0018] The method for introducing these genes is not particularly limited and includes, for example, physical methods (electroporation, microinjection, gene gun, etc.), chemical methods (lipofection, etc.), and methods using viral vectors, among which methods using viral vectors are preferred, such as lentiviral vectors and retroviral vectors.

[0019] The method for obtaining basal cells is not particularly limited. As described below in the Examples, examples include a method of inducing basal cells during the process of immortalizing epithelial cells, and a method of isolating basal cells using an affinity substance for integrin αvβ8, a basal cell marker. Examples of affinity substances include anti-αvβ8 antibodies. Another example is a method of reprogramming epithelial cells into basal cells by treating them with a SMAD signal inhibitor. Examples of SMAD signal inhibitors include Y-27632 (Rock inhibitor), A83-01 (ALK5, ALK4, and ALK7 inhibitor), DMH-1 (ALK2 inhibitor), or a combination thereof.

[0020] In this embodiment, extracellular vesicles (EVs) refer to vesicles secreted from cells. EVs also include vesicles such as exosomes, microvesicles, and apoptotic bodies. The surface of EVs contains lipids and proteins derived from the cell membrane, and the interior contains intracellular substances such as nucleic acids and proteins.

[0021] In this embodiment, EVs are obtained, for example, by culturing basal cells in a medium and recovering them from the culture supernatant. The culture temperature is the same as that for mammalian cells, preferably 30°C to 40°C. The culture period is preferably, for example, 1 to 10 days, more preferably 1 to 5 days. The basal cells are cultured preferably for 1 to 5 days, more preferably 1 to 3 days.

[0022] Methods for recovering EVs from culture medium include ultracentrifugation, immunoprecipitation, gel filtration, ultrafiltration, polymer precipitation, HPLC, and FACS, with ultracentrifugation being preferred. Ultracentrifugation combines crude centrifugation with filtration through a 0.22 μm filter to recover EVs primarily composed of exosomes with diameters of approximately 30–200 nm.

[0023] In this embodiment, the EVs may be solids obtained by removing the water content from EVs recovered from the culture medium by drying.

[0024] The amount of EVs contained in the therapeutic or prophylactic agent of this embodiment can be appropriately determined taking into account various factors such as the sex, weight, age, and symptoms of the subject. In the case of an inhalant, for example, a single inhalation may administer 1 μg to 10 g of the active ingredient in solid form, for example, 10 μg to 2 g of the active ingredient in solid form. In the case of oral administration, for example, a single inhalation may administer 1 μg to 10 g of the active ingredient in solid form per day, for example, 10 μg to 2 g of the active ingredient in solid form per day. In the case of an injection, for example, a single inhalation may administer 0.1 μg to 1 g of the active ingredient in solid form per day, for example, 1 μg to 200 mg of the active ingredient in solid form per day. In the case of a suppository, for example, a single inhalation may administer 1 μg to 10 g of the active ingredient in solid form per day, for example, 10 μg to 2 g of the active ingredient in solid form per day. In the case of an external preparation for skin, for example, 1 μg to 10 g of the active ingredient may be administered per day in solid form, for example, 10 μg to 2 g per day.

[0025] The dosage form is not particularly limited and may be selected appropriately as needed. Generally, the formulation may be administered as an oral preparation such as aerosol, tablet, capsule, granule, fine granule, powder, liquid, syrup, suspension, emulsion, or elixir, or as an injection, infusion, suppository, inhalant, or transmucosal absorbent. Inhalants are administered using a device such as a nebulizer, metered-dose inhaler, or dry powder inhaler. Injections are administered intravenously, either alone or mixed with conventional fluids such as glucose or amino acids, and may also be administered intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally, as needed. Suppositories are administered rectally. Topical skin preparations are applied, pasted, or sprayed onto the affected area.

[0026] The therapeutic or prophylactic agent of this embodiment may be administered, for example, via the respiratory tract, inhalation, aerosol administration, injection, instillation, oral, transdermal, nasal, topical, vaginal, or rectal routes.

[0027] The frequency of administration may be three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, etc. The administration period may be one day, two days, three days, one week, two weeks, one month, six months, one year, or longer.

[0028] [Therapeutic or Preventive Composition] In one embodiment, the present invention provides a composition comprising the above-described therapeutic or prophylactic agent and a pharmaceutically acceptable excipient.

[0029] The composition of the present embodiment can be administered parenterally in the form of, for example, a spray, injection, infusion, suppository, inhalant, transmucosal absorbent, etc., or orally in the form of, for example, a tablet, capsule, granule, fine granule, powder, liquid, syrup, suspension, emulsion, elixir, etc.

[0030] The composition of this embodiment can contain, without limitation, pharmaceutically acceptable additives typically used in the formulation of pharmaceutical compositions. More specifically, examples include excipients such as starch and crystalline cellulose; binders such as gelatin, corn starch, tragacanth gum, and gum arabic; swelling agents such as alginic acid; injectable solvents such as water, ethanol, and glycerin; adhesives such as rubber-based adhesives and silicone-based adhesives; lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavorings such as peppermint and benzyl alcohol; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives, and the like. The additives can be used alone or in combination. The additives can be used alone or in combination as a carrier for a therapeutic or prophylactic drug.

[0031] The composition of this embodiment preferably contains trehalose and poloxamer. Examples of trehalose include α,α-trehalose, α,β-trehalose, and β,β-trehalose, with α,α-trehalose being preferred. The composition of this embodiment preferably contains trehalose dihydrate at 0.1 wt% to 20 wt%, more preferably 0.5 wt% to 10 wt%, and particularly preferably 1 wt% to 8 wt%. Furthermore, the poloxamer is preferably Poloxamer 188. The composition of this embodiment preferably contains poloxamer at 0.01 wt% to 1 wt%, more preferably 0.02 wt% to 0.5 wt%, and particularly preferably 0.08 wt% to 0.2 wt%.

[0032] The composition of the present embodiment preferably contains 10 mM to 100 mM of a buffering agent, and the buffer preferably has a pH of 7.0 to 8.5. Examples of the buffer include MOPS, HEPES, Tris, MES, citrate, and phosphate buffers, with phosphate buffers being preferred.

[0033] The composition of the present embodiment may be for freeze-drying, i.e., before being freeze-dried, or may be freeze-dried, i.e., a freeze-dried product, and is particularly preferably a freeze-dried product.

[0034] [Lyophilization] The lyophilized product, which is the composition of this embodiment, may be a vial-filled product obtained by filling EVs recovered from a culture medium into a vial and removing water by drying to form a solid.

[0035] The freeze-drying cycle of the composition of this embodiment includes a pre-freezing step, a primary drying step, and a secondary drying step. In the pre-freezing step, the composition of this embodiment is hardened by being exposed to low temperatures. In the primary drying step, vacuum control is performed, and heating is performed to deliver enough energy to sublimate the ice in the frozen composition. After the ice is distilled off, the secondary drying step begins. In the secondary drying step, vacuum control is performed, allowing for progressive extraction of bound water above zero. If the sample temperature exceeds the set shelf temperature, drying at that temperature is complete, and in each step, it is preferable to proceed to the next drying step when the sample temperature exceeds the set shelf temperature by 5°C. Furthermore, in the secondary drying step, the drying time has the greatest impact on over-drying, so a short drying time is preferable. Specifically, the freeze-drying cycle for the composition of this embodiment preferably includes a pre-freezing step under atmospheric pressure, starting at 25°C, gradually increasing the temperature to -40°C over 1 to 4 hours, and maintaining -40°C for 0.2 to 1 hour; a primary drying step under vacuum control of 1 Pa or less, starting at -45°C, gradually decreasing the temperature to -25°C over 0.5 to 2 hours, maintaining -25°C for 1.5 to 3.5 hours, starting at -25°C, gradually decreasing the temperature to 0°C over 0.5 to 2 hours, and maintaining 0°C for 0.5 to 2 hours; and a secondary drying step under vacuum control of 1 Pa or less, maintaining 30°C for 3 to 8 hours.

[0036] [Drug Delivery System] In one embodiment, the present invention provides a pulmonary drug delivery system comprising human bronchial basal cell-derived extracellular vesicles and a drug. As described in the Examples below, HBBC-derived EVs were highly uptaken by the lungs. Therefore, the drug delivery system of this embodiment can efficiently deliver drugs to the lungs. Examples of drugs include small molecules, nucleic acids, proteins, peptides, etc. Nucleic acids include cDNA, mRNA, siRNA, shRNA, miRNA, antisense RNA, etc. Proteins include enzymes, antibodies, etc. Peptides include ligands, vaccines, etc. The drug may contain two or more of these, for example, a nucleic acid and a protein, such as CRISPR-Cas9. By delivering CRISPR-Cas9 to the lungs, target genes distributed in the lungs can be efficiently cleaved at specific DNA sites. The drug may be HBBC-derived EVs themselves. These drugs are mixed with HBBC-derived EVs and used in the drug delivery system of this embodiment.

[0037] [Other Embodiments] In one embodiment, the present invention provides basal cell-derived EVs for the treatment or prevention of at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases.

[0038] In one embodiment, the present invention provides a method for treating or preventing at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases, comprising administering an effective amount of basal cell-derived EVs to a patient in need of such treatment or prevention.

[0039] In one embodiment, the present invention provides use of basal cell-derived EVs for producing a therapeutic or preventive agent for at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases, or a therapeutic or preventive composition for at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases.

[0040] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0041] [Preparation of Bronchial Basal Cells] Cells isolated from lung specimens using the first- to fourth-order bronchi were immortalized. Specifically, the following three lentiviral vectors, CSII-CMV-TERT, CSII-CMV-CDK4R24C, and CSII-CMV-cyclinD1, were transfected into 293T cells by lipofection to obtain culture supernatants containing the recombinant viruses. A concentrated solution containing polyethylene glycol 6000 was added to the resulting viral supernatant and incubated at 4°C for 16 hours to form a precipitate. The virus solution was then centrifuged in a swinging bucket at 3500 rpm at 4°C to obtain a viral pellet. The resulting viral pellet was suspended in 1.5 ml of the cell-optimized medium, and polybrene was added to a final concentration of 10 μg / ml. The cells were infected with the recombinant viruses in a 35 mm dish for 48 hours to obtain immortalized cells.

[0042] Immunocytochemistry was performed on HBEC (P1; Passage 1), HBEC (P6), and immortalized cells (P25). The results are shown in Figure 1. The immortalized cells were p63+ / KRT5+. Immortalization induced human bronchial basal cells (HBBCs) with basal cell phenotypes.

[0043] To measure cell proliferation activity in primary cultured HBEC and HBBC, serial cell passages were performed. Specifically, 5 × 10 cells were cultured at the start of each passage. 4 Cells were seeded into three 35-mm dishes. When one well reached confluence, the cells in all wells were treated with trypsin and detached. The number of cells in each well was counted. The PDL value was calculated from the measured cell counts using the formula PDL = log2(A / B), where B represents the number of cells seeded at the beginning of each passage, and A represents the number of cells obtained at the end of each passage. The PDL value was calculated as a cumulative value. The results are shown in Figure 2. While primary cultured HBECs stopped proliferating at P7, HBBCs maintained high cell proliferation capacity even beyond P20.

[0044] SA-β-gal staining was performed on HBEC (P1), HBEC (P7), and HBBC (P25). The results are shown in Figure 3. Senescent primary cultured HBEC (P7) showed a decrease in proliferation capacity and numerous senescent cells. On the other hand, HBBC (P25) did not show a significant increase in senescent cells compared to primary cultured HBEC (P1).

[0045] [Characterization of HBBC-EVs] EVs were isolated from the culture supernatant of primary cultured HBECs and HBBCs by ultracentrifugation and characterized. NanoSight tracking analysis showed that the most common size of each EV was approximately 90-100 nm (A: 98 nm, B: 106 nm), with no significant difference (see Figure 4).

[0046] [Analysis of prepared EVs] The secretion amount (particles / ml) of isolated EVs was compared using a Nanosight NS300. The results are shown in Figure 5. Compared to primary cultured HBECs, the secretion amount was significantly increased by approximately three-fold in HBBCs.

[0047] Anti-fibrotic effect of HBBC-derived EVs. Primary lung fibroblasts were stimulated with TGF-β1 and / or EVs, and the expression of various myofibroblast markers was examined by Western blotting (see Figure 6). Specifically, the expression of collagen and α-SMA was assessed by adding HBEC-EVs or HBBC-EVs (10 μg / ml) to primary lung fibroblasts treated with TGF-β1 (2 ng / ml). HBEC-EVs were collected from early passage (P1-P3) and late passage (P6-P7) cells. HBBCs were used at P25. Late passage primary cultured HBEC-EVs exhibited a reduced inhibitory effect on the induction of α-SMA and Type I collagen expression compared to early passage primary cultured HBEC-EVs. However, HBBC-EVs exhibited an inhibitory effect on the induction of TGF-β1 expression equivalent to that of early passage primary cultured HBEC-EVs, demonstrating their anti-fibrotic effect against TGF-β1.

[0048] Anti-aging effect of HBBC-derived EVs. Human primary airway epithelial cells (HBECs) were stimulated with TGF-β1 and / or EVs, and the expression of senescent cell markers was examined by Western blot (see Figure 7). Specifically, p21 expression was assessed by adding HBEC-EVs or HBBC-EVs (10 μg / ml) to primary lung fibroblasts treated with TGF-β1 (2 ng / ml). HBEC-EVs were collected from early passage (P1-P3) and late passage (P6-P7) cells. HBBCs from P25 were used. Late passage primary cultured HBEC-EVs exhibited a weaker inhibitory effect on p21 induction compared to early passage primary cultured HBEC-EVs. However, HBBC-EVs exhibited the same inhibitory effect as early passage primary cultured HBEC-EVs, demonstrating their anti-aging effect.

[0049] Anti-inflammatory Effect of HBBC-Derived EVs. NF-κB pathway activation was examined in PMA-treated THP-1 cells after stimulation with LPS and / or EVs. HBBC-EVs were prepared using HBBC at P25. First, Western blotting was performed to assess the effect of HBBC-EVs (10 μg / mL) on NF-κB p65 in THP-1 cells treated with LPS (1 μg / mL). Exposure to LPS in THP-1 cells increased the phosphorylation level of NF-κB p65, which was reduced by HBBC-EV treatment (Figure 8). Next, qRT-PCR was used to confirm the expression of NF-κB pathway-related inflammatory markers in THP-1 cells treated with LPS (1 μg / mL) after treatment with HBBC-EVs (10 μg / mL). Exposure of THP-1 cells to LPS increased the expression levels of pro-inflammatory cytokines IL-6, IL-8, and TNF-α, but treatment with HBBC-EVs reduced these elevated levels (see Figure 9).

[0050] [MiRNA Expression in HBBC-Derived EVs] Next, we examined the expression of miRNAs (miR-16, miR-26a, miR-26b, miR-141, miR-148a, and miR-200a) in HBBC-EVs. qRT-PCR revealed decreased miRNA expression in late-passage primary-cultured HBEC-EVs compared to early-passage primary-cultured HBEC-EVs, whereas expression in HBBC-EVs was comparable to that in early-passage primary-cultured HBEC-EVs (see Figure 10).

[0051] [Anti-fibrotic effect of HBBC-derived EVs] Animal experiments were conducted in accordance with the guidelines of the Experimental Animal Research Institute at the Jikei University School of Medicine. Figure 11 shows the experimental scheme. 2.5 U / kg bleomycin (Nippon Kayaku Co., 4234400D4032) was dissolved in 50 μl of saline and administered intratracheally to 8-12 week-old C57BL / 6J mice as a pulmonary fibrosis model. On days 7 and 14 after bleomycin administration, 2x10 HBBC-EVs were injected. 9 Particles (50 μl) were administered intratracheally. The mice were sacrificed 17 days after administration, and tissue staining was performed. Figure 12 shows the results of HE staining and Masson trichrome staining (see Figure 12(A)) in untreated mice (control), a pulmonary fibrosis model mouse created by bleomycin administration (BLM control), and a pulmonary fibrosis model mouse administered HBBC-derived EVs (BLM+EVs), as well as the results of lung fibrosis scoring (Ashcroft score) (see Figure 12(B)). HBBC-derived EVs were confirmed to significantly suppress pulmonary fibrosis.

[0052] [Pulmonary inhalation delivery of HBBC-derived EVs] DiR-labeled EVs derived from each cell type were administered intratracheally to 8-12 week-old C57BL / 6J mice. Post-administration imaging was used to assess pulmonary uptake of EVs. As shown in Figure 13, HBBC-derived EVs were most suitable for pulmonary delivery.

[0053] [Comparison of 2D culture of HBEC and HBBC] 2D culture of HBEC usually requires a substrate. For example, rat collagen type 1 or vitronectin is coated on the dish for culture. HBEC and HBBC were cultured in non-coated 6-well dishes, and cell proliferation with passage was evaluated. Specifically, 1 × 10 5 Cells were seeded per well, counted after 5 days, and then passaged four times. HBECs were passaged from P1 to P4, and HBBCs from P25 to P28. The results are shown in Figure 14. HBECs showed a significant decrease in cell proliferation with passage, whereas HBBCs could be cultured without a substrate. The amount of EV secretion was also compared between HBEC (P4) and HBBC (P28). The results are shown in Figure 15. Under substrate-free culture conditions, HBECs produced a decrease in EV production, whereas HBBCs maintained EV production under the same conditions.

[0054] [Comparison of 3D culture of HBEC and HBBC] The cell proliferation ability in 3D culture of HBEC and HBBC was evaluated. Specifically, microcarrier beads (Cytodex3) were added to a spinner flask and the cells were grown at 4000 cells / cm. 2 Suspension culture was performed for 10 days at a cell density of 100 μg / ml. Figure 16 shows a photograph of cells attached to microcarrier beads after 10 days of culture. HBECs did not adhere to the microcarrier beads, indicating that cell proliferation was not observed in the suspension culture system. HBBCs, on the other hand, were observed to be distributed on the microcarrier beads, indicating that cell proliferation was also observed in 3D culture.

[0055] [Confirmation of SOX2 and SOX9 expression in HBBCs] HBECs (P1), HBECs (P6), and HBBCs (P25) were subjected to immunocytochemistry to confirm the expression of SOX2 and SOX9. The results are shown in Figures 17 and 18. HBBCs were confirmed to be SOX2+ / SOX9-.

[0056] [Storage and Lyophilization of EV Therapeutic Formulations] In recent years, research and development of EVs, including exosomes, as therapeutic agents has progressed. EV preparations purified by methods such as ultracentrifugation and tangential flow filtration (TFF) are typically resuspended in PBS(-) and stored. However, refrigerated or frozen storage presents the drawback of reduced particle count and aggregation within approximately one month, resulting in reduced efficacy. While the effectiveness of the combination of adding stabilizers (sugars or surfactants) to EV preparations and freeze-drying preservation has been demonstrated, evaluations have focused only on particle count reduction and suppression of aggregation, and have not confirmed the maintenance of efficacy. Therefore, there is a need for formulation compositions and manufacturing methods that can guarantee the quality and functional stability of EV preparations as pharmaceuticals. To stabilize EVs, the inventors optimized the buffer composition and freeze-drying (hereinafter also referred to as "FD") protocol.

[0057] [Optimization of buffer composition] Sugars such as sucrose and trehalose are commonly used as cryoprotectants. For the EVs of the present invention, 6 wt% trehalose was optimal; lower concentrations resulted in degradation and aggregation. Poloxamer, a commonly used surfactant stabilizer, has been reported at 0.02%, but for the EVs of the present invention, 0.1% was optimal, suppressing degradation and aggregation.

[0058] [Freeze-Drying Protocol Optimization] Because excessive drying can adversely affect quality and functionality, we investigated shortening the drying time in the freeze-drying protocol. If the sample temperature exceeds the set shelf temperature, drying at that temperature is terminated. Therefore, under the new conditions, the program was modified to automatically proceed to the next drying step when the sample temperature exceeds the set shelf temperature by 5°C. The conventional freeze-drying protocol is shown in Figure 19(A), and the freeze-drying protocol of the present invention is shown in Figure 19(B). Due to the automatic setting, the actual operating time at each set shelf temperature differs from this protocol. Specifically, during the primary drying step after pre-freezing, the processing time at a shelf temperature of -25°C was shortened from 29 hours under normal conditions to 2.5 hours. The subsequent processing time at a shelf temperature of 0°C was also shortened from 3 hours to 1 hour. Furthermore, because the final drying time at 30°C has the greatest impact on excessive drying, the drying time was changed to 5 hours (previously 10 hours). Specifically, the secondary drying shelf temperature was optimized by shortening the processing time from 11 hours to 5 hours at a fixed temperature of 30°C and from 0 to 30°C. The drying time, which was required for a conventional freeze-drying protocol to a total of approximately 65 hours (the conventional conditions or samples obtained using this method are also referred to as "FD65h"), was shortened to a total of approximately 30 hours using the freeze-drying protocol of the present invention (the new conditions or samples obtained using this method are also referred to as "FD30h").

[0059] Below, the effect of shortening the drying time was confirmed for rehydrated samples of freeze-dried products obtained using each freeze-drying protocol, compared with the sample before freeze-drying (100%).

[0060] As shown in Figure 20, compared to before freeze-drying, the particle count retention rate for the rehydrated sample of "0.1% Poloxamer + FD65h" was in the 60% range, while the particle count retention rate for the rehydrated sample of "0.1% Poloxamer + FD30h" increased to approximately 80% / approximately 75%. As shown in Figure 21, in the rehydrated sample of "0.1% Poloxamer + FD30h," the frequency of the particle size distribution decreased with the decrease in particle count, but the peak position of the particle size distribution did not change before and after freeze-drying, suggesting that aggregation did not occur. Similar results were also obtained for the rehydrated sample of "0.02% Poloxamer + FD30h" (see Figure 22).

[0061] Multi-angle light scattering (MALS) was used to confirm the change in EV elution position before and after lyophilization. As shown in Figure 23, the retention time of the EV-derived peak (8.5 min) in the rehydrated sample of "Poloxamer 0.1% / 0.02% + FD30h" did not change before and after lyophilization. Therefore, it was considered that aggregation did not occur. As shown in Figure 24, the CD9 protein retention rate was less than 90% in the rehydrated sample of "Poloxamer 0.1% + FD65h," but improved to over 90% in the rehydrated sample of "Poloxamer 0.1% / 0.02% + FD30h." This suggests that shortening the drying time is effective in maintaining particle number and CD9 protein.

[0062] [Stability Evaluation of Lyophilized Products] Rehydrated samples of lyophilized products stored at room temperature for 3 weeks (after 3W) were evaluated for particle number retention, particle size distribution, and CD9 marker protein retention. As shown in Figure 25, the particle number retention of the rehydrated samples after 3W and immediately after lyophilization (immediately after FD) was approximately 80% compared to before lyophilization. As shown in Figure 26, the peak position of the particle size distribution did not change between the rehydrated samples immediately after FD and after 3W compared to before lyophilization. Therefore, it was considered that storage at room temperature for 3 weeks did not affect the aggregation of the rehydrated samples. As shown in Figure 27, the CD9 protein retention was over 90% for both the rehydrated samples immediately after FD and after 3W, and no change was observed after storage at room temperature for 3 weeks.

[0063] [Stability evaluation of freeze-dried product] A rehydrated sample of filter-sterilized "Poloxamer 0.1% + FD30h" freeze-dried product was added to TGF-β1-treated MRC-5, and the expression of collagen type-1 and α-SMA was evaluated. As shown in Figure 28, the rehydrated freeze-dried product exhibited an inhibitory effect on expression induction, demonstrating an anti-fibrotic effect against TGF-β1. It was determined that freeze-drying did not affect functionality.

[0064] [Evaluation of long-term stability of freeze-dried product] The particle count was measured for rehydrated samples of freeze-dried product stored for a long period at room temperature. The results are shown in Figure 29. No change in particle count over time was observed up to 12 months (12M) after freeze-drying. In addition, the anti-fibrotic effect was examined for rehydrated samples of freeze-dried product stored at room temperature for 12 months (12M). The results are shown in Figure 30. After 12 months (12M) of storage, an inhibitory effect on expression induction was observed, indicating an anti-fibrotic effect against TGF-β1. It was determined that long-term storage did not affect functionality.

[0065] [Effect of EV formulation rehydrated after freeze-drying as an inhalant] Freeze-dried EVs were rehydrated with distilled water (before nebulization), and the aerosol generated using an ultrasonic nebulizer was collected (after nebulization), and its quality was evaluated (see Figure 31). As shown in Figure 32, there was no significant change in the peak top of the particle size distribution before and after nebulization, suggesting that no aggregation occurred. As shown in Figure 33, the 2 × 10 9 The pre-nebulizer ("Before" in the figure) and post-nebulizer ("After" in the figure) samples of particles / ml showed similar anti-fibrotic effects on TGF-β1.

[0066] [Advantages over the prior art] The inventors optimized the buffer (trehalose dihydrate 6 wt%, poloxamer 0.1 wt%, 10 mM phosphate buffer, pH 7) for the EV formulation and the freeze-drying protocol (shortening the freeze-drying time to prevent overdrying). As a result, the EV formulation rehydrated after freeze-drying maintained approximately 80% of the particle number compared to before freeze-drying, with no change in the peak position of the particle size distribution and suppression of degradation and aggregation. Furthermore, CD9 marker protein was maintained at over 90%, and a similar level of anti-fibrotic activity was observed in a fibrosis model using TGF-β1-treated MRC-5 mice. Furthermore, the particle size distribution and anti-fibrotic activity of the rehydrated EV formulation were similar before and after aerosolization with an ultrasonic nebulizer. The EV formulation rehydrated after storing the freeze-dried product at room temperature for 3 weeks maintained approximately 80% of the particle number compared to before freeze-drying, with no change in the peak position of the particle size distribution, and CD9 marker protein retention of over 90%. The particle number and anti-fibrotic activity of the EV preparation rehydrated after 12 months of storage at room temperature were similar. Based on the above, we established a stable EV preparation composition and freeze-drying technology that maintains the quality and function of the EV preparation (freeze-dried product) stored at room temperature for 12 months and rehydrated at the same level as before freeze-drying.

[0067] [Isolation of Human Bronchial Basal Cells (HBBCs)] P0 cells isolated from the airways were sorted by FACS using an antibody against integrin αvβ8, a basal cell marker (see Figure 34). The sorted cells were cultured as αvβ8+ cells (HBBCs) and αvβ8- cells, respectively. As shown in Figure 35, αvβ8+ cells (HBBCs) and αvβ8- cells exhibited macroscopic morphological differences, with αvβ8+ cells exhibiting particularly spherical cell clusters, consistent with basal cell formation. The sorted cells were then cultured without further passage, and the culture supernatant was collected to evaluate the function of EVs. Specifically, primary lung fibroblasts treated with TGF-β1 (2 ng / ml) were cultured with αvβ8+ cell-EVs or αvβ8- cell-EVs (10 μg / ml) (EVs at 2 × 10 9The expression of collagen type-1 and α-SMA was evaluated by αvβ8 cell-EVs (administered at 100 μg / mL). The results are shown in Figure 36. αvβ8 cell-EVs were observed to have an inhibitory effect on the induction of αSMA and collagen type-1 expression, demonstrating an anti-fibrotic effect against TGF-β1.

[0068] [Confirmation of the Differentiation Potential of Bronchial Basal Cells] We examined whether human bronchial basal cells (HBBCs) retained the differentiation potential similar to that of human bronchial epithelial cells (HBECs) by air-liquid interface culture (ALI). For ALI, HBECs or HBBCs were seeded onto 12-mm transwell inserts (apical chambers) in 12-well plates (basal chambers). Cnt-PR-A medium was added to the basal and apical chambers and grown to confluence. To initiate differentiation, Cnt-PR-A medium was aspirated from both chambers. PneumaCult-ALI maintenance medium was prepared and added only to the basal chamber. The medium in the basal chamber was replaced every 2–3 days for a total of 23 days (see Figure 37). Cells on the transwell membrane were lysed for RNA expression analysis. The results of the RNA expression analysis are shown in Figure 38. On the horizontal axis of each graph, HBBCs after air-liquid interface culture are labeled HBBC ALI, and HBECs after air-liquid interface culture are labeled HBEC ALI. The vertical axis shows the names of genes whose expression levels were measured. Increased expression of each gene was observed in HBBC ALI and HBEC ALI after air-liquid interface culture. Furthermore, cells on the transwell membrane were fixed with 4% paraformaldehyde (PFA) and analyzed by immunocytochemistry (ICC) / immunofluorescence (IF). Alternatively, for cross-sectional analysis, cells were fixed with 4% PFA for 15 minutes at room temperature, incubated with 30% and 15% sucrose, embedded in Tissue-Tek® OCT compound, and snap-frozen in 2-methylbutane using liquid nitrogen. Frozen sections were cut at 7 μm using a cryostat and analyzed by ICC / IF. The results are shown in Figure 39. Similar expression of marker proteins was observed in HBBC ALI and HBEC ALI. These results confirm that human bronchial basal cells (HBBC) retain the differentiation potential similar to that of human bronchial epithelial cells (HBEC).

[0069] According to the present invention, a therapeutic or prophylactic agent whose effect is maintained can be provided.

Claims

1. A therapeutic or preventive agent for at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases, the therapeutic or preventive agent comprising basal cell-derived extracellular vesicles as an active ingredient.

2. The therapeutic or prophylactic agent according to claim 1, wherein the basal cells are human bronchial basal cells.

3. The therapeutic or prophylactic agent according to claim 1, which is administered via the airway.

4. A composition for treating or preventing at least one disease selected from the group consisting of fibrotic diseases, inflammatory diseases, and aging diseases, comprising the therapeutic or prophylactic agent according to any one of claims 1 to 3 and a pharmaceutically acceptable additive.

5. A pulmonary drug delivery system comprising human bronchial basal cell-derived extracellular vesicles and drugs.

Images