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WO2017105081A1 - Composition pharmaceutique pour le traitement de maladies pulmonaires ou l'atténuation des symptômes de maladies pulmonaires - Google Patents

Composition pharmaceutique pour le traitement de maladies pulmonaires ou l'atténuation des symptômes de maladies pulmonaires Download PDF

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WO2017105081A1
WO2017105081A1 PCT/KR2016/014654 KR2016014654W WO2017105081A1 WO 2017105081 A1 WO2017105081 A1 WO 2017105081A1 KR 2016014654 W KR2016014654 W KR 2016014654W WO 2017105081 A1 WO2017105081 A1 WO 2017105081A1
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atpase
protein
lung
present
pharmaceutical composition
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박성우
이지민
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Industry Academy Cooperation Foundation of Soonchunhyang University
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Industry Academy Cooperation Foundation of Soonchunhyang University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • the present invention relates to a pharmaceutical composition for treating or symptomatic pulmonary disease comprising V-atpase B2 protein as an active ingredient.
  • Vacuolar type H + -ATPase (V- ATPase) has to flow into a hydrogen ion (H + ion) cell is used as a transporter (transporter) of the cell membrane and the function of the intracellular pH at which the sustain acidified maintain homeostasis of the environmental cell It is a protein that plays a role.
  • the V-ATPase protein has isoforms depending on the tissue in which it is located, but basically consists of two different functional domains, VO and V1.
  • the V0 domain is present in the plasma membrane and the V1 domain is present in the cytoplasm.
  • the V0 domain is the portion where H ions are translocated and is composed of three subunits of c, d, and e.
  • the V1 domain consists of eight subunits of A3, B3, C, D, E2, F, G2, H.
  • the site where ATP binds is A3B3, which is known to cause hydrolysis of ATP.
  • V-ATPase The most basic role of V-ATPase in cells is to provide a proton pump that maintains an optimal acidic pH to process or degrade intracellular delivery of endosome and lysosomes. pumping) role.
  • pumping the most basic role of V-ATPase in cells is to provide a proton pump that maintains an optimal acidic pH to process or degrade intracellular delivery of endosome and lysosomes. pumping
  • An object of the present invention is to provide a pharmaceutical composition for treating pulmonary disease or alleviating symptoms comprising V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) as an active ingredient.
  • V-ATPASE B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • Another object of the present invention is to provide a food composition for preventing or ameliorating lung disease, comprising V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient.
  • V-Etipiase B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • Another object of the present invention is to provide a method for treating or alleviating pulmonary disease, comprising administering to a subject V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein.
  • Another object of the present invention is a method for suppressing or alleviating lung injury by smoking or secondhand smoke, comprising administering to a subject a V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein.
  • V-atpase B2 V-atpase B2, Vacuolar-type H + -ATPase B2 protein.
  • Another object of the present invention is to provide a use for the use of the V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein in the manufacture of a medicament for the treatment of pulmonary disease or alleviation of symptoms. will be.
  • V-Etipiase B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • Another object of the present invention is to use a V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein for the manufacture of a medicament for inhibiting or alleviating lung injury by smoking or secondhand smoking. It is to provide a use.
  • V-ATPASE B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • the pharmaceutical composition of the present invention contains the V-atpase B2 protein as an active ingredient, and is useful for treating lung diseases or alleviating symptoms, and in particular, for treating or alleviating symptoms of acute lung injury, pulmonary fibrosis or emphysema among lung diseases. Can be. In addition, it can be utilized as a pharmaceutical composition for suppressing or alleviating lung damage by smoking or secondhand smoke.
  • FIG. 1 is a diagram showing the sequence information of the V-Etipiase B2 protein represented by SEQ ID NO: 1 of the present invention.
  • Figure 2 is IF staining results confirming whether Vatpase B2 protein expression control of the genetically modified mouse prepared in Example 1 of the present invention.
  • Figure 3 is a Western blotting (Western blotting) results confirming whether the control of Vatpase B2 protein expression of the genetically modified mouse prepared in Example 1 of the present invention.
  • Figure 4 is a schematic diagram illustrating the manufacturing process of a mouse model induced acute lung injury pulmonary fibrosis induced in V-atpase B2 TG mice in Example 2 of the present invention.
  • Figure 5 is a graph showing the results of measuring the total lung inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes from bronchoalveolar lavage fluid (BALF) of acute lung injury pulmonary fibrosis mouse model induced in Example 2 of the present invention.
  • BALF bronchoalveolar lavage fluid
  • Figure 6 is a result of H & E staining showing the degree of lung tissue damage in a lung lung fibrosis mouse model induced acute lung injury in Example 2 of the present invention.
  • Figure 7 is a trichrome staining result of the degree of collagen accumulation in the lung tissue of a mouse model of acute lung injury pulmonary fibrosis induced in Example 2 of the present invention.
  • Figure 8 is a graph showing the results of measuring the total pulmonary inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes count from bronchoalveolar lavage fluid (BALF) of CS-induced emphysema mouse model in Example 3 of the present invention.
  • BALF bronchoalveolar lavage fluid
  • Example 9 is a result of measuring lung mechanics (left: lung purity, right: lung elasticity) using the lung tissue of the CS-induced emphysema mouse model in Example 3 of the present invention.
  • Figure 10 is the result of observing the alveolar tissue of the CS-induced emphysema mouse model in Example 3 of the present invention by H & E staining.
  • Figure 11 shows the results confirmed by IF staining the influx of Vatpase B2 protein using 293T cells in Example 4 of the present invention.
  • Example 12 is a result confirmed by Immunoblotting of the cellular influx of Vatpase B2 protein using 293T cells in Example 4 of the present invention.
  • Figure 13 shows the results of localization of the V-atpaseB2 protein in 293T cells in Example 4 of the present invention using double immunofluorescence staining.
  • FIG. 14 is a schematic diagram illustrating a procedure for testing the effect of V-atpase B2 protein nasal administration in a mouse lung disease model in Example 4 of the present invention.
  • Example 15 is a graph showing the results of measuring the total pulmonary inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes from bronchoalveolar lavage fluid (BALF) of the mouse lung disease model induced in Example 4 of the present invention.
  • BALF bronchoalveolar lavage fluid
  • Example 16 is an immunofluorescence staining result confirming that Vatpase B2 is introduced into the tissue of the mouse lung disease model induced in Example 4 of the present invention.
  • Figure 17 is the result of observing the tissue of the mouse lung disease model induced in Example 4 of the present invention by H & E staining.
  • Example 18 is a result of confirming the degree of collagen accumulation by massons trichrome stain the tissue of the mouse lung disease model induced in Example 4 of the present invention.
  • Example 19 is a result of confirming and quantifying the amount of collagen fibers accumulated in the tissue of the mouse lung disease model induced in Example 4 of the present invention by a sircol assay (* p ⁇ 0.05 by Mann-Whitney U test).
  • a pharmaceutical composition for treating or symptomatic pulmonary disease is V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein active ingredient Include as.
  • the V-Etipiase B2 protein may be hV-atpase B2 represented by SEQ ID NO: 1.
  • the lung disease may be acute lung injury, pulmonary fibrosis or emphysema.
  • the pharmaceutical composition for inhibiting or alleviating pulmonary injury by smoking or indirect smoking includes V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient. Include.
  • V-ATPase B2 protein which is a lung disease, especially acute lung injury, pulmonary fibrosis, emphysema.
  • the present invention was completed by using an animal model to confirm that it is effective in treating and relieving symptoms of lung injury diseases.
  • the pharmaceutical composition according to an embodiment of the present invention may include V- ATPASE B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient and may be applied for treating lung disease or alleviating symptoms. have.
  • V-ATPASE B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • the V-Etipiase B2 protein may be a V-Etipiase B2 protein of a mammal, including human, preferably a human V-Etipiase B2 protein, specifically 511 shown in SEQ ID NO: 1 and FIG. V-Etipiaze B2 protein having 4 amino acid sequences.
  • the lung disease may be acute lung injury, pulmonary fibrosis or emphysema, and in particular, the pharmaceutical composition may be useful for lung disease accompanied by cell damage related to oxidative stress.
  • the lung disease may be emphysema induced by tobacco smoke.
  • treatment of the present invention means any action that improves or advantageously changes the symptoms of the lung disease by administration of the composition according to the present invention.
  • the term “relaxation” means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.
  • the pharmaceutical composition of the present invention may be applied to suppress or alleviate lung damage by smoking or secondhand smoke, including V-atpase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient. .
  • the lung injury may be emphysema.
  • Administration of the pharmaceutical composition may be by a method generally accepted as a method of administering the above-mentioned pharmacological activity, and specifically to oral, parenteral or topical administration, systemic administration by other methods, etc. You can.
  • the pharmaceutical composition is preferably administered to the patient by the method of intranasal administration or bronchial administration in a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also be formulated as a pharmaceutical composition suitable for parenteral administration to a patient, eg, intravenous, arterial, spinal or intraperitoneal.
  • the pharmaceutical composition may be used in the form of a solid, semi-solid or liquid formulation, preferably for example as a pill, capsule, powder, liquid, suspension, etc., suitable for the administration of the correct dose.
  • the pharmaceutical composition may include conventional pharmaceutical carriers or excipients, and may also include other pharmaceutical agents, pharmaceutical agents, carriers, additives, and the like.
  • the excipient may include, but is not limited to, other proteins such as, for example, human serum albumin or plasma proteins.
  • the pharmaceutical composition may be administered at the same time or separately from the existing therapeutic agent in the treatment of the lung disease.
  • the administered pharmaceutical composition or formulation thereof contains an amount of the active ingredient in an amount effective to achieve a desired effect in the subject being treated, and the amount of the active ingredient administered to the patient is determined by the characteristics of the subject, the severity of the disease, Depending on the mode of administration and the judgment of the doctor, it is better to administer a relatively low concentration of protein.
  • the pharmaceutical composition may be 1 x 10 5 IU / person to 9 x 10 6 IU / person.
  • the present invention provides a food composition for preventing or ameliorating lung disease, comprising V-Etipiase B2 (V-atpase B2, Vacuolar-type H + -ATPase B2) protein as an active ingredient.
  • V-Etipiase B2 V-atpase B2, Vacuolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • pulmonary disease is as described above.
  • improvement refers to any action that improves or benefits the suspicion of a disease and the onset of a pulmonary disease to be prevented or treated using a food composition comprising V-Etipiase B2 as an active ingredient.
  • prevention of the present invention means any action that inhibits or delays the onset of the lung disease by administration of the composition according to the present invention.
  • V-Etipiaze B2 of the present invention can be added to food compositions for the purpose of preventing or ameliorating lung diseases.
  • the food composition of the present invention may include the form of pills, powders, granules, acupuncture, tablets, capsules or liquids, etc., and there is no particular limitation on the kind of foods to which V-Etipiaze B2 of the present invention may be added. Examples include various beverages, gums, teas, vitamin complexes, and dietary supplements.
  • V-Etipia agent B2 In addition to the V-Etipia agent B2, other ingredients may be added to the food composition, and the kind thereof is not particularly limited.
  • various herbal extracts, food acceptable food additives, natural carbohydrates, and the like may be included as additional ingredients, such as conventional foods, but are not limited thereto.
  • the term "food supplement” means a component that can be added to food supplements, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation.
  • food additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like, but is not limited to the kind of food additives of the present invention by the above examples.
  • Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol, and as flavoring agents other than those described above, natural flavoring agents (such as taumartin), stevia extract (rebaudioside A, glycyr) Higin and the like) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • monosaccharides such as glucose and fructose
  • Disaccharides such as maltose and sucrose
  • polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol
  • sugar alcohols such as xylitol, sorbi
  • the food composition of the present invention may include a health functional food.
  • the term "health functional food” refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body.
  • functional means to obtain a useful effect for health use such as nutrient control or physiological action on the structure and function of the human body.
  • the health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art.
  • unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug with food as a raw material, can be excellent in portability.
  • the mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
  • the V-Etipiaze B2 of the present invention may be added in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight, in the manufacture of food, but is not limited thereto.
  • the amount may be used below the above range.
  • Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes and the like, and include all of the dietary supplements in the conventional sense.
  • the present invention provides a method of treating or alleviating pulmonary disease, comprising administering to a subject a V-atpase B2 (Vacuolar-type H + -ATPase B2) protein. do.
  • V-atpase B2 Vauolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • pulmonary disease is as described above.
  • the term "individual” means all animals including a human having a lung disease or a lung disease. Mammals, birds, and the like, including cattle, pigs, sheep, chickens, dogs, humans, and the like, and include, without limitation, individuals whose lung diseases are treated or alleviated by the protein of the present invention.
  • the present invention provides a method for inhibiting lung injury by smoking or secondhand smoke, comprising administering to a subject a V-atpase B2 (Vacuolar-type H + -ATPase B2) protein. Or provide mitigation.
  • V-atpase B2 Vauolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • lung damage is as described above.
  • the term "individual” means all animals including a human in which lung injury has occurred or a lung injury has occurred. Mammals, birds, and the like, including cattle, pigs, sheep, chickens, dogs, humans, and the like, and include, without limitation, individuals whose lung injury is inhibited or alleviated by the protein of the present invention.
  • the present invention provides the use of the V- atpase B2 (Vacuolar-type H + -ATPase B2) protein for use in the manufacture of a medicament for the treatment of pulmonary disease or alleviation of symptoms. to provide.
  • V- atpase B2 Vauolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • pulmonary disease is as described above.
  • the present invention uses the V-atpase B2 (Vacuolar-type H + -ATPase B2) protein in the manufacture of a medicament for inhibiting or alleviating lung injury by smoking or secondhand smoking. It provides a use for.
  • V-atpase B2 Vauolar-type H + -ATPase B2
  • V-atpase B2 (Vacuolar-type H + -ATPase B2)
  • lung damage is as described above.
  • Example 1 alveoli (type II pneumocyte )on Vatpase Preparation of engineered mice overexpressing B2 protein
  • type II pneumocyte Vatpase B2 protein was overexpressed, and double transgenic mice were prepared to control the overexpression time by DOXY.
  • hVatpase B2 construct was constructed by subcloning the VATPaseB2 cDNA construct containing 1x flag sequene in the TRE-tight vector using RT-PCR, and it was possible to accurately detect V-atpase B2 protein expression as Flag Ab.
  • Double transgenic mice which were made by injecting two constructs into the eggs of C57 / BL6 mice, were applied to the following experiments.
  • mice were confirmed by IF staining and Western blotting using Flag Ab. Whether Vatpase B2 protein expression was regulated by DOXY, and the results are shown in FIGS. 2 and 3, respectively.
  • mice prepared for the following experiments were well expressed in the flag tagged hVatpase B2 protein depending on whether DOXY was introduced.
  • Example 2 Acute induction with engineered mice Lung injury Experimental Effects of Overexpressed V-atpase B2 in a Mouse Model
  • Acute lung injury / pulmonary fibrosis was induced in experimental animals by administering bleomycin into the bronchus (intratracheal) of V-atpase B2 TG mice prepared above. Specifically, as shown in FIG. 4 below, 5 days before bleomycin administration, Doxy water was administered to induce overexpression of V-atpase B2 and sacrifice at 14 days after bleomycin administration to bronchoalveolar lavage fluid (BALF), bronchus and pesos. After the organization was conducted, the experiment was conducted.
  • BALF bronchoalveolar lavage fluid
  • Bronchoalveolar labage fluid (BALF) in mice was obtained by carefully inhaling 1000 ⁇ l of PBS into the airway of the disease-induced mice repeatedly with a syringe at 37 ° C. After washing with Han's balanced salt soulution (HBSS) containing 2% FCS (fetal calf serum), erythrocytes were removed using lysis buffer, and the supernatants were collected separately by centrifugation.
  • HBSS Han's balanced salt soulution
  • FCS fetal calf serum
  • Sacred mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 ⁇ m intervals to prepare tissue samples.
  • the tissue samples were deparaffinized and stained with 200 ⁇ l of hematocyline for 1 minute and washed with water for 10 minutes. Subsequently, 1 minute staining with 200 ⁇ l of Eosin was followed by washing with water for 10 minutes, followed by mounting through a dehydration process. The results are shown in FIG. 6.
  • tissue damage was significantly reduced in samples of the experimental group overexpressed with Doxy (+) V-atpase B2 protein.
  • Sacred mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 ⁇ m intervals to prepare tissue samples.
  • Tissue samples were washed with running water after deparaffinization and treated with 200 ⁇ l of Bouin solution for 1 hour in a 56 ° incubator. It was washed with running water and stained with Weigrt iron hematoxylin 200 ⁇ l at room temperature for 10 minutes.
  • the treated tissue samples were washed with running water, dehydrated, and then mounted, and the results are shown in FIG. 7.
  • Example 3 Experimental Effects of V-atpase B2 Overexpressed in a Mouse Model of Cigarette Smoke Induced Emphysema
  • mice Genetically modified mice (Vatpase B2 overexpressing mice) prepared in Example 1 above were exposed to cigarette smoke five times a week for three months for five months to prepare a CS (cigarette smoke) induced emphysema mouse model.
  • CS cigarette smoke
  • Vatpase B2 overexpressing mice were exposed to CS in a plexiglass chamber (16x 25x 16cm) for 6 months using a smoking tester system (ThreeShineCom, Daejeon, Korea). Overexpression of V-atpase B2 was induced by the administration of Doxy containing water (50mg / ml) from the first day of exposure to CS. CS was used with a 3R4F burning reference cigarette for 5 hours, 3 times a week, for 6 months. Exposure to tobacco smoke.
  • VaTPase B2 overexpression test group VATPaseB2 air
  • WT_CS Vatpase B2 overexpression test group
  • the CS-induced emphysema mouse model was prepared, and then sacrificed to obtain bronchoalveolar lavage fluid (BALF), bronchial and alveolar tissues, and then experiments were performed.
  • BALF bronchoalveolar lavage fluid
  • Vatpase B2 overexpression may be the result of treating or preventing CS-induced emphysema.
  • Emphysema is characterized by increased lung compliance (static compliance) and reduced elasticity (elastance), the lung mechanics of the sacrificed mouse lung tissue was measured using a Flexi-vent device, the results are shown in FIG.
  • Observations of hematoxylin and eosin (H & E) staining were performed using lung tissue of a cigarette smoke induced emphysema mouse model as described above, and the results are shown in FIG. 10.
  • Doxy (+) / CS that is, normal alveoli without air space enlargement (Doxy (-) CS) does not appear when smoking Vatpase B2 overexpressing mice. It was confirmed that the appearance of.
  • Vatpase B2 significantly reduced animal emphysema induced by CS.
  • VatpaseB2 has a pulmonary protective action against CS and can be used for the prevention or treatment of emphysema. Is thought to be the result showing.
  • Example 4 V- in mouse lung disease model atpase B2 protein nasal administration and acute lungs Damage / Fibrosis Treatment Effect
  • Vatpase B2 protein (ORIGENE, USA, flag tagged recombinant protein, 56K.D.) was administered to 293T cells to confirm that the protein was introduced into the cell during the endocytosis process as follows.
  • 293T cells (ATCC No .: CRL-3216) were treated with 10% (v / v) heat-inactivated fetal bovine serum, 10,000 U / mL Penicillin, 10,000 ⁇ g / mL Streptomycin at 37 ° C. in a humidified atmosphere of 5% CO 2 . Incubated in Dulbecco's modified Eagle's medium, and the cultured 293T cells were administered with Vatpase B2 at 2 ng / well, followed by incubation for 17 hours under the same conditions, followed by IF staining.
  • the culture medium was removed, washed with DPBS, fixed with 15% of 4% paraformaldehyde for 15 minutes, washed with water, and then subjected to permiabilization using Triton X-100. Washed with DPBS and blocked for 1 hour at room temperature with 5% goat normal serum, the first anti-flag was diluted in a blocking solution at 10ug / ml and incubated at 4 °C 17 hours. Subsequently, the secondary antibody (Anti-Rabbit FITC) was diluted 1: 1000, treated, incubated at room temperature for 1 hour while blocking light, and nuclear stained with DAPI. The samples thus treated were mounted with a mounting solution for fluorescence staining, and the results were confirmed with a fluorescence microscope, and are shown in FIG. 11.
  • Vatpase B2 protein was well introduced into the cytoplasm of 293T cells by the method.
  • Vatpase B2 protein (2 ⁇ g / ml) was introduced into the same 293T cells as used in the above experiments, lysis buffer was added to prepare cell lysates, and proteins were extracted. The protein was developed using 10% SDS-PAGE, broken with 5% skim milk and then anti-FLAG antibody (SIGMA, 1: 5000) as the primary antibody and anti-rabbit as the secondary antibody. After the reaction using IgG (1: 5000) to perform a detecting process, the results are shown in Figure 12 below. Beta actin was applied as a positive control.
  • mice Male C57BL / 6 mice were distributed in Oriental Bio, followed by a five-day acclimation period in the laboratory, followed by induction of pulmonary damage with bleomycine and then applied to experiments confirming the therapeutic effect of Vatpase B2 protein (FIG. 14).
  • mice undergoing the adaptation period were administered bleomycin into the airways to induce acute lung injury and fibrosis, and 500 ng of V-atpase B2 protein was administered through the nasal cavity from 4 days to 7 days.
  • mice Two weeks after the administration of bleomycin, mice were sacrificed, and bronchial alveolar lavage fluid (BALF), bronchial and alveolar tissues were obtained, and then experiments were performed.
  • BALF bronchial alveolar lavage fluid
  • bronchoalveolar lavage fluid obtained from the above experimental animals. Obtaining BALF and measuring the number of pulmonary inflammatory cells, macrophages, neutrophils, eosinophils, lymphocytes and the like was applied to the same method as described above, the results are shown in Figure 15 below.
  • FIG. 15 it was confirmed that inflammation due to lung injury induced by v-atpase B2 protein administration was alleviated or suppressed. Specifically, the number of total pulmonary inflammatory cells, neutrophils, and macrophages was induced in the lung injury-inducing group. Compared with the experimental group was significantly reduced.
  • Sacred mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 ⁇ m intervals to prepare tissue samples and subjected to immunofluoroscence staining.
  • Tissue samples were subjected to deparaffinization, washed with running water, and subjected to permiabilization using Triton X-100. Washed with DPBS, blocked for 1 hour at room temperature with 5% goat normal serum, diluted the anti-flag at 10 ug / ml in a blocking solution and incubated at 4 ° C for 17 hours, followed by secondary antibody ( Anti-Rabbit FITC) was diluted 1: 1000, incubated for 1 hour at room temperature with light blocking, and nuclear stained with DAPI. Mounting with a mounting solution for fluorescence staining and observing with a fluorescence microscope to confirm the results are shown in Figure 16.
  • Vatpase B2 was well introduced into the bronchus and alveolar cells of the lung through an immunofluoroscence stain using Flag-FITC Ab.
  • Tissue samples were prepared from the sacrificed mouse lung tissues as described above, followed by staining with hematoxylin and eosin (H & E), and the results are shown in FIG. 17.
  • Trichrome staining was used to determine the effect of inhibiting pulmonary fibrosis of V-atpase B2 protein in mouse lung tissue. Specifically, the sacrificed mouse lung tissue was fixed with 4% paraformaldehyde, prepared with paraffin blocks, and cut at 4 ⁇ m intervals to prepare tissue samples. Tissue samples were washed with running water after deparaffinization and treated with 200 ⁇ l of Bouin solution and embedded in an incubator at 56 ° C. for 1 hour. It was washed with running water and stained with 200 ⁇ l of Weigrt iron hematoxylin at room temperature for 10 minutes.
  • the Sircol assay confirmed the effect of inhibiting pulmonary fibrosis of V-atpase B2 protein in mouse lung tissue.
  • pulmonary tissue lysate was prepared by adding lysis buffer to the sacrificed mouse lung tissue, and the protein was extracted. Protein lysate and standard solution were added to each test tube, sircol dye was added, and reacted at room temperature for 30 minutes. After centrifugation at 14000 rpm for 15 minutes, the supernatant was removed, and the pellet was washed with wasing buffer. After centrifugation at 14000 rpm for 15 minutes, the supernatant was removed, and the pellet was sufficiently released with alkari reagent.
  • the collagen amount was measured at a wavelength of 540 nm with an ELISA reader machine, and the value quantified and corrected for the protein is shown in FIG. 19.
  • V-atpase B2 protein is introduced into the intracellular lysosome through endocytosis when administered in vitro or in mice.
  • Animal models of injury and pulmonary fibrosis were found to have a function of effectively inhibiting inflammation and fibrosis of the lungs.

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  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Zoology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Marine Sciences & Fisheries (AREA)

Abstract

Une composition pharmaceutique pour le traitement de maladies pulmonaires ou l'atténuation des symptômes de maladies pulmonaires, selon la présente invention, comprend une protéine H+-ATPase B2 de type vacuolaire (V-atpase B2) utilisée comme principe actif. La composition pharmaceutique est utile pour le traitement de maladies pulmonaires, en particulier d'une lésion pulmonaire aiguë, d'une fibrose pulmonaire ou d'un emphysème pulmonaire, ou d'atténuer les symptômes de celles-ci.
PCT/KR2016/014654 2015-12-14 2016-12-14 Composition pharmaceutique pour le traitement de maladies pulmonaires ou l'atténuation des symptômes de maladies pulmonaires Ceased WO2017105081A1 (fr)

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KR10-2015-0178303 2015-12-14

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KR101665269B1 (ko) * 2015-12-14 2016-10-12 순천향대학교 산학협력단 폐질환 치료용 또는 증상완화용 약학조성물
KR102868970B1 (ko) 2022-07-28 2025-10-13 주식회사 우리기술 인디루빈 함량을 증대시키는 대청엽의 수경재배 방법 및 그에 따라 제조된 대청엽

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US6982252B2 (en) * 2000-12-04 2006-01-03 Memorial Sloan-Kettering Cancer Center Inhibition of vacuolar proton ATPase activity and/or the modulation of acidic organelle function sensitizes cells to radiation, chemotherapy and biological agents
EP1779848A1 (fr) * 2005-10-28 2007-05-02 Nikem Research S.R.L. Inhibiteurs de la V-ATPase pour le traitement des maladies inflammatoires et autoimmunes
US20110189306A1 (en) * 2010-01-13 2011-08-04 Norbert Kartner COMPOUNDS, COMPOSITIONS AND TREATMENTS FOR V-ATPase RELATED DISEASES
KR101665269B1 (ko) * 2015-12-14 2016-10-12 순천향대학교 산학협력단 폐질환 치료용 또는 증상완화용 약학조성물

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US6982252B2 (en) * 2000-12-04 2006-01-03 Memorial Sloan-Kettering Cancer Center Inhibition of vacuolar proton ATPase activity and/or the modulation of acidic organelle function sensitizes cells to radiation, chemotherapy and biological agents
EP1779848A1 (fr) * 2005-10-28 2007-05-02 Nikem Research S.R.L. Inhibiteurs de la V-ATPase pour le traitement des maladies inflammatoires et autoimmunes
US20110189306A1 (en) * 2010-01-13 2011-08-04 Norbert Kartner COMPOUNDS, COMPOSITIONS AND TREATMENTS FOR V-ATPase RELATED DISEASES
KR101665269B1 (ko) * 2015-12-14 2016-10-12 순천향대학교 산학협력단 폐질환 치료용 또는 증상완화용 약학조성물

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