KR102651607B1 - Composition for preventing, treating, or alleviating eosinophilic inflammatory diseases or Th2 hypersensitivity immune diseases comprising vesicle derived from lactococcus lactis - Google Patents

Abstract

The present invention relates to a composition for preventing, treating, or improving eosinophilic inflammatory diseases and Th2 hypersensitivity immune diseases containing Lactococcus lactis-derived vesicles. The Lactococcus lactis-derived vesicles according to the present invention are used in a Th2 hypersensitivity immune disease mouse model. It was confirmed that when administered, it suppresses eosinophilic inflammation and the functional and histopathological changes that occur as a result. In addition, it suppressed the secretion of Th2 cytokines such as IL-5 and IL-13 from T cells in a mouse model of Th2 hypersensitivity immune disease, and this induced the secretion of IL-12, which suppresses the Th2 immune response, in dendritic cells, which are antigen-presenting cells. As confirmed, it is expected that the Lactococcus lactis-derived vesicles according to the present invention can be usefully used as a composition for preventing, treating, or improving eosinophilic inflammatory diseases and Th2 hypersensitivity immune diseases.

Description

Composition for preventing, treating, or alleviating eosinophilic inflammatory diseases or Th2 hypersensitivity immune diseases comprising vesicle derived from lactococcus lactis}

The present invention relates to a composition for preventing, treating, or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease containing Lactococcus lactis-derived vesicles.

Eosinophilic inflammatory diseases are a group of diseases in which a large number of eosinophils are found within the lesion or have characteristics in which eosinophils are presumed to play an important pathophysiological role in the development of the disease. Eosinophilic inflammatory disease can occur due to a variety of causes, and the disease may vary depending on the site of inflammation. For example, in eosinophilic pneumonia, a representative eosinophilic inflammatory disease, infiltration of numerous eosinophils can be seen in the lung parenchyma, whereas allergic bronchopulmonary aspergillosis may occur localized in the respiratory tract.

Among eosinophilic inflammatory diseases, the cause of eosinophilic pneumonia can be known, such as parasitic infection or medication, and the cause can be unknown, such as Leffler syndrome, chronic eosinophilic pneumonia, or Schöck-Strauss allergic granulomatosis. When inflammation occurs, inflammatory cells such as monocytes, giant cells, and eosinophils infiltrate into the alveoli, and interstitial pulmonary infiltration is sometimes accompanied. In addition, allergic asthma is a chronic inflammatory disorder of the airways associated with Th2 (type 2 helper T) cell-dependent immune response, eosinophilia, and IgE production. In the development of allergic asthma, Th2 cells produce interleukin (IL)-which mediates the immune response. 5, produces various cytokines including IL-9 and IL-13. Among these factors, IL-5 and IL-9 contribute to eosinophilia and mast cell proliferation, while IL-13 is involved in mucus hypersecretion, and dendritic cells are important for antigen presentation and T cell differentiation in lymphoid organs as a result of allergen exposure. It plays a role.

Dendritic cells can generally regulate the Th1/Th2 balance by producing the cytokine IL-4, which induces differentiation into Th2 cells, as well as IL-12, which induces differentiation of Th1 cells. Among many factors, bacteria are also known to interact with dendritic cells to regulate allergic airway inflammation.

Changes in the abundance and diversity of commensal bacteria may influence asthma exacerbations by contributing to inflammation and remodeling of the lung. In particular, probiotics (defined as live microorganisms that exert beneficial effects on the host) have been proposed to prevent allergic reactions through several mechanisms. These are primarily associated with the development of regulatory T cells that produce IL-10, also known as an immunosuppressive cytokine. Among probiotics , Bifidobacterium breve has been shown to attenuate airway inflammation by inducing IL-10-producing T cells. These non-pathogenic and non-invasive bacteria are already being used in biotechnology applications, and probiotics are safe in the long term. It has been shown that there are advantages with

Meanwhile, the number of microorganisms living in the human body reaches 100 trillion, which is 10 times more than human cells, and the number of genes in microorganisms is known to be more than 100 times that of humans. Microbiota (microbiota or microbiome) refers to the microbial community including bacteria, archaea, and eukaryotes that exist in a given habitat, and the intestinal microflora plays an important role in human physiology. It is known to have a significant impact on human health and disease through interactions with human cells. Bacteria that live in our body secrete nanometer-sized vesicles to exchange information such as genes and proteins with other cells. The mucosa forms a physical barrier that prevents particles larger than 200 nanometers (nm) from passing through the mucosa, so bacteria that live in the mucosa cannot pass through the mucosa. However, vesicles derived from bacteria are usually less than 200 nanometers in size, so they are relatively easy to pass through. It freely passes through mucous membranes and is absorbed into our body. Bacterial-derived vesicles are secreted from bacteria, but their composition, body absorption rate, risk of side effects, etc. are different from each other. As a result, using bacterial-derived vesicles has a completely different or significant effect than using live bacteria.

Extracellular vesicles (EVs; membrane-bound organelles that transport cargoes of proteins, nucleic acids, lipids, and metabolites) are released by all cell types, including eukaryotes and prokaryotes, under physiological and pathological conditions. Because these new molecules have the ability to interact between cells with associated effects on the immune system, EVs are known to be involved in several human diseases such as cancer, metabolic disorders, and allergic diseases.

Bacteria of the genus Lactococcus are gram-positive cocci that secrete lactic acid. Among them, Lactococcus lactis is known to be an important bacterium in the fermentation of dairy products such as cheese, fermented vegetables, and alcoholic beverages. Lactis bacteria can be isolated from milk and fermented plant materials.

However, nothing is yet known about the therapeutic effect of Lactococcus bacteria-derived vesicles on eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.

Korean Patent No. 10-2299252

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, which contains vesicles derived from Lactococcus lactis as an active ingredient.

Another object of the present invention is to provide an inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, which contains vesicles derived from Lactococcus lactis as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, which contains vesicles derived from Lactococcus lactis as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, which contains vesicles derived from Lactococcus lactis as an active ingredient.

Another object of the present invention is to provide a composition for delivering a drug for treating respiratory diseases, comprising vesicles derived from Lactococcus lactis as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, which contains vesicles derived from Lactococcus lactis as an active ingredient.

In addition, the present invention provides an inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient.

In addition, the present invention provides a quasi-drug composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient.

In one embodiment of the present invention, the eosinophilic inflammatory disease is an allergic inflammatory disease,

The allergic inflammatory disease may include, but is not limited to, eosinophilic drug allergy, eosinophilic asthma, allergic rhinitis, and atopic dermatitis.

In another embodiment of the present invention, the eosinophilic inflammatory disease is an eosinophilic inflammatory disease with unknown causative agent,

The eosinophilic inflammatory diseases of unknown cause include eosinophilic cardiomyopathy, eosinophilic colitis, eosinophilic enteritis, eosinophilic esophagitis, eosinophilic gastritis, Includes eosinophilic pneumonia, eosinophilic bronchitis, eosinophilic fasciitis, hypereosinophilic syndrome, and Churg-Strauss syndrome or eosinophilic granulomatosis with polyangiitis. It can be done, but is not limited to this.

As another embodiment of the present invention, the Th2 hypersensitivity immune disease includes atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, and hypersensitivity pneumonitis ( Hypersensitivity pneumonitis, food allergy, drug allergy, and anaphylaxis may include, but are not limited to.

In another embodiment of the present invention, the eosinophilic inflammatory disease may be a respiratory disease showing Th2 (type 2 helper T) cell hypersensitivity, but is not limited thereto.

As another embodiment of the present invention, the respiratory diseases showing Th2 cell hypersensitivity include atopic asthma, allergic rhinitis, eosinophilic bronchitis, and hypersensitivity pneumonitis. It may include, but is not limited to.

As another embodiment of the present invention, the eosinophilic inflammatory disease or Th2 hypersensitivity immune disease may be a respiratory disease characterized by excessive secretion of mucus in the respiratory tract, but is not limited thereto.

As another embodiment of the present invention, respiratory diseases characterized by excessive secretion of mucus in the respiratory tract may include, but are not limited to, chronic rhinitis, chronic sinusitis, and chronic bronchitis.

As another embodiment of the present invention, the Lactococcus lactis-derived vesicles can suppress airway hyperresponsiveness, but are not limited thereto.

As another embodiment of the present invention, the vesicles may have an average diameter of 10 to 200 nm, but are not limited thereto.

As another embodiment of the present invention, the vesicles may be naturally or artificially secreted from Lactococcus lactis , but are not limited thereto.

Additionally, the present invention provides a composition for delivering a drug for treating respiratory diseases, comprising vesicles derived from Lactococcus lactis as an active ingredient.

Additionally, the present invention provides a method for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising administering to an individual a composition containing vesicles derived from Lactococcus lactis as an active ingredient.

In addition, the present invention provides the use of a composition containing vesicles derived from Lactococcus lactis as an active ingredient for the prevention or treatment of eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.

Additionally, the present invention provides the use of Lactococcus lactis -derived vesicles for the production of a drug for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.

In addition, the present invention provides a method of delivering a drug for treating respiratory diseases, comprising the step of administering to an individual a composition containing vesicles derived from Lactococcus lactis carrying the desired drug for treating respiratory diseases as an active ingredient. .

Additionally, the present invention provides the use of a composition containing Lactococcus lactis -derived vesicles as an active ingredient for drug delivery for the treatment of respiratory diseases.

Additionally, the present invention provides the use of vesicles derived from Lactococcus lactis for the production of a medicament for preventing or treating respiratory diseases.

When the Lactococcus lactis-derived vesicles according to the present invention were administered to a mouse model of Th2 hypersensitivity immune disease, the secretion of IFN-γ was increased in Th1 cells and the secretion of IL-5 and IL-13 in Th2 cells was decreased. It was confirmed that increasing the secretion of IL-12p70 from dendritic cells induced a shift in immune response from Th2 cell immune response to Th1 cell immune response. In addition, it exhibits effects such as suppressing airway hyperresponsiveness, reducing eosinophil infiltration into lung tissue, and suppressing mucus production in the lung, and the Lactococcus lactis-derived vesicles according to the present invention are used to prevent eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, It is expected that it can be usefully used as a treatment or improvement composition.

Figures 1A and 1B confirm the characteristics of Lactococcus lactis-derived vesicles according to an embodiment of the present invention. Figure 1A is a diagram confirming the vesicle image using a transmission electron microscope (scale bar: 50 nm), and Figure 1B is a diagram confirming the protein component in the vesicle.
Figures 2a and 2b confirm the characteristics of vesicles derived from Bifidobacterium brevi according to an embodiment of the present invention. Figure 2a is a diagram confirming the vesicle image using a transmission electron microscope (scale bar: 50 nm). Figure 2b is a diagram confirming protein components in vesicles.
Figure 3 is a diagram showing an experimental protocol for evaluating the therapeutic effect of Lactococcus lactis-derived vesicles and Bifidobacterium brevi-derived vesicles in a Th2 hypersensitivity immune disease mouse model according to an embodiment of the present invention.
Figures 4a and 4b show the effects of Lactococcus lactis-derived vesicles ( L. lactis ) and Bifidobacterium brevi-derived vesicles ( B. breve ) on eosinophil infiltration in the Th2 hypersensitivity immune disease mouse model according to one embodiment of the present invention. As confirmation of the impact, Figure 4a is a diagram showing an image of H&E-stained cells in bronchoalveolar lavage fluid (BALF), and Figure 4b is a diagram showing the number of eosinophils in bronchoalveolar lavage fluid (BALF) (n=5, * *P<0.01, ***P<0.001, ns means not significant).
Figure 5 shows that in the Th2 hypersensitivity immune disease mouse model according to one embodiment of the present invention, Lactococcus lactis-derived vesicles ( L. lactis ) and Bifidobacterium brevi-derived vesicles ( B. breve ) cause lung inflammation due to eosinophilic inflammation. This is a diagram confirming the effect on functional disability (airway hyperresponsiveness) (n=5, *P<0.05, **P<0.01).
Figure 6 shows the effects of Lactococcus lactis-derived vesicles ( L. lactis ) and Bifidobacterium brevi-derived vesicles ( B. breve ) on histological changes in the lung in a Th2 hypersensitivity immune disease mouse model according to an embodiment of the present invention. As an evaluation of the impact, Figure 6a is a diagram confirming the change in mucus production in the lung (scale bar: 50 μm), and Figure 6b is a diagram quantifying the results of Figure 6a (*P<0.05, **P< 0.01, ***P<0.001, ns means not significant).
Figures 7a and 7b show Th1 and Th2 immunity of Lactococcus lactis-derived vesicles ( L. lactis ) and Bifidobacterium brevi-derived vesicles ( B. breve ) in a Th2 hypersensitivity immune disease mouse model according to an embodiment of the present invention. As an evaluation of the response control effect, Figure 7a shows the results of measuring the Th1 cytokine, IFN-γ, in bronchoalveolar lavage fluid (BALF), and Figure 7b shows the results of IL-5 and IL-13, the Th2 cytokines that induce eosinophil infiltration. This is a drawing measuring (n=5, *P<0.05, **P<0.01, ***P<0.001, ns means not significant).
Figures 8a and 8b show Th1 and Th2 immunity of Lactococcus lactis-derived vesicles ( L. lactis ) and Bifidobacterium brevi-derived vesicles ( B. breve ) in a Th2 hypersensitivity immune disease mouse model according to one embodiment of the present invention. As an evaluation of the response modulation effect, Figure 8a shows the results of measuring the secretion of Th1 cytokine, IFN-γ, in T cells isolated from lung tissue, and Figure 8b shows IL-, a Th2 cytokine, in T cells isolated from lung tissue. 5 and a diagram measuring IL-13 secretion (n=5, *P<0.05, **P<0.01, ***P<0.001, ns means not significant).
Figures 9a to 9c show evaluation of the Th1 and Th2 immune response regulation mechanisms by Lactococcus lactis-derived vesicles ( L. lactis ) and Bifidobacterium brevi-derived vesicles ( B. breve ) according to an embodiment of the present invention. As such, Figure 9a is an experimental protocol for evaluating cytokine secretion patterns in T cells in peripheral blood isolated from healthy controls, and Figure 9b shows Th1 secreted from T cells in peripheral blood by stimulation with anti-CD3/28 antibody. This is the result of measuring the secretion of cytokine, IFN-γ, and Figure 9c shows the secretion levels of IL-4 and IL-5, which are Th2 cytokines secreted from T cells in peripheral blood by stimulation with anti-CD3/28 antibody. Figure (n=6, ***P<0.001, ns means not significant).
Figures 10a and 10b show evaluation of the Th1 and Th2 immune response regulation mechanisms by Lactococcus lactis-derived vesicles ( L. lactis ) and Bifidobacterium brevi-derived vesicles ( B. breve ) according to one embodiment of the present invention. Figure 10a is an experimental protocol for evaluating the cytokine secretion pattern involved in T cell differentiation in dendritic cells in peripheral blood isolated from healthy controls, and Figure 10b shows the level of IL-12p70 that induces Th1 immune response. This is a figure (n=6, *P<0.05, ***P<0.001, ns means not significant).

In one experimental example of the present invention, vesicles derived from Lactococcus lactis form a double-layered membrane, and vesicles derived from Bifidobacterium brevi show a single protein band, while vesicles derived from Lactococcus lactis show multiple protein bands. Confirmed (see Experimental Example 1).

In another experimental example of the present invention, eosinophil infiltration into lung tissue and airway hyperresponsiveness were not suppressed by treatment of Bifidobacterium brevi-derived vesicles in a Th2 hypersensitivity immune disease animal model, but treatment of Lactococcus lactis-derived vesicles did not inhibit eosinophil infiltration into lung tissue and airway hyperreactivity. It was confirmed that eosinophil infiltration into lung tissue and airway hyperresponsiveness were suppressed, and although treatment of Bifidobacterium brevi-derived vesicles had no effect on mucus production in a Th2 hypersensitivity immune disease animal model, the treatment of Lactococcus lactis-derived vesicles was confirmed to be suppressed. It was confirmed that mucus production was significantly reduced by treatment (see Experimental Example 2).

In another experimental example of the present invention, in an animal model of Th2 hypersensitivity immune disease, treatment of Lactococcus lactis-derived vesicles inhibits the secretion of Th2 cytokines such as IL-5 and IL-13, which induce eosinophilic inflammation, and inhibits Th1 cytokines. It was confirmed that the secretion of kine, IFN-γ, was induced, and this immunomodulatory effect was not observed by treatment of Bifidobacterium brevi-derived vesicles. Through this, it was found that Lactococcus lactis-derived vesicles induce immunological homeostasis by inducing an immune response from Th2 to Th1 in a pathological situation where the Th2 immune response is dominant (see Experimental Example 3).

In another experimental example of the present invention, rather than directly acting on T cells to regulate Th1 and Th2 immune responses, Lactococcus lactis-derived vesicles act on dendritic cells, which are antigen-presenting cells, to induce a Th1 immune response. It was found to have an immune regulatory mechanism that suppresses Th2 hypersensitivity response by significantly increasing the secretion of cytokines such as 12p70 (see Experimental Example 4).

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, which contains vesicles derived from Lactococcus lactis as an active ingredient.

In the present invention, “eosinophil” is a type of white blood cell and a member of the immune system that plays a role in fighting multicellular parasites and specific infections in mammals. They are involved in the pathogenesis of eosinophilic inflammatory diseases through cytokines such as IL-5 and IL-13 secreted by the Th2 immune response. They develop through hematopoiesis in the bone marrow and are sent to the blood, where they undergo final differentiation and no longer proliferate. These are cells that have acid-friendly granules in their cytoplasm and appear brick red when stained with the dye eosin. Small granules in the cytoplasm contain several chemical mediators such as peroxidase, RNase, DNase, lipase, and plasminogen. These mediators are secreted through the degranulation process following activation of eosinophils, which causes histological and functional changes and causes disease.

In the present invention, “eosinophilic inflammatory disease” refers to an inflammatory disease caused by eosinophils infiltrating tissues due to various causes. The eosinophilic inflammatory disease may be an inflammatory disease caused by allergic causative factors, including drug allergy, eosinophilic asthma, allergic rhinitis, and atopic dermatitis. It is not limited to this.

In addition, the eosinophilic inflammatory diseases include eosinophilic cardiomyopathy, eosinophilic colitis, eosinophilic enteritis, eosinophilic esophagitis, eosinophilic gastritis, and eosinophilic inflammatory diseases. Causes include eosinophilic pneumonia, eosinophilic bronchitis, eosinophilic fasciitis, hypereosinophilic syndrome, and Churg-Strauss syndrome or eosinophilic granulomatosis with polyangiitis. It may be an eosinophilic inflammatory disease with an unknown cause, but is not limited to this.

In the present invention, “Th2 hypersensitivity immune diseases” refers to IL-4, IL-5, IL-9, IL- secreted by Th2 (type 2 helper T) cell hypersensitivity reaction to a specific antigen. It refers to an immune disease mediated by cytokines such as 13. Th2 hypersensitivity immune diseases caused by Th2 cell hypersensitivity reactions include atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, and hypersensitivity pneumonitis. , food allergy, drug allergy, and anaphylaxis, but are not limited thereto.

In the present invention, the eosinophilic inflammatory disease may be a respiratory disease that exhibits Th2 (type 2 helper T) cell hypersensitivity, and in this case, the respiratory disease that exhibits Th2 cell hypersensitivity is atopic asthma and allergic rhinitis. It may include, but is not limited to, allergic rhinitis, eosinophilic bronchitis, and hypersensitivity pneumonitis.

In the present invention, the eosinophilic inflammatory disease or Th2 hypersensitivity immune disease may be a respiratory disease characterized by excessive mucus secretion in the respiratory tract. In this case, the respiratory disease characterized by excessive mucus secretion in the respiratory tract is chronic rhinitis and chronic sinusitis. , and chronic bronchitis, etc., but is not limited thereto.

In the present invention, “asthma” is a disease of the ‘bronchial tubes’, which are passages connected to the lungs. When exposed to a specific causative agent, the bronchial tubes become severely narrowed due to inflammation of the bronchial tubes, causing coughing, wheezing (wheezing sound when breathing), A disorder of the lungs characterized by recurrent episodes of shortness of breath and chest tightness, whatever the cause (endogenous, exogenous, or both; allergic or non-allergic), characterized by changes in lung airflow associated with airway constriction. refers to a disease. The term asthma may be used with one or more adjectives indicating the cause.

As used in the present invention, the term “extracellular vesicle or vesicle” refers to a nano-sized membrane structure secreted by various bacteria. Gram-positive bacteria such as Lactococcus or Bifidobacterium. Vesicles derived from (gram-positive bacteria) contain, in addition to proteins and nucleic acids, peptidoglycan and lipoteichoic acid, which are components of the bacterial cell wall, and various low-molecular-weight compounds within the vesicles. In the present invention, , vesicles are naturally secreted from Lactococcus lactis bacteria or artificially produced, such as 10 to 200 nm, 10 to 180 nm, 10 to 150 nm, 10 to 120 nm, 10 to 100 nm, 10 to 80 nm. , may have an average diameter of 20 to 200 nm, 20 to 180 nm, 20 to 150 nm, 20 to 120 nm, 20 to 100 nm, or 20 to 80 nm, but is not limited thereto.

The vesicles are obtained by centrifuging the culture medium containing Lactococcus lactis bacteria, ultra-high-speed centrifugation, high-pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thawing, electroporation, mechanical digestion, chemical treatment, and filtering. It can be separated using one or more methods selected from the group consisting of filtration, gel filtration chromatography, free-flow electrophoresis, and capillary electrophoresis. Additionally, processes such as washing to remove impurities and concentrating the obtained vesicles may be additionally included.

In the present invention, the Lactococcus lactis-derived vesicles increase secretion of interferon-γ (IFN-γ) in T cells; By reducing the secretion of one or more selected from the group consisting of IL-5 and IL-13 in T cells and increasing the secretion of IL-12p70 in dendritic cells, the Th2 immune response can be suppressed, thereby preventing eosinophilic inflammatory disease or It may be used as a composition for preventing, treating, or improving Th2 hypersensitivity immune disease, but is not limited thereto.

The pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen. Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

Additives for the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

A solution of white sugar, other sugars, or sweeteners, etc. may be used in the syrup according to the present invention, and if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

Suspensions according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil. , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; Isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Sulfurizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75(S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massaupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease.

In another aspect of the present invention, the present invention provides an inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient.

In the inhalant composition of the present invention, the active ingredient can be added as is to the inhalant or used together with other ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or treatment).

Inhalation agents for parenteral administration include aerosols, powders for inhalation, or liquids for inhalation. These inhalation liquids may be dissolved or suspended in water or another suitable medium at the time of use. These inhalants are manufactured according to known methods. For example, in the case of liquid preparations for inhalation, preservatives (benzalkonium chloride, parabens, etc.), colorants, buffering agents (sodium phosphate, sodium acetate, etc.), isotonic agents (sodium chloride, concentrated glycerin, etc.), thickeners (carboxyvinyl polymer, etc.), absorption agents, etc. It is prepared by appropriately selecting accelerators, etc. according to need.

In the case of powder for inhalation, lubricants (stearic acid and its salts, etc.), binders (starch, dextrin, etc.), excipients (lactose, cellulose, etc.), colorants, preservatives (benzalkonium chloride, parabens, etc.), absorption accelerators, etc. are required. It is appropriately selected and prepared according to the following.

The inhalant composition can be administered through an inhalant device, which is a device capable of delivering the composition to an individual's lung tissue, such as an inhaler, nebulizer, or ventilator. When administering a liquid medication for inhalation, a nebulizer (atomizer, nebulizer) is usually used, and when administering a powder medication for inhalation, an inhalation dispenser for powder medication is usually used.

In another aspect of the present invention, the present invention provides a food composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient.

In the present invention, the food composition may be a health functional food composition, but is not limited thereto.

When using the Lactococcus lactis-derived vesicles of the present invention as a food additive, the Lactococcus lactis-derived vesicles can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the Lactococcus lactis-derived vesicle of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There are no special restrictions on the types of foods above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, These include alcoholic beverages and vitamin complexes, and include all health functional foods in the conventional sense.

The health drink composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional drinks. The above-mentioned natural carbohydrates include 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. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of natural carbohydrates is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain carbonating agents used in carbonated drinks. In addition, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

In another aspect of the present invention, the present invention provides a quasi-drug composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient.

In the present invention, "quasi-drugs" refers to products with a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans or animals. For example, according to the Pharmaceutical Affairs Act, Quasi-drugs exclude products used for medicinal purposes and include products used to treat or prevent diseases in humans and animals, and products that have a mild or no direct effect on the human body.

When the composition of the present invention is included in a quasi-drug for the purpose of preventing or improving eosinophilic inflammatory disease, the composition can be included as is or used together with other quasi-drug ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use.

The quasi-drug of the present invention may contain various bases and additives necessary for formulation depending on its formulation, and the types and amounts of these ingredients can be easily selected by a person skilled in the art.

The quasi-drug composition of the present invention includes, for example, disinfectant cleaners, detergents, kitchen cleaners, cleaning detergents, wet tissues, detergents, soaps, hand washes, humidifier fillers, masks, ointments, filter fillers, and temporary inhalation of air or oxygen directly or indirectly. It can be manufactured in a formulation selected from the group consisting of portable products that supply air or oxygen, but is not limited thereto.

In another aspect of the present invention, the present invention provides a composition for delivering a drug for treating respiratory diseases, comprising vesicles derived from Lactococcus lactis as an active ingredient.

In the present invention, “drug delivery” refers to any means of loading and delivering a drug for treating respiratory diseases into the Lactococcus lactis-derived vesicle according to the present invention in order to deliver the drug to a specific organ, tissue, cell, or organelle, or means action.

In the composition for delivering a drug for treating respiratory diseases of the present invention, the respiratory diseases may include respiratory diseases showing Th2 cell hypersensitivity and respiratory diseases characterized by excessive secretion of mucus in the respiratory tract, but are not limited to the types thereof.

In the present invention, there is no limitation to the type of drug for treating respiratory diseases.

In another aspect of the present invention, the present invention provides a method for preventing eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising administering to an individual a composition containing vesicles derived from Lactococcus lactis as an active ingredient. Provides treatment methods.

In another aspect of the present invention, the present invention provides the use of a composition containing vesicles derived from Lactococcus lactis as an active ingredient for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.

In another aspect of the present invention, the present invention provides the use of Lactococcus lactis -derived vesicles for the production of a medicament for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.

In the present invention, “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of

In the present invention, “administration” means providing a given composition of the present invention to an individual by any appropriate method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of the desired disease, and “treatment” refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed, and “improvement” refers to all actions that reduce parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.

In the present invention, when a part “includes” a certain component, this means that it may further include other components rather than excluding other components, unless specifically stated to the contrary.

Below, preferred examples and experimental examples are presented to aid understanding of the present invention. However, the following examples and experimental examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples and experimental examples.

[Example]

Example 1. Bacterial culture and isolation of extracellular vesicles

Lactococcus lactis and Bifidobacterium breve strains were cultured in self-prepared medium under anaerobic conditions until the optical density reached 1.5 at 600 nm, respectively. To isolate extracellular vesicles (EVs), the bacterial culture medium was centrifuged at 10,000 g for 20 minutes, and the supernatant was filtered through a 0.45 μm vacuum filter. The filtrate was concentrated using QuixStand (GE Healthcare, UK) and then filtered through a 0.22 μm bottle-top filter (Sigma-Aldrich, USA). Then, the filtrate was pelleted by ultracentrifugation in a 45 Ti rotor (Beckman Coulter, USA) at 150,000 g for 2 hours at 4°C. The final pellet was resuspended in phosphate-buffered saline, stored at -80 °C, and a JEM1011 microscope (JEOL, Japan) was used to observe EV morphology. Additionally, EV size was measured using Zetasizer Nano S (Malvern Instruments, UK). EV protein patterns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Example 2. Th2 hypersensitivity immune disease mouse model

To induce a Th2 hypersensitivity immune disease mouse model, female 6-week-old BALB/c mice (Jackson Laboratory, USA) were administered 75 μg of ovalbumin (OVA; Sigma-Aldrich) and 2 mg of aluminum hydroxide (alum; Thermo FisherScientific) for sensitization. , USA) was treated intraperitoneally. Then, for the challenge, 50 μg of OVA was injected intranasally five times. During the challenge, mice were treated intraperitoneally with 10 μg of dexamethasone (Dexa; Sigma-Aldrich) or intranasally with 10 μg of EV. To measure airway hyperresponsiveness to inhaled methacholine (Sigma-Aldrich), the flexiVent System (SCIREQ, Canada) was used, and to measure the number of immune cells in bronchoalveolar lavage fluid (BALF), Diff-quick staining (Dade Behring) was used. , Switzerland) was performed. For histology, lung tissues stained with H&E or PAS were examined using ImageJ (National Institutes of Health, Bethesda, USA).

Example 3. Mouse lung T cell isolation and stimulation

Lung cells were isolated using immunomagnetic cell sorting (Miltenyi Biotec Inc, USA). These cells (5 × 10 ⁵ ) were seeded in 24-well plates (TPP, Switzerland) and stimulated with mouse anti-CD3/CD28 antibodies (1 μg/mL each; eBioscience, USA) for 24 hours.

Example 4. Stimulation of Immune Cells in Human Peripheral Blood

To isolate immune cells, venous blood from asthma patients was collected in vacuum tubes containing acid citrate dextrose solution (BD Biosciences, USA). Blood was layered in Lymphoprep solution (Axis-Shield, Norway) and centrifuged at 800 × g for 25 minutes at 20°C. Then, the layer containing peripheral blood mononuclear cells was separated and red blood cells were removed by hypotonic lysis. Finally, immunomagnetic cell separation (Miltenyi Biotec Inc, USA) was used to isolate dendritic cells and CD4+ T cells, respectively. The dendritic cells were treated with 100 ng/mL human recombinant IL-1β (R&D Systems, USA), 10-6 M Dexa (Sigma-130 Aldrich), or 1 μg/mL EV for 24 hours, and CD4+ T cells were treated with 10 Stimulation was performed with human anti-CD3/CD28 antibodies (1 μg/mL each; Thermo FisherScientific) with or without −6 M Dexa (Sigma-Aldrich) or 1 μg/mL EV for 24 h.

Example 5. Enzyme immunosorbent assay (ELISA)

The levels of several cytokines, such as IFN-γ, IL-4, IL-5, IL-6, IL-10, IL-12p70, and IL-13, in BALF or cell culture supernatants were measured using kits (R&D Systems). Measurements were made according to the manufacturer's recommendations.

Example 6. Statistical analysis

All statistical analyzes were performed using IBM SPSS software version 25.0 (IBM Corp., USA), and P<0.05 was considered statistically significant. Graphs were generated using GraphPad Prism 8.0 software (GraphPad Inc., USA).

[Experimental example]

Experimental Example 1. Isolation and characterization of probiotic-derived EVs

Lactococcus lactis-derived vesicles and Bifidobacterium brevi-derived vesicles were purified from bacterial culture medium, and EVs were observed using transmission electron microscopy to determine whether EVs formed spherical lipid bilayers with intact morphology. As a result, as shown in Figures 1a and 2a, both Lactococcus lactis-derived vesicles and Bifidobacterium brevi-derived vesicles showed well-structured membranes composed of double layers.

Additionally, when comparing the protein patterns of EVs, vesicles from Lactococcus lactis showed multiple protein bands, as shown in Figure 1b, whereas vesicles from Bifidobacterium brevi showed a single band, as shown in Figure 2b. indicated.

Experimental Example 2. Therapeutic effect of Lactococcus lactis-derived vesicles in a Th2 hypersensitivity immune disease mouse model

To investigate the role of EV compared to Dexa in the Th2 hypersensitivity immune disease model, mice were treated with several drugs as shown in Figure 3. When Lactococcus lactis-derived vesicles, Bifidobacterium brevii-derived vesicles, or Dexa were administered during challenge, eosinophil numbers in the BALF increased significantly compared to dexamethasone (Dexa) and Lactococcus lactis-derived vesicles, as shown in Figures 4A and 4B. It was significantly reduced by vesicles, but not by Bifidobacterium brevi-derived vesicles.

In addition, as shown in Figure 5, in the Th2 hypersensitivity immune disease model, changes in lung function (airway hyperresponsiveness) induced by Th2 hypersensitivity reaction were significantly suppressed by dexamethasone (Dexa) and Lactococcus lactis-derived vesicles. However, it was not reduced by Bifidobacterium brevi-derived vesicles.

In addition, as shown in Figures 6a and 6b, in the Th2 hypersensitivity immune disease model, histological changes such as mucus secretion induced by the Th2 hypersensitivity reaction were significantly observed by dexamethasone (Dexa) and Lactococcus lactis-derived vesicles. However, it was not reduced by Bifidobacterium brevi-derived vesicles.

The above results showed that the eosinophilic inflammatory reaction caused by immunological hypersensitivity reaction and the functional and histopathological changes that occur as a result thereof can be efficiently treated using Lactococcus lactis-derived vesicles. .

Experimental Example 3. Immunomodulatory effect of Lactococcus lactis-derived vesicles in a Th2 hypersensitivity immune disease mouse model

To evaluate the effect of probiotic-derived EVs and dexamethasone, a representative corticosteroid as a control drug, on Th2 hypersensitivity response, cytokines secreted from Th1 and Th2 cells were measured in a mouse model of Th2 hypersensitivity immune disease.

As a result, as shown in Figure 7a, the concentration of IFN-γ, a Th1 cytokine, in the airway washing fluid was significantly increased by the administration of dexamethasone (Dexa) and Bifidobacterium brevi-derived vesicles ( B. breve ) compared to the disease group. There was no difference, but it was significantly increased compared to the disease group by administration of Lactococcus lactis-derived vesicles ( L. lactis ).

On the other hand, as shown in Figure 7b, there was no significant difference in the concentration of Th2 cytokines IL-5 and IL-13 in the airway washing fluid by administration of Bifidobacterium breve-derived vesicles ( B. breve ) compared to the disease group. It was significantly reduced compared to the disease group by administration of dexamethasone (Dex) and Lactococcus lactis-derived vesicles ( L. lactis ).

In addition, T cells were isolated from the lung tissue of mice with Th2 hypersensitivity immune disease, stimulated with anti-CD3/28, and the cytokine secretion pattern was evaluated. As shown in Figure 8a, the T cells secreted IFN-γ. There was no significant difference by administration of Bifidobacterium brevi-derived vesicles ( B. breve ) compared to the disease group, but it was suppressed by administration of dexamethasone (Dexa), and the effect of Lactococcus lactis-derived vesicles ( L. lactis) ) was significantly increased by administration. On the other hand, as shown in Figure 8b, there was no significant difference in the secretion of Th2 cytokines IL-5 and IL-13 in T cells by administration of Bifidobacterium brevi-derived vesicles ( B. breve ) compared to the disease group. , was significantly reduced by the administration of dexamethasone (Dexa) and Lactococcus lactis-derived vesicles ( L. lactis ).

Based on the above results, while dexamethasone (Dexa) suppresses eosinophilic inflammation through a mechanism of suppressing overall immune function, Lactococcus lactis-derived vesicles increase Th1 immune response, while Th2 immune response It was found that the inhibitory action suppresses eosinophilic inflammation.

Experimental Example 4. Th2 hypersensitivity response inhibition mechanism of Lactococcus lactis-derived vesicles

It is well known that during the differentiation of naïve T cells into Th1 or Th2 cells, IL-12 secreted by dendritic cells, which are antigen-presenting cells, induces differentiation into Th1 cells, and IL-4 induces differentiation into Th2 cells. In this experimental example, to evaluate the mechanism by which Lactococcus lactis-derived vesicles suppress Th2 hypersensitivity response, an experiment was conducted by isolating T cells and dendritic cells from the peripheral blood of normal people.

As an experimental example, as shown in Figure 9a, the cytokine secretion pattern was evaluated after anti-CD3/28 stimulation of T cells isolated from peripheral blood. As shown in Figure 9b, dexamethasone (Dexa) ) inhibited IFN-γ secretion in T cells, but Lactococcus lactis-derived vesicles ( L. lactis ) and Bifidobacterium brevi ( B. breve )-derived vesicles had no significant effect on IFN-γ secretion. In addition, as shown in Figure 9c, the secretion of Th2 cytokines IL-4 and IL-5 in T cells was also suppressed by dexamethasone, but there was no change by Lactococcus- and Bifidobacterium-derived vesicles.

In another experimental example, as shown in Figure 10a, the secretion pattern of IL-12, which induces differentiation into Th1 cells in dendritic cells isolated from peripheral blood, was evaluated. As a result, as shown in Figure 10b, the secretion of IL-12p70 was not significantly affected by dexamethasone (Dexa) and Bifidobacterium-derived vesicles, but was affected by Lactococcus-derived vesicles and the control drug IL-1β. increased significantly.

Based on the above results, Lactococcus lactis-derived vesicles act on dendritic cells, which are antigen-presenting cells, rather than directly on T cells, and induce the secretion of IL-12, which induces a Th1 immune response, suppressing the Th2 hypersensitivity response. Could know.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims (28)

A pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient,
The composition is one in which Lactococcus lactis cells have been removed,
The Th2 hypersensitivity immune diseases include atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, hypersensitivity pneumonitis, and food allergy. ), drug allergy, and anaphylaxis. A pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.
According to paragraph 1,
The eosinophilic inflammatory disease is an allergic inflammatory disease,
The allergic inflammatory disease is an eosinophilic inflammatory disease, characterized in that it includes eosinophilic drug allergy, eosinophilic asthma, allergic rhinitis, and atopic dermatitis. Or a pharmaceutical composition for preventing or treating Th2 hypersensitivity immune disease.
According to paragraph 1,
The eosinophilic inflammatory disease is an eosinophilic inflammatory disease with unknown causative agent,
The eosinophilic inflammatory diseases of unknown cause include eosinophilic cardiomyopathy, eosinophilic colitis, eosinophilic enteritis, eosinophilic esophagitis, eosinophilic gastritis, Includes eosinophilic pneumonia, eosinophilic bronchitis, eosinophilic fasciitis, hypereosinophilic syndrome, and Churg-Strauss syndrome or eosinophilic granulomatosis with polyangiitis. A pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.
delete According to paragraph 1,
A pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitive immune disease, wherein the eosinophilic inflammatory disease is a respiratory disease that exhibits Th2 (type 2 helper T) cell hypersensitivity reaction.
According to clause 5,
Respiratory diseases that exhibit Th2 cell hypersensitivity include atopic asthma, allergic rhinitis, eosinophilic bronchitis, and hypersensitivity pneumonitis. Pharmaceutical composition for preventing or treating constitutive inflammatory diseases or Th2 hypersensitivity immune diseases.
A pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient,
The composition is one in which Lactococcus lactis cells have been removed,
A pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitive immune disease, wherein the eosinophilic inflammatory disease or Th2 hypersensitive immune disease is a respiratory disease characterized by excessive secretion of mucus in the respiratory tract.
In clause 7,
Respiratory diseases characterized by excessive secretion of mucus in the respiratory tract include chronic rhinitis, chronic sinusitis, and chronic bronchitis. A pharmaceutical composition for preventing or treating eosinophilic inflammatory diseases or Th2 hypersensitivity immune diseases.
According to paragraph 1,
The Lactococcus lactis-derived vesicle is a pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitive immune disease, characterized in that it suppresses airway hyperresponsiveness.
According to paragraph 1,
A pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, wherein the vesicles have an average diameter of 10 to 200 nm.
According to paragraph 1,
A pharmaceutical composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, wherein the vesicles are naturally or artificially secreted from Lactococcus lactis .
An inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient,
The composition is one in which Lactococcus lactis cells have been removed,
The Th2 hypersensitivity immune diseases include atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, hypersensitivity pneumonitis, and food allergy. ), drug allergy, and anaphylaxis. An inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.
According to clause 12,
The eosinophilic inflammatory disease is an allergic inflammatory disease,
The allergic inflammatory disease is an eosinophilic inflammatory disease, characterized in that it includes eosinophilic drug allergy, eosinophilic asthma, allergic rhinitis, and atopic dermatitis. Or an inhalant composition for preventing or treating Th2 hypersensitivity immune disease.
According to clause 12,
The eosinophilic inflammatory disease is an eosinophilic inflammatory disease with unknown causative agent,
The eosinophilic inflammatory diseases of unknown cause include eosinophilic cardiomyopathy, eosinophilic colitis, eosinophilic enteritis, eosinophilic esophagitis, eosinophilic gastritis, Includes eosinophilic pneumonia, eosinophilic bronchitis, eosinophilic fasciitis, hypereosinophilic syndrome, and Churg-Strauss syndrome or eosinophilic granulomatosis with polyangiitis. An inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.
delete According to clause 12,
An inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, wherein the eosinophilic inflammatory disease is a respiratory disease that exhibits Th2 (type 2 helper T) cell hypersensitivity reaction.
According to clause 16,
Respiratory diseases that exhibit Th2 cell hypersensitivity include atopic asthma, allergic rhinitis, eosinophilic bronchitis, and hypersensitivity pneumonitis. Inhalant composition for preventing or treating inflammatory diseases or Th2 hypersensitivity immune diseases.
An inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient,
The composition is one in which Lactococcus lactis cells have been removed,
An inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitive immune disease, wherein the eosinophilic inflammatory disease or Th2 hypersensitive immune disease is a respiratory disease characterized by excessive secretion of mucus in the respiratory tract.
According to clause 18,
Respiratory diseases characterized by excessive secretion of mucus in the respiratory tract include chronic rhinitis, chronic sinusitis, and chronic bronchitis. An inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.
According to clause 12,
An inhalant composition for preventing or treating eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, wherein the Lactococcus lactis-derived vesicles suppress airway hyperresponsiveness.
A food composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient,
The composition is one in which Lactococcus lactis cells have been removed,
The Th2 hypersensitivity immune diseases include atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, hypersensitivity pneumonitis, and food allergy. ), drug allergy, and anaphylaxis. A food composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.
According to clause 21,
The eosinophilic inflammatory disease is an allergic inflammatory disease,
The allergic inflammatory disease is an eosinophilic inflammatory disease, characterized in that it includes eosinophilic drug allergy, eosinophilic asthma, allergic rhinitis, and atopic dermatitis. Or a food composition for preventing or improving Th2 hypersensitivity immune disease.
According to clause 21,
The eosinophilic inflammatory disease is an eosinophilic inflammatory disease with unknown causative agent,
The eosinophilic inflammatory diseases of unknown cause include eosinophilic cardiomyopathy, eosinophilic colitis, eosinophilic enteritis, eosinophilic esophagitis, eosinophilic gastritis, Includes eosinophilic pneumonia, eosinophilic bronchitis, eosinophilic fasciitis, hypereosinophilic syndrome, and Churg-Strauss syndrome or eosinophilic granulomatosis with polyangiitis. A food composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.
delete According to clause 21,
A food composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, wherein the eosinophilic inflammatory disease is a respiratory disease that exhibits Th2 (type 2 helper T) cell hypersensitivity reaction.
According to clause 25,
Respiratory diseases that exhibit Th2 cell hypersensitivity include atopic asthma, allergic rhinitis, eosinophilic bronchitis, and hypersensitivity pneumonitis. Food composition for preventing or improving inflammatory diseases or Th2 hypersensitivity immune diseases.
A quasi-drug composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease, comprising vesicles derived from Lactococcus lactis as an active ingredient,
The composition is one in which Lactococcus lactis cells have been removed,
The Th2 hypersensitivity immune diseases include atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, hypersensitivity pneumonitis, and food allergy. ), drug allergy, and anaphylaxis. A quasi-drug composition for preventing or improving eosinophilic inflammatory disease or Th2 hypersensitivity immune disease.
delete