Immunomodulators

Abstract

The present application relates to an immunomodulator comprising a yeast deposited with the National Institute of Technology and Evaluation as International Depositary Authority under International Depositary Accession Number NITE BP-04012. Preferably, it is for modulation of cytokine production. More preferably, it is an immunomodulator that modulates cytokine production in macrophages or dendritic cells. More preferably, it is an immunomodulator that modulates immunity via TLR2. The cytokine is preferably at least one selected from the group consisting of IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α.

Description

Technical Field

[0001] This invention relates to an immunomodulator that uses yeast. Background Technology

[0002] Macrophages and dendritic cells are the frontline cells of the innate immune system. Besides phagocytosis, macrophages also transmit antigen information to T cells, which act as coordinators. Dendritic cells are antigen-presenting cells (cells that present substances identified as foreign to other cells) and also have phagocytic activity. It is well known that macrophages and dendritic cells produce various cytokines in response to stimulation by pathogens, which are crucial in the immune response.

[0003] For example, it is known that bacterial cell components (such as lipopolysaccharide LPS) activate TLRs (toll-like receptors) present on the surface of macrophages and dendritic cells, resulting in the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, thus initiating inflammation. IL-8 is also a pro-inflammatory cytokine, which, in addition to inducing the migration of neutrophils or other granulocytes to the site of infection, also plays a role in inducing phagocytosis.

[0004] On the other hand, IL-10 and IL-12 are also known to be produced by macrophages stimulated by LPS, etc. These cytokines possess immunostimulatory properties, while also exhibiting anti-inflammatory effects. For example, IL-10 has shown immunostimulatory properties, including stimulating the proliferation of thymocytes treated with IL-2 and IL-4, increasing B cell survival, and stimulating the expression of MHC class II molecules. Furthermore, IL-10 can suppress the immune response by inhibiting the expression of IL-1α, IL-1β, IL-6, IL-8, TNF-α, GM-CSF, and G-CSF in activated monocytes and activated macrophages, and also inhibit the production of interferon-γ (IFN-γ) by NK cells.

[0005] Furthermore, IL-12 is a heterodimeric cytokine encoded by two separate genes, IL-12A and IL-12B, which are composed of p35 and p40 subunits, respectively. The main function of IL-12 is to induce the production of interferon-γ by NK cells and T cells, and to enhance the activity of NK cells and cytotoxic T cells. In addition, it is known to act on naive T cells (Th0) to induce their differentiation into Th1 cells, thereby promoting the production of interferon-γ and preferentially activating cellular immunity through Th1 cells. Furthermore, IL-12 is said to activate cellular immunity by preferentially activating Th1 cells and inhibiting Th2 cells mediated by IgE suppression, etc. Based on this, there is interest in applying IL-12 to the treatment of Th2-dominant immune abnormalities such as allergies, and in the treatment of cancer and infectious diseases based on the activation of cellular immune function.

[0006] As mentioned above, TLRs play a central role in the recognition of various pathogens and the activation of the immune system. Furthermore, among TLRs, TLR2 is expressed on the surface of specific cells such as macrophages, playing a fundamental role in pathogen recognition and the activation of innate immunity.

[0007] Previously, yeast was known to be used as an immune activator (see Patent Documents 1 and 2). Existing technical documents Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 2004-107281 Patent Document 2: Japanese Patent Application Publication No. 2006-75039 Summary of the Invention

[0009] Immunomodulatory function refers to the function of regulating the immune state to a favorable state. Excellent immunomodulatory function can achieve both immune enhancement and / or immunosuppression to maintain a favorable immune state. Therefore, immunomodulators with superior immunomodulatory effects are required.

[0010] The inventors conducted in-depth research and discovered that specific strains have excellent immunomodulatory effects, thus completing this invention. This invention is based on the above-mentioned insights and provides an immunomodulator deposited with the Technical Base for Evaluation of Pharmaceutical Products under the international accession number NITE BP-04012 at the independent administrative agency. Invention Effects

[0011] According to the present invention, an immunomodulatory agent capable of exerting excellent immunomodulatory effects can be provided. The immunomodulatory agent of the present invention can exert excellent immune-enhancing and / or immunosuppressive effects. Attached Figure Description

[0012] Figure 1 This is a graph showing the results of evaluating the gene expression of various cytokines in macrophage-like cells using powdered yeast from a specific yeast strain. Figure 2 This is a graph showing the results of evaluating the gene expression of various cytokines in macrophage-like cells using extracts from specific yeasts. Figure 3 This is a graph showing the results of evaluating the gene expression of IL-6 in dendritic-like cells using extracts from specific yeasts. Figure 4 This is a graph showing the results of evaluating the production of various cytokines in macrophage-like cells using powdered yeast of a specific yeast. Figure 5This is a graph showing the results of evaluating the production of various cytokines in macrophage-like cells using extracts from specific yeasts. Figure 6 This is a graph representing the results of evaluating the gene expression of various cytokines in macrophage-like cells under LPS-added conditions using powdered yeast of a specific yeast. Figure 7 This is a graph representing the results of evaluating the gene expression of various cytokines in macrophage-like cells under LPS-added conditions using extracts from specific yeasts. Figure 8 This is a graph representing the results of evaluating the production of various cytokines in macrophage-like cells under LPS-added conditions using powdered yeast of a specific yeast. Figure 9 This is a graph showing the results of evaluating the production of various cytokines in macrophage-like cells under LPS-added conditions using extracts from specific yeasts. Figure 10 This is a graph showing the results of evaluating the protein expression levels of TLR in genetically modified HEK293 cells using powder from a specific yeast strain. Figure 11 This is a graph showing the results of evaluating the protein expression levels of TLRs in genetically modified HEK293 cells using extracts from a specific yeast. Detailed Implementation

[0013] The present invention will now be described based on its preferred embodiments. This invention relates to an immunomodulator comprising yeast deposited with the Technical Base for Product Evaluation (TDE) under international accession number NITE BP-04012. This specific yeast was deposited with TDE on November 14, 2023, under accession number NITE P-04012, and transferred to international deposit on September 11, 2024. The yeast (hereinafter referred to as "specific yeast") deposited with the Technical Base for Product Evaluation, an independent administrative agency, under the international accession number NITE BP-04012, is scientifically named *Saccharomyces cerevisiae*. Its scientific properties are described below. 1. Scientific properties… Forms white to pale yellow colonies in a nutrient medium containing carbon and nitrogen sources. As a division morphology observed under an optical microscope, proliferation via budding is observed. Mother cells and daughter cells can be distinguished. 2. Taxonomic position… Yeast: Edible yeast (Saccharomyces cerevisiae) 3. Cultivation conditions (1) Culture medium name...YPD culture medium (2) Composition of the culture medium… per 1000ml of culture medium Yeast extract 10g, peptone 20g, glucose 20g

[0014] (3) pH of the culture medium…4~7 (optimal pH is 5~6) (4) Sterilization conditions of the culture medium…121℃, 10 minutes (5) Incubation temperature…30℃ (6) During the cultivation period…2~3 days (7) Oxygen requirement...facultative anaerobic

[0015] 4. Storage conditions It can be preserved by freezing. (1) Freezing conditions... -80℃ (2) Protectant…10~20% aqueous glycerol solution (15% is optimal) (3) Recovery rate after freezing...40-80% over 2 years.

[0016] 5. Conditions for survival testing (1) Microbial recovery…30℃ (2) Inoculation, culture and confirmation method... carried out under the same conditions as culture.

[0017] As described above, the specific yeast of the present invention is preferably cultured in YPD medium with a pH of 4 to 7, more preferably 5 to 6. Cultivation can be carried out under aerobic conditions such as atmospheric conditions.

[0018] The yeast of the present invention can be in the form of a yeast cell wall, yeast contents, or the yeast cell itself. The yeast cell wall is a fraction insoluble in water or other aqueous liquid media. Proteins, sugars, amino acids, nucleic acids, organic acids, and other contents soluble in aqueous liquid media are removed from the yeast cell. The method for preparing the yeast cell wall is not particularly limited; it can be achieved by breaking or destroying the yeast cell to make the water-soluble contents soluble, followed by solid-liquid separation to obtain the water-insoluble fraction, which is then dried. Examples of breaking or destroying methods include physical breaking methods such as ultrasonic treatment, grinding using a bead mill, and pressurized liquid shearing using a Freund's crusher, or chemical destruction methods using surfactants or lysozymes such as cell wall degrading enzymes. Examples of drying methods include freeze-drying, spray drying, etc. Physical breaking methods are preferred as a measure to enable the yeast cell contents to dissolve in the aqueous liquid media. Examples of the aqueous liquid media include water, ethanol, and mixtures thereof, with water being the most preferred. When the aqueous liquid medium is water, the water contains components other than water, and the water content can exceed 50% by mass, preferably 60% by mass or more.

[0019] Alternatively, yeast contents can be obtained by breaking down yeast cells to extract them into an aqueous liquid medium, followed by extraction of the resulting fragments using the aqueous liquid medium. Instead of breaking down yeast cells, the contents can also be extracted into an aqueous solvent by dissolving the yeast cell walls with cell wall-degrading enzymes. Yeast contents are also called yeast extracts. Yeast extracts can be solids obtained by drying using any method. In this invention, from the perspective of superior immunomodulatory effects, specifically immune-enhancing and immunosuppressive effects, yeast components containing yeast contents, including the yeast cell wall and yeast contents, are preferred. The yeast components containing yeast contents can be, in addition to the yeast contents themselves, mixtures in which the yeast cell wall, obtained by breaking or destroying yeast cells, is mixed with the yeast contents. If such a mixture is brought into contact with an aqueous liquid for solid-liquid separation, the yeast extract can be separated from the cell wall. The proportion of yeast extract in the yeast components is preferably 10% by mass or more, more preferably 20% by mass or more, further preferably 50% by mass or more, and particularly preferably 60% by mass or more.

[0020] There is no strict limitation on the dosage of the active ingredient of the immunomodulator of the present invention, namely the specific yeast. The effect varies depending on the target population, the applicable disease, and other usage conditions. Therefore, it is preferable to set an appropriate dosage. Regarding the preferred dosage, for the specific yeast, it is 10 mg to 100 g per day based on the cell weight, more preferably 160 mg to 4 g. The medicine of the present invention can be taken continuously for more than one week, or more than two weeks, or more than four weeks.

[0021] In addition, as a specific method of yeast intake according to the present invention, oral intake, skin intake, mucosal intake, and intestinal intake are mentioned, with oral intake being particularly preferred.

[0022] The immunomodulators of the present invention can be formulated into pharmaceuticals, quasi-pharmaceuticals, or food supplements for animals, including humans, or used in the manufacture of such pharmaceuticals. The immunomodulators of the present invention can be formulated into pharmaceuticals, quasi-pharmaceuticals, or food supplements for direct administration to animals, including humans, or for ingestion. They can also be added to or incorporated into animal feeds such as food supplements or pet food for immunomodulation. In the latter case, there are no particular limitations on the method of adding or incorporating the specific yeast into the food supplement or animal feed. For example, the specific yeast can be directly incorporated into the raw materials before the manufacture of the food supplement or animal feed, can be added during the manufacturing process of the food supplement or animal feed, or can be added to the finished food supplement or animal feed. The term "food and beverage products" refers to substances that can be ingested by humans as food. In addition to general food and beverage products, including so-called health foods, examples include specific health foods, nutritional functional foods, and nutritional supplements as stipulated in the Ministry of Health, Labour and Welfare's Health Functional Foods Regulations. The term "animal feed" refers to substances provided as feed to animals other than humans (animals raised by humans), such as livestock feed and pet food.

[0023] When the immunomodulator of the present invention is used as a pharmaceutical or quasi-pharmaceutical, it may contain a specific yeast as an active ingredient alone, or it may further contain a pharmaceutically acceptable carrier, or it may further contain other active ingredients or pharmacological components without impairing the immunomodulatory effect of the specific yeast. Examples of such carriers include excipients, coating agents, adhesives, expanders, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure regulators, pH regulators, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet absorbers, humectants, thickeners, activity enhancers, bactericides, flavoring agents, and odor-correcting agents.

[0024] When the pharmaceutical agent of the present invention is used as a medicine or quasi-medicine, it can be administered in any manner. The administration method can be oral or non-oral. Examples of oral administration methods include solid administration methods such as tablets, coated tablets, granules, powders, and capsules, and liquid administration methods such as elixirs, syrups, and suspensions. Examples of non-oral administration methods include injection, infusion, transdermal, transmucosal, nasal, enteral, inhalation, suppositories, boluses, and patches. Oral administration is preferred. In the pharmaceutical preparation of the present invention, the amount of the specific yeast is arbitrary as long as it can be an effective ingredient. In the solid components of the preparation, the specific yeast may account for 5% or more by mass, or 10% or more by mass, or 20% or more by mass, or 30% or more by mass, or 40% or more by mass, or 50% or more by mass, or 70% or more by mass. It should be noted that the solid components referred to here are the total amount after removing the solvent. Examples of solvents include water, ethanol, and commonly used organic solvents.

[0025] When the pharmaceutical preparation of the present invention is used as a food or beverage, it may contain only a specific yeast as an active ingredient, or it may further contain various additives that can be used in the manufacture of food or beverages, within a range that does not impair the immunomodulatory effect of the specific yeast. Examples of such additives include various oils, raw pharmaceutical materials, amino acids, polyols, natural polymers, vitamins, dietary fiber, surfactants, purified water, excipients, stabilizers, pH adjusters, antioxidants, sweeteners, flavoring agents, acidulants such as organic acids, stabilizers, flavorings, colorings, and fragrances.

[0026] As a food and beverage, its uses include, for example, oral care products (chewing gum, candy, etc.), processed seafood products such as fish cakes and fish rolls, animal products such as sausages and ham, bread, Western-style pastries, Japanese sweets, fresh noodles, Chinese noodles, lo mein, buckwheat noodles, sauces, soy sauce, condiments, sugar, honey, powdered syrups, water syrups, spices such as curry powder, mustard powder, pepper, jams, citrus marmalades, chocolate spreads, pickles, prepared dishes, seaweed salt, or various canned or bottled processed vegetables and fruits, dairy products such as cheese, butter, and yogurt, miso soup, soup, fruit juice, vegetable juice, whey drinks, soft drinks, alcoholic beverages, and health foods, as well as other general food and beverage uses.

[0027] The present invention includes a package comprising a package body and an immunomodulatory agent or food or animal feed containing the present invention, as described above, contained within the package body. The packaging body only needs to be able to hold the pharmaceuticals, food and beverages, or animal feed of the present invention and be able to print their ingredient information, etc., and there are no particular restrictions on its shape and material. Examples of packaging body shapes include boxes and bags. Examples of raw materials for the packaging body include paper, plastic, fabric, and metal. The packaging clearly displays various information, such as the content of specific yeasts in the pharmaceutical, food, or animal feed contained within it. There are no particular limitations on how this information is presented; for example, 1) it can be printed on the outer or inner surface of the packaging; 2) for immunomodulatory purposes, it can be printed on a printing medium, such as printing paper, inside the packaging along with the food or animal feed; 3) a QR code (registered trademark) is included on the packaging or its inner printing medium, containing an internet URL and information about a website that allows access to the pharmaceutical, food, or animal feed containing the present invention, which is then accessed via the URL.

[0028] As described in the examples below, specific yeasts exhibit excellent effects in regulating the production of cytokines by cells such as macrophages or dendritic cells. Macrophages and dendritic cells are preferably derived from mammals. Examples of mammals include non-human mammals such as humans, rats, mice, rabbits, cattle, pigs, dogs, cats, sheep, and monkeys. Once mature, macrophages and dendritic cells can perform their inherent functions, such as phagocytosis and antigen presentation.

[0029] For example, as described in Examples 1-5 below, the specific yeast of the present invention promotes the production of cytokines by administration to macrophages or dendritic cells, such as macrophages or dendritic cells in a state of near-unstimulation by antigens after differentiation. For example, it effectively enhances the gene expression of at least one cytokine selected from interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), and TNF-α, and promotes their production.

[0030] IL-1β, IL-6, IL-8, and TNF-α are pro-inflammatory cytokines. IL-1β is a potent pro-inflammatory cytokine. IL-1β stimulates CD4+. +TNF-α plays a crucial role in both local and hematogenous differentiation, including inflammatory responses. It not only induces expression in vascular endothelial cells but also enhances leukocyte adhesion molecules that stimulate immune cell infiltration, promoting lymphocyte infiltration to the site of infection. IL-6 is important for inducing the differentiation of B cells into antibody-producing cells (plasma cells). IL-8 induces chemotaxis of neutrophils or other granulocytes to the site of infection, and upon arrival, IL-8 further induces phagocytosis.

[0031] In addition to suppressing the immune response, IL-10 also exhibits immunostimulatory properties, including stimulating the proliferation of thymocytes treated with IL-2 and IL-4, increasing the survival rate of B cells, and stimulating the expression of MHC class II molecules.

[0032] Furthermore, IL-12 induces natural killer cells (NK cells) and T cells to produce interferon-γ, enhancing the activity of NK cells and cytotoxic T cells. The promotion of IL-12 production is clearly demonstrated by facilitating the production of its subunit, IL-12A.

[0033] As can be seen from the above, by utilizing the specific yeast of the present invention to promote the production of various cytokines, T cells, B cells, NK cells, etc., can be activated, and the immune response caused by the action of the various cytokines can protect the organism from pathogens, viruses, etc.

[0034] On the other hand, the specific yeast of the present invention can exhibit immunosuppressive effects on macrophages and dendritic cells that have been activated by antigens (such as lipopolysaccharide (LPS)). For example, in macrophages, it can inhibit the production of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. While LPS is an antigen that strongly induces the expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, the excessive production of these pro-inflammatory cytokines can sometimes cause tissue damage and is associated with various inflammatory diseases, such as rheumatoid arthritis and osteoarthritis, Alzheimer's disease, adult respiratory distress syndrome, allergies, asthma, eating disorders, atherosclerosis, muscle contusions, and cancer. Therefore, by using the specific yeast of this invention, these diseases can be prevented.

[0035] Furthermore, LPS is known to possess endotoxic activity, and it is known that after reacting with large amounts of LPS, the action of TNF-α and IL-1 produced by macrophages leads to blood coagulation, increased vascular permeability, and decreased blood pressure, resulting in peripheral circulatory failure and shock (endotoxin shock). Additionally, it is known that when IL-1, IL-6, TNF-α, etc., produced by various cells under the influence of LPS are transported to the brain via the bloodstream, prostaglandin E2 is produced from brain cells, acting on the hypothalamus of the pituitary gland, which has thermoregulatory functions, causing fever. Therefore, by using the specific yeast of this invention, it may be possible to prevent or improve these intravascular shock effects and fever.

[0036] Furthermore, it is known that if fat cells become excessively large due to obesity, these fat cells will die, and immune cells such as macrophages that engulf these fat cells will accumulate in visceral adipose tissue, causing inflammation. This inflammation is said to have systemic effects, leading to lifestyle-related diseases such as diabetes. Therefore, by using the specific yeast of this invention, it is possible to prevent diseases such as inflammation caused by obesity.

[0037] Furthermore, the specific yeast of this invention can increase IL-12 in macrophages and dendritic cells that have already been activated by antigens (such as LPS). It is believed that increased IL-12 production in macrophages and dendritic cells is related to enhanced IL-10 expression in T cells, thereby being associated with anti-inflammatory activity. In addition, IL-12 is known to inhibit IgE production, thereby suppressing the Th2 immune response, and is therefore considered to be related to the suppression of the Th2 immune response.

[0038] Th2 immune abnormalities are considered to include allergic diseases (atopic dermatitis), myasthenia gravis, chronic active hepatitis (HBsAg-negative), membranous glomerulonephritis, pemphigus vulgaris, hyperthyroidism or hypothyroidism, nodules, etc. Additionally, it is associated with chronic rheumatoid arthritis, SLE, urticaria, bronchial asthma, etc. Therefore, by using the specific yeast of this invention, these diseases can be prevented.

[0039] As described in Examples 10 and 11 below, the specific yeast of the present invention selectively increases the expression of TLR2 on the cell surface, whether it is a mixture of cell wall and contents or the contents themselves. TLR2 has been reported to function not only in cells responsible for innate immunity, such as dendritic cells and macrophages, but also in T cells, which are central to acquired immunity, and plays an important role in the induction of infection immunity, anti-tumor immunity, and autoimmune diseases. Therefore, the cytokine production regulation function of the specific yeast of the present invention should be understood to be mediated by TLR2.

[0040] Based on the above, the immunomodulator of the present invention can be expected to provide an immunomodulatory effect suitable for the body's state at the time of intake. For example, it is believed that by taking it when fatigued or after exercise, an immune-enhancing effect can be obtained, suppressing infections by viruses, bacteria, etc., when immunity is reduced. Therefore, the immunomodulator of the present invention can also be used as an immune enhancer. Furthermore, by ingesting the immunomodulator of the present invention during a cold, or by having obese or elderly individuals take the immunomodulator of the present invention, it is expected to exert immunosuppressive effects such as anti-inflammatory effects, thereby alleviating inflammation or chronic inflammation, autoimmune diseases, etc., caused by colds. Therefore, the immunomodulator of the present invention can also be used as an anti-inflammatory agent and an immunosuppressant. For the same individual, whether it exerts an immune-enhancing or immunosuppressive effect may correspond to the degree of inflammation in that individual, such as the degree and state of inflammation at the site of administration. In mammals such as humans, methods for assessing the degree and state of inflammation include, for example, identifying inflammatory markers in the blood.

[0041] Generally, existing reports indicate that microbial strains affecting immunity only exhibit one of the effects of immunosuppression or immunostimulation, while the specific yeast of this invention possesses either immunostimulatory or immunosuppressive effects. Therefore, this specific yeast of the present invention has the potential to exert an effect adapted to the individual's physical condition. The immunomodulator of the present invention is believed to have the effect of increasing immunity in a state of weakened immune function and decreasing it in a state of enhanced immune function. It is believed that this immunomodulator of the present invention can exert immunosuppression without reducing immunity. Furthermore, it is expected that it can control abnormalities, prevent the occurrence of pathological conditions, or restore normalcy to existing pathological conditions in any situation of weakened or enhanced immune status in an individual. Therefore, even if the body's immune status changes during continuous intake, it is possible to exert an effect adapted to that state, making it an excellent immunomodulator.

[0042] For example, the present invention provides the following. [1] An immunomodulator comprising yeast deposited with the Technical Base for Evaluation of Pharmaceutical Products under the international accession number NITE BP-04012 at the independent administrative agency. [2] The immunomodulator described in [1] is used to regulate cytokine production. [3] According to [2], the immunomodulator regulates the production of cytokines in macrophages or dendritic cells. [4] The immunomodulatory agent according to any one of [1] to [3] mediates immunity via TLR2. [5] The immunomodulatory agent according to any one of [2] to [3], wherein the cytokine is at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α. [6] The immunomodulatory agent according to any one of [1] to [5] is for both immune enhancement and immunosuppression. [7] According to [6], the immunomodulators are used to exert an immune-enhancing or immunosuppressive effect depending on the state or degree of inflammation in the body. [8] The immunomodulatory agent according to any one of [1] to [7] is used to increase the production of at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α. [9] The immunomodulatory agent according to any one of [1] to [7] is used to inhibit the production of at least one of IL-1β, IL-6 and TNF-α under inflammation, or to promote the production of IL-12 under inflammation.

[10] An immunomodulatory agent according to any one of [1] to [7], which is used to regulate the production of at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α in macrophages, or to regulate the production of IL-6 in dendritic cells.

[11] The immunomodulator according to any one of [1] to

[10] comprises the cell wall component of the yeast.

[12] The immunomodulatory agent according to any one of [1] to

[10] comprises the contents of the yeast.

[13] An immunomodulatory food or beverage comprising any one of [1] to

[12] .

[14] Use of yeast in the manufacture of immunomodulators, deposited at the Technical Base for Product Evaluation, an independent administrative agency, with international accession number NITE BP-04012. Example

[0043] The present invention will be described in more detail below with examples, but the invention is not limited to the examples described below. It should be noted that in the following experiments, macrophage-like cells were used as macrophage models, and dendritic cell-like cells were used as dendritic cell models. The macrophages and dendritic cells in the present invention respectively include the macrophage-like cells and dendritic cell-like cells described below.

[0044] <Yeast Cultivation and Drying> The yeast strain (International Accession No. NITE BP-04012) was cultured using the following method. First, the strain, preserved under the above conditions, was collected and inoculated onto YPD agar medium using the streak method. After incubating the agar medium at 30°C under atmospheric conditions for 3 days, multiple colonies appearing on the agar medium were cultured under the above conditions. After culturing the yeast, it was dried by spray drying.

[0045] <Making Yeast Powder> 150 mg of the dried powder obtained in the above <Yeast Culture and Drying> section was suspended in 1.5 mL of sterile water and placed in a test tube. The cell walls were broken up using a bead-type cell disruption device (TOMY Medical, Micro Smash MS-100R), followed by freeze-drying to obtain a fragmented product from which the contents were dissolved. 10 mg of the obtained solid component was suspended in 10 mL of each experimental culture medium to obtain yeast powder from which the yeast contents were dissolved (concentration in the culture medium was 1 w / v%, sometimes referred to simply as "yeast powder"). The obtained yeast powder contained both yeast cell walls and yeast extract, with the yeast extract accounting for approximately 60% by mass.

[0046] <Preparation of Yeast Extract> Add 10 mg of the yeast powder (crushed material) obtained in the above <Preparation of Yeast Powder> to 10 mL of each test culture medium, stir thoroughly, and allow it to precipitate. Collect the supernatant obtained as the extract (the concentration in the culture medium is 1 w / v%, sometimes referred to as "yeast extract" below).

[0047] <Evaluation 1: Evaluation of cytokine production (gene expression, macrophage-like cells)> THP-1 cells (human monocyte-derived cells) were cultured in proliferation medium (10% FBS (fetal bovine serum)-RPMI 1640 medium) for 4 days, and then cultured in differentiation induction medium (10% FBS, 0.5 μg / mL PMA (Phorbol 12-myristate 13-acetate)-RPMI 1640 medium) to reach 1×10⁻⁶ cells / year. 4Cells were seeded at a rate of 0.1 mL / well in 96-well plates. After 4 days of culture and confirmation of differentiation into macrophage-like cells, the medium was replaced with test medium containing various concentrations of the test substance or LPS (Sigma-Aldrich) and the test substance (10% FBS, 100 Units / mL penicillin, 100 Units / mL streptomycin-RPMI 1640 medium). After 1 hour of culture at 37°C and 5% CO2, the medium was replaced again with test medium containing the test substance or LPS and the test substance and cultured for 5 hours. Cells were washed twice with PBS, and cDNA was recovered using the FastLane cDNA kit (QIAGEN). Real-time PCR was performed using the recovered cDNA with TBGreen Premix Ex Taq II (Takara). Relative gene expression levels were calculated by comparing the Ct values ​​with those of internal standard genes using the ΔΔCt method. Primer sequences for each gene are shown in Table 1 below.

[0048] Table 1

[0049] <Evaluation 2: Evaluation of cytokine production (gene expression, dendritic cell-like cells)> THP-1 cells (human monocyte-derived cells) were cultured in proliferation medium (10% FBS (fetal bovine serum)-DMEM medium) for 2-3 days, and then cultured in differentiation induction medium (200 ng / mL ionomycin, 100 ng / mL GM-CSF, 200 ng / mL IL-4, 20 ng / mL TNF-α-DMEM medium) to reach a density of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a rate of 0.5 mL / well in a 24-well plate. After 48 hours of culture to allow differentiation into dendritic cell-like cells, the diluent was added to each well at a rate of 3.3 μL / well to achieve the specified concentration of the test substance. The plates were then incubated at 37°C and 5% CO2 for 24 hours. Sterile water was used as the diluent. Cells were washed with PBS, and RNA was extracted using a commercially available kit (Takara). cDNA was recovered using the PrimeScript RT reagent Kit with gDNA Eraser (Takara), and real-time PCR was performed using TB Green Premix Ex Taq II. Relative gene expression levels were calculated by comparing Ct values ​​with internal standard genes using the ΔΔCt method. Primer sequences for the genes are shown in Table 2 below.

[0050] Table 2

[0051] <Evaluation 3: Evaluation of cytokine production levels (ELISA analysis, macrophage-like cells)> After culturing THP-1 cells in proliferation medium, they were then induced to differentiate into macrophages using macrophage-like differentiation induction medium to reach a cell size of 6.3 × 10⁶. 4 Cells were seeded at a rate of 0.5 mL / well in 24-well plates. After incubation at 37°C and 5% CO2 for 4 days, once differentiation into macrophage-like cells was confirmed, the medium was replaced with test medium (10% FBS, 100 Units / mL penicillin, 100 Units / mL streptomycin-RPMI 1640 medium) containing various concentrations of the test substance or containing LPS (Nacalai Tesque) and the test substance. After incubation at 37°C and 5% CO2 for 1 hour, the medium was again replaced with test medium containing the test substance or containing LPS and the test substance, and incubated for 24 hours. The culture supernatant from each well was transferred to a 1.5 mL tube and centrifuged at 3000×g for 5 minutes. The supernatant was then used for ELISA analysis. In the ELISA analysis, IL-1β was measured using the IL-1β ELISA Kit, Human (ProteinTech), IL-6 was measured using the Levis (registered trademark) Human IL-6 ELISA Kit (Fujifilm and Hikari Pure Chemicals), IL-8 was measured using the Levis (registered trademark) Human IL-8 ELISA Kit (Fujifilm and Hikari Pure Chemicals), IL-10 was measured using the AuthentiKine (registered trademark) IL-10 ELISA Kit, Human (ProteinTech), IL-12A was measured using the Human IL-12 ELISA Kit (R&D Systems), and TNF-α was measured using the AuthentiKine (registered trademark) TNF-alpha ELISA Kit, Human (ProteinTech).

[0052] <Evaluation 4: Evaluation of TLR receptor activation> In this evaluation, HEK293 cells were used as the cells capable of highly efficient gene delivery. The HEK293 cell line containing NF-κB receptor luciferase was increased to 1×10⁻⁶. 4Cells were seeded in 96-well plates using proliferation medium (10% FBS (fetal bovine serum), 100 U / mL penicillin, 100 U / mL streptomycin, 100 μg / mL hygromycin B-DMEM) at a rate of 100 μL / well. After 24 hours of culture, plasmid vectors containing the TLR2 or TLR4 gene (TLR2: pRP[Exp]-Neo-CMV>hTLR2 (VectorBuilder), TLR4: pRP(Exp)-Neo-CMV>hTLR4 (VectorBilder)) were introduced into cells using liposome transfection. A transfection solution was prepared by mixing 100 ng of Plasmid DNA, 0.1 μL of X-tremeGENE HP DNA transfection reagent (MERCK), and 10 μL of DMEM medium in each well. The transfection solution was added at a rate of 10 μL / well, slowly mixed, and the cells were cultured again. On the second day, as confirmation of vector introduction, the cells were cultured for another day in test medium containing 200 μg / mL G418 (10% FBS, 100 Units / mL penicillin, 100 Units / mL streptomycin, 200 μg / mL G418-RPMI1640 medium). Replace the culture supernatant in each well with test medium containing 100 μL / well of the test substance or positive control (10 μg / mL Zymosan or 1 μg / mL LPS). Then, incubate for 6 hours. After incubation, evaluate the activity of intracellular luciferase using the Bright-Glo Luciferase Assay System (Promega). After incubation, remove 30 μL of culture supernatant from each well, add 70 μL of luciferase assay reagent, stir gently, and let stand for 2 minutes. Measure the relative luminescence value of each well using a microplate reader. The relative luminescence value is the ratio to the control (without test substance or positive control).

[0053] (Example 1: Increased expression of cytokine genes (macrophage-like cells)) For macrophage-like cells, the above-mentioned yeast powder was added as the test substance, and the above-mentioned <Evaluation 1: Evaluation of Cytokine Production (Gene Expression, Macrophage-like Cells)> was performed to determine the cytokine gene expression levels (relative to internal standards). For the obtained gene expression levels, the ratio was calculated to the cytokine gene expression levels of the control obtained in the same manner as above, excluding the addition of yeast powder. The results are presented in... Figure 1 . like Figure 1 As shown, the addition of yeast powder confirmed that the expression levels of various cytokine genes increased compared to the absence of yeast powder.

[0054] (Example 2: Increased expression of cytokine genes (macrophage-like cells)) For macrophage-like cells, the above-mentioned yeast extract was added as the test substance, and the above-mentioned <Evaluation 1: Evaluation of Cytokine Production (Gene Expression, Macrophage-like Cells)> was performed to evaluate cytokine gene expression levels (relative to internal standards). For the obtained gene expression levels, the ratios to the cytokine gene expression levels of the control obtained in the same manner as above, excluding the addition of yeast powder, were calculated. The results are presented as follows: Figure 2 . like Figure 2 As shown, the addition of yeast extract confirmed that the expression levels of various cytokine genes increased compared to the absence of yeast extract.

[0055] (Example 3: Increased expression of cytokine genes (dendritic cell-like cells)) For dendritic cell-like cells, the yeast extract described above was added as the test substance, and the IL-6 cytokine gene expression level was evaluated using <Evaluation 2: Evaluation of Cytokine Production (Gene Expression, Dendritic Cell-like Cells)>. The ratio of cytokine gene expression levels to the control obtained in the same manner (excluding the addition of yeast extract) was calculated. The results are presented in... Figure 3 . like Figure 3 As shown, this confirmed that the expression levels of cytokine genes were increased in dendritic-like cells as well, compared to the absence of addition.

[0056] (Example 4: Increased production of cytokines) For macrophage-like cells, the above-mentioned yeast powder was added as the test substance, and <Evaluation 3: Evaluation of Cytokine Production (ELISA Analysis, Macrophage-like Cells)> was performed. Cytokine production was evaluated using ELISA analysis. The ratio of cytokine gene expression levels to the control obtained in the same manner (excluding the addition of yeast powder) was calculated. The results are presented as follows. Figure 4 .like Figure 4 As shown, it was confirmed that the addition of yeast powder increased the levels of various cytokines compared to the absence of yeast powder.

[0057] (Example 5: Increased production of cytokines) For macrophage-like cells, the above-mentioned yeast extract was added as the test substance in <Evaluation 3: Evaluation of Cytokine Production (ELISA Analysis, Macrophage-like Cells)>, and the cytokine production was evaluated by ELISA analysis. The results are shown below. Figure 5 .like Figure 5As shown, the addition of yeast extract confirmed that the levels of various cytokines increased compared to the control group without it.

[0058] (Example 6: Regulation of gene expression levels for cytokine production) For macrophage-like cells, the inflammatory condition with 1 μg / mL of LPS was used as a control. 1 μg / mL of LPS was added, and the above-mentioned yeast powder was added as the test substance. The cytokine gene expression level (relative to the internal standard) was evaluated by <Evaluation 1: Evaluation of Cytokine Production (Gene Expression, Macrophage-like Cells)>. Figure 6 The figure shows the gene expression ratio when the values ​​without LPS and the test substance are set to 1. It was confirmed that the addition of yeast powder reduced the gene expression levels of inflammatory cytokines (IL-6, TNF-α) that are increased under inflammation.

[0059] (Example 7: Regulation of gene expression levels for cytokine production) For macrophage-like cells, the inflammatory condition with LPS 1 μg / mL was used as a control. The cytokine gene expression levels when LPS 1 μg / mL and yeast extract were added were evaluated using <Evaluation 1: Evaluation of cytokine production (gene expression, macrophage-like cells)>. Figure 7 The figure shows the gene expression ratio when the values ​​without LPS and the test substance are set to 1. It was confirmed that the addition of yeast extract reduced the expression levels of inflammatory cytokine genes that are increased under inflammation.

[0060] (Example 8: Regulation of cytokine production) For macrophage-like cells, the inflammatory condition with LPS 1 μg / mL was used as a control. LPS 1 μg / mL was added along with the aforementioned yeast powder as the test substance. Cytokine production was evaluated using ELISA analysis based on <Evaluation 3: Evaluation of Cytokine Production (ELISA Analysis, Macrophage-like Cells)>. The results are shown below. Figure 8 .like Figure 8 As shown, the addition of yeast powder confirmed that the production of inflammatory cytokines (IL-6, TNF-α) under inflammation was inhibited.

[0061] (Example 9: Regulation of cytokine production) For macrophage-like cells, the inflammatory condition with LPS 1 μg / mL was used as a control. LPS 1 μg / mL was added along with the aforementioned yeast extract as the test substance. Cytokine production was evaluated using ELISA analysis based on <Evaluation 3: Evaluation of Cytokine Production (ELISA Analysis, Macrophage-like Cells)>. The results are shown below. Figure 9 .like Figure 9 As shown, the addition of yeast extract confirmed that the production of inflammatory cytokines (IL-6, TNF-α, IL-1β) under inflammation was inhibited.

[0062] (Example 10: Activation of TLR receptor) The activities of TLR2 and TLR4 receptors were evaluated using Evaluation 4: TLR Receptor Activation Evaluation, respectively, when the above-mentioned yeast powder was added as the test substance. As positive controls (PCs), Zymosan (Nova Biologicals) was used for TLR2, and LPS-EB (Nacalai tesque) was used for TLR4. The results are presented below. Figure 10 .like Figure 10 As shown, it was confirmed that the addition of yeast powder mediated by TLR2 increased luciferase activity, which in turn mediated by TLR2 increased cytokine production.

[0063] (Example 11: Activation of TLR receptor) The activities of TLR2 and TLR4 receptors with the addition of yeast extract alone were evaluated using Evaluation 4: Evaluation of TLR Receptor Activation. Zymosan (Nova Biologicals) was used as the PC (positive control) for TLR2, and LPS-EB (Nacalai tesque) was used for TLR4. The results are presented below. Figure 11 .exist Figure 11 In this study, it was confirmed that the addition of yeast extract mediated by TLR2 increased luciferase activity, which in turn mediated by TLR2 increased cytokine production.

Claims

1. An immunomodulator comprising yeast deposited with the Technical Base for Evaluation of Pharmaceutical Products under the international accession number NITE BP-04012 at the independent administrative agency.

2. The immunomodulator according to claim 1, wherein it is used to regulate cytokine production.

3. The immunomodulator according to claim 2, which regulates the production of cytokines in macrophages or dendritic cells.

4. The immunomodulatory agent according to claim 1 or 2, wherein it mediates immunity via TLR2.

5. The immunomodulatory agent according to claim 2, wherein, The cytokine is at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α.

6. The immunomodulatory agent according to claim 1 or 2, which is for both immune enhancement and immunosuppression.

7. The immunomodulator according to claim 6, which is used to exert an immune-enhancing or immunosuppressive effect according to the state or degree of inflammation in the body.

8. The immunomodulator according to claim 1 or 2, which is used to increase the production of at least one selected from IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α.

9. The immunomodulator according to claim 1 or 2, which is used to inhibit the production of at least one selected from IL-1β, IL-6 and TNF-α under inflammation, or to increase the production of IL-12 under inflammation.

10. The immunomodulator according to claim 1 or 2, comprising the cell wall components of the yeast.

11. The immunomodulator according to claim 1 or 2, comprising the contents of the yeast.

12. An immunomodulatory food or beverage comprising the immunomodulator as described in claim 1 or 2.

13. Use of yeast deposited with the Technical Base for Evaluation of Pharmaceutical Products, Inc. (NITE BP-04012) in the manufacture of immunomodulators.

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