Immunotherapy modifiers containing proteoglycans

Abstract

[Problem] To examine the influence proteoglycans exhibit on immune responses induced by a variety of immunogenic factors that act on dendritic cells, macrophages, and other such immune cells, and provide an agent or composition that is useful for modifying immune response. [Solution] An agent of modifying immune response, the agent containing proteoglycan as an active ingredient.

Description

Cross-reference

[0001] This application claims priority based on Japanese Patent Application No. 2022-002171 filed on January 11, 2022 in Japan, and all of the contents described in the application are incorporated herein by reference as they are.

Technical Field

[0002] The present invention relates to an immunomodulator containing proteoglycan (including a composition for administration to humans, etc.).

Background Art

[0003] Proteoglycan (hereinafter sometimes referred to as "PG") is one of the complex carbohydrates and consists of a core protein and glycosaminoglycan (acid mucopolysaccharide) bound thereto. PG exists in the skin, cartilage, bone, blood vessel wall, etc. as a main component of the extracellular matrix and is involved in cell growth and adhesion together with collagen, laminin, fibronectin, etc. Together with chondroitin sulfate that constitutes PG, PG is a main component of articular cartilage and is considered important for preventing joint disorders in the elderly. Furthermore, it has been reported that PG suppresses the inflammatory reaction of macrophages stimulated by heat-treated bacteria (see Non-Patent Document 1), and that administration of PG suppresses the progression of colitis (see Non-Patent Document 2).

[0004] Patent Document 1 discloses the pharmacological action of PG on the immune system, but the full extent of the action of PG is still unclear.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] [Patent Document 1] Japanese Patent Publication No. 2007-131548 [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, the problem that the present invention aims to solve is to investigate the effect of proteoglycans on immune responses induced by various immunogenic factors acting on immune cells such as dendritic cells and macrophages, and to provide agents useful for regulating immune responses, compositions containing such agents, and the like. [Means for solving the problem]

[0008] The present invention was made to solve the aforementioned problems and is based on the discovery that proteoglycans have the effect of modulating the immune response by coordinating with various immunogenic factors to enhance or suppress their activity. That is, the present invention includes the following embodiments.

[0009] [1] A composition for activating an immune response, comprising a proteoglycan and a bacterial component of Enterococcus faecalis strain EC-12 (accession number FERM BP-10284) and / or Bifidobacterium longum strain BR-108 (accession number: NITE P-1317, deposited under Bifidobacterium longum strain BR-108), either together or separately. [2] The composition according to [1], wherein the immune response is activated via innate and / or adaptive immunity. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide agents useful for regulating immune responses induced by various immunogenic factors that act on immune cells such as macrophages, as well as compositions containing such agents. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the results of measuring the amount of TNF-α secreted into the culture medium when various immunogenic factors (Pam, LPS, ODN, or Zymosan) were added to the culture medium of mouse macrophage cells, with or without the presence of prostaglandins (PG). [Figure 2] Figure 2 shows the results of measuring the amount of IL-6 secreted into the culture medium when various immunogenic factors (LPS or Zymosan) were added to the culture medium of mouse macrophage cells, with or without the presence of prostaglandins (PG). [Figure 3] Figure 3 shows the results of measuring the amount of IL-10 secreted into the culture medium when various immunogenic factors (Pam, LPS, ODN, or Zymosan) were added to the culture medium of mouse macrophage cells, with or without the presence of prostaglandins (PG). [Figure 4] Figure 4 shows the results of measuring the amount of IL-1β secreted into the culture medium when various immunogenic factors (Pam, LPS, Imiquimod, or Zymosan) were added to the culture medium of mouse macrophage cells, with or without the presence of prostaglandins (PG). [Figure 5]Figure 5 shows the results of measuring the amount of TNF-α secreted into the medium when various immunogenic factors (shiitake mushroom extract or kawaratake mushroom extract) were added to the culture medium of mouse macrophage cells in the presence or absence of PG. [Figure 6] Figure 6 shows the results of measuring the amount of IL-6 secreted into the medium when various immunogenic factors (shiitake mushroom extract or kawaratake mushroom extract) were added to the culture medium of mouse macrophage cells in the presence or absence of PG. [Figure 7] Figure 7 shows the results of measuring the amount of IL-10 secreted into the medium when various immunogenic factors (shiitake mushroom extract or kawaratake mushroom extract) were added to the culture medium of mouse macrophage cells in the presence or absence of PG. [Figure 8] Figure 8 shows the results of measuring the amount of IL-1β secreted into the medium when various immunogenic factors (shiitake mushroom extract or kawaratake mushroom extract) were added to the culture medium of mouse macrophage cells in the presence or absence of PG. [Figure 9] Figure 9 shows the results of measuring the amount of IL-1β secreted into the medium when various immunogenic factors (Pam, Poly(I:C), LPS, CpG DNA, Loxoribine, Zymosan, shiitake mushroom extract or kawaratake mushroom extract) were added to the culture medium of mouse macrophage cells in the presence or absence of PG. [Figure 10] Figure 10 shows the results of measuring the amount of TNF-α secreted into the medium when Lactobacillus Enterococcus faecalis EC-12 was added to the culture medium of mouse bone marrow-derived dendritic cells in the presence or absence of PG. [Figure 11] Figure 11 shows the results of measuring the amount of IL-6 secreted into the medium when Lactobacillus Enterococcus faecalis EC-12 was added to the culture medium of mouse bone marrow-derived dendritic cells in the presence or absence of PG. [Figure 12]Figure 12 shows the results of measuring the amount of IL-12p70 secreted into the medium when Enterococcus faecalis EC-12, a lactic acid bacterium, was added to the culture medium of mouse bone marrow-derived dendritic cells in the presence or absence of PG. [Figure 13] Figure 13 shows the results of measuring the amount of TNF-α secreted into the medium when heat-killed Listeria monocytogenes (HK-LM) as a test sample, Houttuynia cordata extract or Hericium erinaceus extract were added to the culture medium of mouse bone marrow-derived dendritic cells in the presence or absence of PG. [Figure 14] Figure 14 shows the results of measuring the amount of IL-12p70 secreted into the medium when heat-killed Listeria monocytogenes (HK-LM) as a test sample, Houttuynia cordata extract or Hericium erinaceus extract were added to the culture medium of mouse bone marrow-derived dendritic cells in the presence or absence of PG. [Figure 15] Figure 15 shows the results of measuring the amount of IL-6 secreted into the medium when heat-killed Listeria monocytogenes (HK-LM) as a test sample, Houttuynia cordata extract or Hericium erinaceus extract were added to the culture medium of mouse bone marrow-derived dendritic cells in the presence or absence of PG. [Figure 16] An experimental protocol for examining the effect (innate immunity) of oral administration of Hericium erinaceus extract on Listeria infection in mice is shown. [Figure 17] Figure 17 shows the results of measuring the amount of IFN-γ secreted into the medium when the spleen on the 4th day was removed and cultured after infecting mice with Listeria while feeding them a diet with or without Hericium erinaceus extract added. [Figure 18] Figure 18 shows the results of measuring the amount of TNF-α secreted into the medium when the spleen on the 4th day was removed and cultured after infecting mice with Listeria while feeding them a diet with or without Hericium erinaceus extract added. [Figure 19]Figure 19 shows the results of measuring the amount of IL-12p70 secreted into the culture medium when mice were infected with Listeria monocytogenes while being fed diets with or without Chaga mushroom extract, and then their spleens were removed and cultured on the fourth day. [Figure 20] This document presents an experimental protocol investigating the effects of oral administration of Chaga mushroom extract on Listeria infection in mice (acquired immunity). [Figure 21] Figure 21 shows the results of measuring the amount of IFN-γ secreted into the culture medium when mice were infected with Listeria monocytogenes while being fed diets with or without Chaga mushroom extract, and then their spleens were removed and cultured after 4 weeks. [Figure 22] Figure 22 shows the results of measuring the amount of TNF-α secreted into the culture medium when mice were infected with Listeria monocytogenes while being fed diets with or without Chaga mushroom extract, and then their spleens were removed and cultured after 4 weeks. [Figure 23] Figure 23 shows the results of measuring the amount of IL-12p70 secreted into the culture medium when mice were infected with Listeria monocytogenes while being fed diets with or without Chaga mushroom extract, and then their spleens were removed and cultured after 4 weeks. [Modes for carrying out the invention]

[0012] Next, each embodiment of the present invention will be described with reference to the drawings. It should be noted that the embodiments described below are not intended to limit the invention as defined in the claims, and not all of the elements and combinations thereof described in each embodiment are necessarily essential to the solution of the present invention.

[0013] (An agent for regulating the immune response) In this specification, “agent for modulating immune response” means any agent having one or more of the following effects: repairing a reduced immune response, suppressing an abnormal immune response, and enhancing a normal immune response. The immune response includes, for example, one or more of humoral (antibody-mediated) immunity, cellular immunity, adaptive immunity, and innate immunity. In one embodiment, a composition for activating an immune response is provided by combining such an agent with an immunogenic factor. “Immunogenic factor” means any factor that can stimulate the immune system of a human or animal. Such immunogenic factors include, for example, ligands for pathogen-associated pattern recognition receptors (PRRs). Examples of PRRs include, but are not limited to, Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG-I-like receptors, C-type lectin receptors, scavenger receptors, and complement receptors.

[0014] TLRs are a class of non-catalytic receptors with a single transmembrane domain that recognize structurally conserved molecules called pathogen-associated molecular patterns (PAMPs). PAMPs are present on microorganisms and are distinguishable from host molecules. TLRs are present in all vertebrates, and in humans, they include TLR1-10. TLR ligands are compounds that bind to these TLRs and include pathogenic molecules associated with threats to host survival, such as pathogens or cellular stress. Exemplary pathogenic molecules include, but are not limited to, lipopolysaccharides (LPS), lipoproteins, lipoarabinomannan, flagellin, double-stranded RNA, and unmethylated CpG islands of DNA.

[0015] Examples of C-type lectin receptors include Dectin 1, Dectin 2, DEC205, Mincle, and DC-SIGN. Among these, Dectin 1 is a membrane protein expressed in dendritic cells and macrophages, and is known to be an extremely important receptor for the recognition of (1→3)-β-D-glucan, activation of innate immunity, and as a protective factor against fungi. The agent or composition of this embodiment contains a proteoglycan as an active ingredient and has the effect of regulating various immune responses mediated by the above-mentioned receptors, or promoting immune responses in cooperation with these receptor ligands.

[0016] (Proteoglycan) Proteoglycans (PGs) are glycoproteins in which sugar chains called glycosaminoglycans (hereinafter referred to as GAGs), such as chondroitin sulfate and dermatan sulfate, are covalently bonded to a core protein. PGs are one of the major components of the extracellular matrix and are widely distributed throughout the body, including in the skin and cartilage. GAG chains have a long, unbranched linear structure. Because they have numerous sulfate and carboxyl groups, they are negatively charged, and the GAG ​​chains take on an elongated shape due to this electrical repulsion. In addition, PGs can hold a large amount of water due to the water affinity of sugars. The numerous GAG chains contained in PGs flexibly hold water like a sponge, while performing cartilage-specific functions such as elasticity and resistance to impact.

[0017] The core protein of prostaglandins (PGs) has the property of binding to various molecules in the matrix. In the case of cartilage PGs, the N-terminus has a binding domain for hyaluronic acid and link proteins, and can bind to these substances, or associate with other molecules of the same type. The C-terminus has lectin-like and EGF-like domains, which allow it to bind to various other molecules. Due to this property, PGs can build structures that are appropriate for each tissue.

[0018] The PG derived from salmon nasal cartilage is PG obtained by extraction from salmon nasal cartilage. Here, salmon refers to fish belonging to the genus Oncorhynchus, for example, but preferably salmon with the scientific name "Oncorhynchus keta" is selected from the viewpoint of efficiently regulating the immune response. The proteoglycan contained in the agent or composition of this embodiment is prepared, for example, by the method described in the publication (Japanese Patent Publication No. 6317053). Furthermore, the proteoglycan content in the composition (1g) containing the agent of this embodiment is preferably at a lower limit of 0.1 μg / g or more, more preferably at 100 μg / g or more, and even more preferably at 1 mg / g or more, from the viewpoint of effectively regulating the immune response. In this specification, the amount of proteoglycan effective in regulating the immune response, or "effective amount," refers to the amount of proteoglycan that, when administered to a subject in single or multiple doses, is effective in regulating the immune response beyond the state expected without such treatment.

[0019] (Composition for activating the immune response) A composition for activating an immune response in one embodiment comprises an immunogenic factor and a proteoglycan, either together or separately. Examples of immunogenic factors include TLR ligands, dectin-1 ligands, Chaga mushroom extract, Trametes versicolor extract, lactic acid bacteria cell components, and plant extracts (e.g., Houttuynia cordata extract). Specifically, TLR ligands include TLR2 ligands or TLR4 ligands such as lipoproteins and lipopolysaccharides, TLR3 ligands such as double-stranded DNA, TLR5 ligands such as the proteinogenic ligand flagellin, TLR7 ligands or TLR8 ligands such as the imidazoquinoline compounds imiquimod, reximod (R848), and single-stranded RNA, and TLR9 ligands such as the nucleoside ligand CpG sequence.

[0020] More specifically, the following can be mentioned: (TLR2 ligand) TLR2 ligands include synthetic triacylated and diacylated lipopeptides. Non-limiting examples of TLR2 ligands include FSL-1 (a synthetic lipoprotein derived from Mycoplasma salivarium 1), Pam3Cys (tripalmitoyl-S-glycerylcysteine), or S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine, where "Pam3" is "tripalmitoyl-S-glyceryl". Derivatives of Pam3Cys are also suitable TLR2 agonists, and derivatives include, but are not limited to, the following: S-[2,3-bis(palmitoyloxy)-(2-R,S)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(Lys)4-hydroxytrihydrochloride; Pam3Cys-Ser-Ser-Asn-Ala; PaM3Cys-Ser-(Lys)4; Pam3Cys-Ala-Gly; Pam3Cys-Ser-Gly; Pam3Cys-Ser; PaM3Cys-OMe; Pam3Cys-OH; PamCAG, palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-Ala-Gly-OH, etc. Another non-limiting example of a suitable TLR2 agonist is Pam2CSK4;PaM2CSK4 (dipalmitoyl-S-glycerylcysteine-serine-(lysine)4; or Pam2Cys-Ser-(Lys)4), a synthetic diacylated lipopeptide. For example, peptidoglycans and teicoic acid, which make up the cell walls of lactic acid bacteria, are also thought to trigger various immune responses via TLR2.

[0021] (TLR3 ligand) TLR3 ligands include natural double-stranded RNA (dsRNA), synthetic dsRNA, and synthetic dsRNA analogs. Exemplary and non-exclusive examples of synthetic dsRNA analogs include polyrivoinosine and polyribocytidic acid (Poly(I:C)), polyadenosine-polyuridylic acid (PolyAU), poly-L-lysine, and polyinosine-polycytidic acid (Hiltonol®) stabilized with carboxymethylcellulose.

[0022] (TLR4 ligand) TLR4 ligands include naturally occurring lipopolysaccharides (LPS), such as LPS from a wide variety of Gram-negative bacteria; derivatives of natural LPS; synthetic LPS; bacterial heat shock protein-60 (Hsp60); mannuronic acid polymers; flavolipins; tycronate; Streptococcus pneumoniae pneumolysin; bacterial pili; and respiratory syncytial (RS) virus coat proteins. TLR4 ligands also include monophosphoryl lipid A synthesis (MPLA, Invivogen) and phosphorylated hexaacyl disaccharide (PHAD, Avanti Polar Lipids), as well as other synthetic TLR4 agonists.

[0023] (TLR-7 / 8 ligand) Several TLR-7 ligands are known, such as the adenine analog CL264, the guanosine analog loxoribine, or preferably imidazoquinoline compounds (such as reciquimod, Gardiquimod (trademark), or imiquimod (4-amino-1-isobutyl-1H-imidazol[4,5-c]chinolin)). TLR-8 agonists are known to have similar biological effects to TLR-7 agonists and therefore can also be used, or alternatively. Examples of TLR-8 agonists are single-stranded RNA or E. coli RNA. Exemplary TLR-7 / 8 ligands include thiazoloquinoline compound CL075, imidazoquinoline compound R848, or water-soluble R848 imidazoquinoline compound CL097, and thymidine homopolymer phosphorothioate ODN (poly(dT)).

[0024] (TLR-9 ligand) Examples of TLR-9 ligands include CpG oligodeoxyribonucleotides.

[0025] Among these, TLR-3 ligand, TLR-4 ligand, TLR-7 / 8 ligand, and TLR-9 ligand are preferred. The TLR ligands may be naturally derived or obtained synthetically.

[0026] (Chaga mushroom extract and Trametes versicolor extract) Chaga, also known as Inonotus obliqua, is a cold-hardy mushroom widely distributed in cold regions such as Central Europe, Siberia, China, and northern Japan. It commonly parasitizes birch trees, including white birch and Japanese white birch, feeding on their sap. Its hard, black sclerotia are called chaga and have long been consumed as a tea substitute in Russia. Inonotus obliqua is known to possess physiological activities such as antitumor and hypoglycemic effects, and chaga is the sclerotia of the cold-hardy mushroom known as Inonotus obliqua in Japan, which parasitizes white birch trees. Recent research has also confirmed that chaga (Inonotus obliqua) contains abundant amounts of β-glucan, which has immunostimulant effects, and SOD (superoxide dismutase), which has reactive oxygen species scavenging ability, compared to other mushrooms. In particular, it is said to contain about 30 times more SOD than Agaricus or Lion's Mane mushrooms. Furthermore, it has been found to contain a wide variety of other physiologically active substances, and has been shown to have antitumor effects, effects against chronic gastritis and gastric ulcers, and blood circulation-enhancing effects.

[0027] Coriolus versicolor, also known as the Japanese wood turtle mushroom (Trametes versicolor), belongs to the Basidiomycetes and the Polyporaceae family. It is widely distributed throughout the world, with over 120 strains identified in the temperate forest zones of Asia, Europe, and North America. Coriolus versicolor has been reported to possess immunomodulatory, anticancer, antibacterial, and antiviral effects.

[0028] When extracting extracts from Chaga mushrooms and Trametes versicolor, one or more materials selected from the group consisting of fruiting bodies, mycelium, and liquid cultures of mycelium are used as raw materials. However, it is preferable to use fruiting bodies because they allow for the extraction of a large amount of extract and, even when using the same amount of extraction solvent, can result in a higher extract concentration than when using mycelium. The extraction method and conditions for extracting Chaga mushrooms and Trametes versicolor are not particularly limited, but known extraction methods such as water extraction, hot water extraction, warm water extraction, alcohol extraction, aqueous alcohol extraction, acetone extraction, aqueous acetone extraction, and supercritical fluid extraction can be used. If the raw material is a liquid culture of mycelium, it may be dried, cut, and pulverized before extraction. If the raw material is fruiting bodies or mycelium, they may be used as is for extraction, or they may be cut and pulverized before extraction.

[0029] The solvent used for solvent extraction is not particularly limited and includes, for example, water, lower alcohols such as methanol and ethanol, polyhydric alcohols such as propylene glycol and 1,3-butylene glycol, ketones such as acetone, esters such as diethyl ether, dioxane, acetonitrile and ethyl acetate, xylene, hexane, etc. The organic solvent may be an aqueous solution. Preferably, it is water, or acetone, ethanol, methanol, or an aqueous solution thereof. Furthermore, when used in the food industry, water, or ethanol, or an aqueous solution thereof is preferred because it eliminates the need for a step to remove solvents that are harmful to food. In the case of an aqueous solution, the concentration of the organic solvent in the aqueous solution is preferably 10% or more, more preferably 20% or more, even more preferably 30% or more, preferably 90% or less, preferably 70% or less, and preferably 50% or less. Only one of these solvents may be used, or two or more may be used in combination. The temperature during extraction is not particularly limited as long as it is at a temperature at which extracts can be obtained with each solvent. The obtained extract (or its purified or concentrated form) can be used as is, but it may also be dried and used as a powder. This can be done by freeze-drying or by spray-drying with the addition of excipients such as dextrin, corn starch, or gum arabic, according to known methods. Furthermore, it may be dissolved in pure water, ethanol, etc., as needed before use.

[0030] (Cellular components of lactic acid bacteria) According to one embodiment of the present invention, lactic acid bacteria are used as immunogenic factors. Lactic acid bacteria are a general term for bacteria that produce lactic acid by breaking down sugars such as glucose and lactose to obtain the energy necessary for growth. Based on differences in cell shape, they are classified into rod-shaped or cylindrical lactobacilli and spherical lactococci. Bifidobacteria also produce lactic acid and contribute to human health, so they can be considered included in the broader definition of lactic acid bacteria. Examples of lactic acid bacteria include bacteria belonging to the genus Enterococcus and bifidobacteria (for example, Bifidobacterium longum BR-108: Bifidobacterium longum BR-108 strain deposited with accession number: NITE P-1317). An example of bacteria belonging to the genus Enterococcus is Enterococcus faecalis.

[0031] Examples of Enterococcus faecalis include strains such as Enterococcus faecalis EC-12, ATCC19433, ATCC14508, ATCC23655, IFO16803, and IFO16804, or their variants. Here, "variant" means a strain that has been modified by a person skilled in the art using a method well known to a person skilled in the art, to the extent that it does not alter the properties of that strain, or a strain that a person skilled in the art can confirm to be equivalent thereto.

[0032] Enterococcus faecalis EC-12 was deposited with the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (AIST) (1-1-1 Higashi, Tsukuba, Ibaraki Prefecture 305-8566, Japan) on February 25, 2005 (original deposit date). The accession number is FERM BP-10284.

[0033] The lactic acid bacteria used as an immunogenic factor can be live or / or dead cells, but due to the shelf life of the composition, dead cells are preferably used, and more preferably, heat-sterilized cells obtained by sterilizing the above lactic acid bacteria by known heat treatment methods are used. Heat-sterilized cells can be prepared by recovering the cells from a culture obtained by culturing the above lactic acid bacteria according to a conventional method by methods such as filtration and centrifugation, washing with water, suspending in water or the like and heat-treating, and then concentrating and drying as necessary. Alternatively, the cells may be prepared by calcining or steaming. The heat-sterilized bacterial cell powder of the above EC-12 strain is commercially available under the trade names "EC-12" (manufactured by Combi Corporation) or "La Flora EC-12" (manufactured by Ichimaru Falcos Co., Ltd.). Therefore, in the present invention, such commercially available products may be used as the lactic acid bacteria used as an immunogenic factor.

[0034] (Houttuynia cordata extract) The Houttuynia cordata extract used in this invention is, for example, obtained by extracting the flowering aerial parts (crude drug name: Juuyaku) of Houttuynia cordata Thunb. (hereinafter referred to as H. cordata) of the family Houttuyniaceae using a solvent (such as water). The Houttuynia cordata extract used in the following examples is Houttuynia cordata DXP100 (Ichimaru Falcos, an extract obtained by adding the aerial parts of Houttuynia cordata to water and extracting it, with a quercitrin content of 0.15% or more).

[0035] (Dosage form and administration method of compositions for activating the immune response) When using the composition of this embodiment, the dosage form is not particularly limited and can be liquid, solid, semi-solid, or semi-liquid, preferably in liquid form. These dosage forms can be easily manufactured by employing appropriate pharmaceutical carriers as needed, based on methods known to those skilled in the art. When the dosage form is solid, examples include powders, granules, tablets, and capsules containing at least an immunogenic factor and a proteoglycan. When the dosage form is semi-solid or semi-liquid, examples include ointments, lotions, creams, and gels. When the dosage form is liquid, examples of solvents to be used include water, phosphate-buffered saline, Ringer's solution, dextrose solution, serum-containing solution, Hank's solution, other aqueous physiological equilibrium solutions, oils, esters, and glycols. The solvent may include appropriate auxiliary substances necessary to approach the physiological conditions of the recipient, for example, by enhancing chemical stability and isotonicity. Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer. The compositions of the present invention may be sterilized by conventional methods and / or freeze-dried.

[0036] Furthermore, while the administration routes of the composition of this embodiment include, but are not limited to, oral, topical, subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, transmucosal, and transdermal administration, the composition may be administered by known methods, such as direct injection subcutaneously, intradermally, intravenously, intramuscularly, or intraperitoneally; spraying onto mucous membranes such as the nasal cavity, oral cavity, lungs, vagina, or rectum; and oral administration.

[0037] (Target recipients of the composition) Any animal can be administered the composition, with mammals and vertebrates such as birds being preferred, and mammals being more preferred. Furthermore, while the mammalian test subject is, for example, a human, livestock (e.g., chickens, pigs, horses, goats, sheep, and cattle) and pet animals (e.g., cats, dogs, hamsters, rabbits, and guinea pigs) may also be used as test subjects.

[0038] The composition for activating the immune response according to this embodiment is generally administered to subjects such as animals, including humans, in amounts and frequencies sufficient to bring about immune system enhancement. The concentration of immunogenic factors contained in the composition of this embodiment can be appropriately set depending on the type of subject to administration. For example, the concentration of immunogenic factors in the composition (1 g) is preferably in the range of 3 μg / g or more and 50 mg / g or less. Furthermore, from the viewpoint of reducing the volume of the composition, a higher concentration of immunogenic factors contained in the composition is preferable. The upper limit of the concentration of immunogenic factors can also be, for example, 50 mg / g or less, 45 mg / g or less, 40 mg / g or less, 35 mg / g or less, 30 mg / g or less, 25 mg / g or less, 20 mg / g or less, 15 mg / g or less, or 10 mg / g or less. The lower limit of the immunogenicity factor concentration can be, for example, 3 μg / g or higher, and can also be 10 μg / g or higher, 25 μg / g or higher, 50 μg / g or higher, 75 μg / g or higher, or 100 μg / g or higher. Administering at such doses is effective because it allows for sufficient immune response activity while maintaining safety for the body, without causing adverse effects due to an excessive immune response in the target organism.

[0039] The concentration of proteoglycan in the composition (1g) is, for example, within the range of 0.1 μg / g or higher. Furthermore, from the viewpoint of reducing the volume (liquid amount) of the composition, a higher concentration of proteoglycan in the composition is preferable.

[0040] The composition may contain the immunogenic factor and the proteoglycan together, or each active ingredient may be included separately in combination. For example, (a) the immunogenic factor and (b) the proteoglycan for activating the immune response may be administered simultaneously or individually in a specific order. The administration of (a) and (b) may be simultaneous or sequential, preferably (a) and (b) are administered sequentially (or separately). This means that (a) and (b) may be provided as a single unit dosage form for joint administration, or as separate entities (e.g., in separate containers) to be administered simultaneously or with a certain time difference. This time difference may range from one hour to one week, preferably from 12 hours to three days. Furthermore, the proteoglycan may be administered by a method of administration separate from that of the immunogenic factor. In this regard, it may be advantageous to administer either (a) or (b) intravenously and the other systemically or orally. Sequential administration is particularly useful when the active ingredients of this embodiment are in different dosage forms (one drug is a tablet or capsule, and the other is a sterile solution) and / or are administered on different schedules, for example, an active ingredient administered at least daily and an active ingredient administered less frequently, such as once a week, once every two weeks, or once every three weeks.

[0041] (Uses in pharmaceuticals or food products, etc.) The agent or composition of this embodiment has a significant immune response modulating or promoting effect, and is therefore useful, for example, in treating patients with diseases, disorders, or disabilities in which the normal metabolic immune response is reduced or suppressed in the body. Furthermore, the agent or composition of this embodiment can be used to therapeutically or preventively treat subjects such as animals or humans who are at high risk of developing diseases, disorders, or disabilities caused by conditions that adversely affect the immune system.

[0042] Furthermore, the composition of this embodiment has the effect of synergistically activating humoral immunity and cellular immunity. In addition, since immunogenic factors and proteoglycans exhibit distinct adjuvant effects, it can also be used as a vaccine. A vaccine refers to a pharmaceutical composition intended to prevent or treat a specific disease, disorder, or disability by containing the pathogen itself or a specific antigen derived from the pathogen. The concept of a pharmaceutical composition includes not only pharmaceutical compositions for humans but also pharmaceutical compositions for animals. The type of antigen contained in the vaccine is selected according to the specific disease, etc., that is the target of prevention or treatment. In the vaccine according to this embodiment, compared to conventional adjuvants, very high immunostimulatory activity and high neutralizing antibody titers can be obtained even with a small amount of immunostimulant and antigen. Since the amount of adjuvant and antigen can be reduced, excessive stimulation to the body is not generated, resulting in a highly safe and inexpensive vaccine.

[0043] The composition of this embodiment can also be used, for example, as an additive for food and beverages. By using the composition of this embodiment as an additive for food and beverages, it is possible to provide a food and beverage composition with enhanced immune response activity. Examples of food and beverage compositions with enhanced immune response activity include foods and beverages for specified health uses, foods and beverages with functional claims, or foods and beverages with nutritional function claims that are labeled as immune-boosting, immune-activating, or immune-boosting.

[0044] The present invention will now be described in more detail with reference to examples, but the present invention is not limited in any way to these examples. [Examples]

[0045] [Example 1] Effects of various immunogenic factors and proteoglycans on cytokine production in mouse macrophages (Experimental materials) • Proteoglycan: Proteoglycan derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Corporation, Product Code: 162-22131) • TLR2 ligand: Pam3-Cys-Ser-(Lys)4 / 3HCl, ALX-165-066-M002 (Enzo Life Science) • TLR3 ligand: Poly(I:C), ALX-746-021-M005 (Enzo Life Science) • TLR4 / NLR ligand: LPS (E. coli O111:B4), ALX-581-012-L001 (Enzo Life Science) • TLR7 / 8 ligand: Imiquimod (R-837), ALX-420-039-M100 (Enzo Life Science) • TLR9 ligand: ODN1826, ALX-746-052-M001 (Enzo Life Science) • Dectin-1 ligand: Zymosan (yeast β-glucan), SC-25836 (Santa Cruz Biotechnology) • Chaga mushroom extract: Chaga mushroom extract powder • Trametes versicolor extract: Trametes versicolor powder

[0046] (Experimental method) Using a 24-well culture plate, mouse macrophage cell line RAW264.7 was cultured in DMEM containing 100 U / mL penicillin G, 100 μg / mL streptomycin sulfate, 100 μg / mL amphotericin B, and 10% fetal bovine serum at their respective final concentrations, in 2 × 10⁶ cells. 6Cell culture was performed in / well. After culturing for 1 day at 37°C in a CO2 incubator, the culture medium was removed from each well, and the immunogenic factors (each TLR ligand, Dectin-1 ligand, Chaga mushroom extract or Trametes versicolor extract) and proteoglycan (hereinafter abbreviated as PG) were simultaneously added to the fresh culture medium at the specified concentrations as test samples. As controls, only the culture medium and the culture medium with only PG added were used. After culturing for 24 hours at 37°C in a CO2 incubator, the culture medium was collected from each well after confirming that the cells were not dead under a microscope. The amounts of TNF-α, IL-6, IL-10, IL-1β, and IFN-β contained in the culture supernatant collected after centrifugation at 3000 rpm for 20 minutes at 4°C were measured by ELISA. The ELISA used for this measurement is as follows. ·TNF-α:A43658, ThermoFisher ·IL-6:BMS603-2, ThermoFisher ·IL-10:BMS614INST, ThermoFisher ·IL-1β:BMS6002, ThermoFisher ·IFN-β:A47435, ThermoFisher

[0047] (Experimental results) The results are shown in Tables 1 to 3 and Figures 1 to 9 below. In Figures 1 to 9, the horizontal axis of the graphs represents the various additives, specifically as follows (the concentration of the additive is shown in parentheses): • Medium: Culture medium • PG: Proteoglycan (250 μg / mL) ·Pam:Pam3-Cys-Ser-(Lys)4 / 3HCl(250ng / mL) • ODN: ODN1826 (10 μg / mL) • Zymosan: Beta-glucan derived from Saccharomyces cerevisiae (25 μg / mL) ·LPS:LPS (E. coli O111:B4) (10μg / mL) • Kabano: Chaga mushroom extract (500 μg / mL) • Kawara: Kawaratake mushroom extract (500 μg / mL) ·Loxoribine:7-Allyl-8-oxoguanosine(250μM)

[0048] The vertical axis of the graph shows the concentrations (pg / mL) of various cytokines measured by ELISA. These results show that PG itself induced the expression of IL-10, IL-1β, and IFN-β in mouse macrophage cell lines. When PG was added along with various immunogenic factors, PG suppressed the production of various cytokines mediated by TLR4. On the other hand, PG enhanced the production of various cytokines mediated by TLR2, TLR7 / 8, TLR9, and Dectin 1. Chaga mushroom extract and Trametes versicolor extract promoted TNF-α production, and this effect was enhanced by the addition of PG.

[0049] [Table 1]

[0050] [Table 2]

[0051] [Table 3]

[0052] (Consideration) Based on these results, it is thought that the induction of the anti-inflammatory cytokine IL-10 in experiments using mouse macrophage cell lines is related to the anti-inflammatory effect of prostaglandins (PGs). PGs can regulate the innate immune response in a manner dependent on innate immune receptors. Furthermore, it was found that Chaga mushroom extracts and Trametes versicolor extracts can promote the innate immune response, and this effect is enhanced by PGs.

[0053] [Example 2] Effects of lactic acid bacteria and proteoglycans on cytokine production in dendritic cells (Experimental method) Mouse bone marrow-derived dendritic cells (myeloid dendritic cells (mDCs)) were prepared using the following procedure. Bone marrow was extracted from the femur of C57BL / 6 mice, and bone marrow cells were collected by destroying red blood cells with hemolysis buffer. Bone marrow cells were then placed in RPMI1640 medium containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.1% 2-mercaptoethanol, and recombinant mouse GM-CSF (2-20 ng / mL) in a solution of 2 × 10⁶ cells. 5 Cells were cultured at a cell density of cells / mL for 7 days to differentiate into myeloid dendritic cells (mDCs).

[0054] Using a 24-well culture plate, the myeloid dendritic cells (mDCs) prepared above were incubated in DMEM containing 100 U / mL penicillin G, 100 μg / mL streptomycin sulfate, 100 μg / mL amphotericin B, and 10% fetal bovine serum at a final concentration, in a 2 × 10⁶ solution. 6 Culture was performed in / well. After culturing for 1 day at 37°C in a CO2 incubator, the culture medium was removed from each well, and Enterococcus faecalis EC-12 (10 μg / mL) was added alone or together with proteoglycan (250 μg / mL) as the test sample to the fresh culture medium. As controls, only the culture medium and the culture medium with only PG added were used. After culturing for 48 hours at 37°C in a CO2 incubator, the culture medium was collected from each well after confirming that the cells were not dead under a microscope. The culture supernatant was centrifuged at 4°C at 3000 rpm for 20 minutes, and the amounts of TNF-α, IL-6, and IL-12p70 contained in the collected culture supernatant were measured by ELISA. The ELISA used for this measurement is as follows. ·TNF-α:A43658, ThermoFisher ·IL-6:BMS603-2, ThermoFisher ·IL-12p70:BMS6004, ThermoFisher

[0055] (Experimental results) The results are shown in Table 4 and Figures 10-12 below. [Table 4]

[0056] IL-12 is a heterodimer protein composed of IL-12p35 and IL-12p40, and is known as an inflammatory cytokine, similar to TNF-α and IL-6. The IL-12 heterodimer is specifically called IL-12p70. IL-12 is mainly produced by phagocytic cells and dendritic cells that engulf bacteria. In addition to stimulating NK cells, IL-12 is thought to induce the differentiation of naive T cells into Th1 cells, thereby conferring resistance to bacteria and viruses. As shown in Table 4 and Figures 10-12, EC-12 can activate dendritic cells by promoting the production of these cytokines, and this effect was found to be enhanced by prostaglandins (PGs).

[0057] [Example 3] Effects of Chaga mushroom extract and proteoglycans on cytokine production in dendritic cells Using a 24-well culture plate, 2 × 10⁶ of mouse bone marrow-derived myeloid dendritic cells (mDCs) prepared in Example 2 were incubated in DMEM containing 100 U / mL penicillin G, 100 μg / mL streptomycin sulfate, 100 μg / mL amphotericin B, and 10% fetal bovine serum, respectively. 6 Cell culture was performed in one well. After culturing for 1 day at 37°C in a CO2 incubator, the culture medium was removed from each well, and to the fresh culture medium, heat-sterilized Listeria monocytogenes (HK-LM) and either Houttuynia cordata extract or Chaga mushroom extract (500 μg / mL each) were added together, either in the presence or absence of proteoglycan (250 μg / mL, hereafter abbreviated as PG). A control was used in which only HK-LM was added. After culturing for 48 hours at 37°C in a CO2 incubator, the culture medium was collected from each well after confirming that the cells were not dead under a microscope. The amounts of TNF-α, IL-6, and IL-12p70 in the culture supernatant collected after centrifugation at 3000 rpm for 20 minutes at 4°C were measured by ELISA.

[0058] (Experimental results) The results are shown in Table 5 and Figures 13-15 below. [Table 5]

[0059] As shown in Table 5 and Figures 13-15, Chaga mushroom extract enhanced the production of TNF-α and IL-6 by HK-LM, a ligand that primarily activates the immune response via TLR-2, and this effect was further enhanced by PG. Furthermore, while HK-LM alone resulted in almost no secretion of IL-12p70, it was found that co-addition of Chaga mushroom extract, particularly Chaga mushroom extract with proteoglycans, promoted IL-12p70 production in dendritic cells.

[0060] [Example 4] Effects of oral administration of Chaga mushroom extract on Listeria infection in mice (innate immunity) Following the experimental procedure shown in Figure 16, 1 × 10⁶ female C57BL / 6 mice were given 1 × 10⁶ mice each, at 6 weeks of age. 6Mice were infected with Listeria monocytogenes via intraperitoneal administration (IP). From 9 days before infection to 3 or 4 days after infection, the mice were fed a test sample containing 3 mg of Chaga mushroom extract per day or a control sample without it. For each group of mice (N=5), the spleen and liver were removed 3 days after infection. The removed organs were homogenized in PBS (physiological saline phosphate buffer) using a homogenizer. 0.1 mL of the homogenized solution was spread onto tryptosoy agar plates. After spreading, the plates were incubated at 37°C for 24 hours. After incubation, the number of bacteria present on the medium was counted. The counted number was defined as the viable cell count. From another group of mice, spleens were removed 4 days after infection and cultured in the same manner as in Example 3. The culture medium was then immunostimulated by adding heat-sterilized Listeria monocytogenes (HK-LM). After culturing at 37°C in a CO2 incubator for 48 hours, the cells were confirmed to be intact under a microscope, and the culture medium was collected from each well. The culture supernatant was centrifuged at 4°C at 3000 rpm for 20 minutes, and the amounts of TNF-α, IL-6, and IL-12p70 in the collected culture supernatant were measured by ELISA.

[0061] (Experimental results) The results of measuring the number of bacteria present in the spleen and liver are shown in Table 6 below. [Table 6] Mice fed with test samples containing Chaga mushroom extract showed a tendency for a decrease in the number of infecting bacteria in their organs.

[0062] Meanwhile, cytokine production levels are shown in Table 7 and Figures 17-19 below. In the figures, "Control" refers to samples taken from mice fed a diet without Chaga mushroom extract, and "Extract" refers to samples taken from mice fed a diet containing Chaga mushroom extract. [Table 7]

[0063] It was found that the production of TNF-α, IL-6, and IL-12p70 by HK-LM, a ligand that primarily activates the immune response via TLR-2, is enhanced by oral administration of Chaga mushroom extract.

[0064] [Example 5] Effects of oral administration of Chaga mushroom extract on Listeria infection in mice (acquired immunity) Following the experimental protocol shown in Figure 20, 6-week-old female C57BL / 6 mice were subjected to 1 × 10⁶ injections per mouse. 5 After infecting with Listeria monocytogenes (LM) via intraperitoneal administration (ip), 1 x 10⁶ cells were administered again 4 weeks later. 6 A number of mice were infected with LM. From 7 days before the first infection until the end of the experiment, the mice were fed a test sample containing 3 mg of Chaga mushroom extract per day or a control sample without it. For one group of mice, the spleen and liver were removed 2 days after infection. The removed organs were homogenized in PBS (physiological saline phosphate buffer) using a homogenizer. 0.1 mL of the homogenized solution was spread onto tryptosoy agar plates. After spreading, the plates were incubated at 37°C for 24 hours. After incubation, the number of bacteria present in the medium was counted. The counted number was defined as the viable cell count. From another group of mice, the spleen was removed immediately before the second infection and cultured in the same manner as in Example 3. The culture medium was then immunostimulated by adding heat-sterilized Listeria monocytogenes (HK-LM). A similar experiment was performed using mice that had not undergone initial infection with LM as a control. After culturing the cells in a CO2 incubator at 37°C for 48 hours, and confirming that the cells were still alive under a microscope, the culture medium was collected from each well. The culture supernatant was then centrifuged at 3000 rpm for 20 minutes at 4°C, and the amounts of TNF-α, IL-6, and IL-12p70 in the collected supernatant were measured by ELISA.

[0065] (Experimental results) The results of measuring the number of bacteria present in the spleen and liver are shown in Table 8 below. [Table 8] In mice fed with test samples containing Chaga mushroom extract, the bacterial count in the organs was dramatically reduced to a level where the infecting bacteria could not be detected.

[0066] On the other hand, cytokine production levels are shown in Table 9 and Figures 21-23 below. [Table 9] Without immunization with HK-LM, cytokine production was not observed regardless of whether or not the cells were fed Chaga mushroom extract. However, four weeks after HK-LM stimulation, spleen cells produced TNF-α, IL-6, and IL-12p70, and this effect was enhanced by feeding with Chaga mushroom extract.

[0067] Formulation example 1: Tablets Each 200 mg tablet contains 5 mg of proteoglycan and is manufactured by thoroughly mixing the ingredients and then compressing them into tablets. Proteoglycan 5mg Chaga mushroom extract 10mg Lactose 127mg Starch 45mg Carboxymethylcellulose 10mg Talc 2mg Magnesium stearate 1 mg Total 200mg / tablet

[0068] Formulation example 2: Capsule Each 100 mg capsule contains 20 mg of proteoglycan and has the following component composition. It was manufactured by thoroughly mixing each component and then filling it into capsules. Proteoglycan 20mg Trametes versicolor extract 10mg Lactose 43mg Starch 25mg Magnesium stearate 2mg Total 100mg / capsule [Industrial applicability]

[0069] The present invention has industrial applicability in that it can provide a novel use for proteoglycans as immunomodulators.

[0070] TIFF0007854000000010.tif230152

Claims

[Claim 1] A combination for promoting the production of IL-6 and / or IL-12p70, The aforementioned combination comprises salmon nasal cartilage-derived proteoglycan and the cellular components of Enterococcus faecalis strain EC-12 (accession number FERM BP-10284), either together or separately.

Images