Composition comprising bryostatin or use

Abstract

The present disclosure relates to a method and composition for modulating Th2-dominant immune balance and improving intestinal flora, and more particularly relates to use of bryostatin in the prevention or treatment of a condition, disorder or disease in which suppression of Th2 reaction and / or improvement in intestinal flora is beneficial, or a composition comprising bryostatin for use in the prevention or treatment of a condition, disorder or disease in which suppression of Th2 reaction or improvement in intestinal flora is beneficial.

Description

Compositions or uses containing bryostatin

[0001] The present disclosure relates to methods and compositions for adjusting the Th2-dominant immune balance and improving the intestinal flora, and more particularly to the use of bryostatin in the prevention or treatment of conditions, disorders, or diseases in which suppression of a Th2 response and / or improvement of the intestinal flora is beneficial, or compositions comprising bryostatin for use in the prevention or treatment of conditions, disorders, or diseases in which suppression of a Th2 response or improvement of the intestinal flora is beneficial.

[0002] Immune regulation is thought to involve a homeostasis between type 1 helper T cell (Th1) and type 2 helper T cell (Th2) activity. Th1 cells drive the type 1 pathway ("cell-mediated immunity") to combat viruses and other intracellular pathogens, eliminate cancer cells, and stimulate delayed-type hypersensitivity (DTH) skin reactions. Th2 cells drive the type 2 pathway ("humoral immunity"), upregulating antibody production to combat extracellular microorganisms. Th2 dominance is thought to contribute to xenograft and fetal tolerance during pregnancy. Overactivation of either pathway can cause disease, and either pathway can downregulate the other. Experimentally, a Th1-dominant state can easily be converted to a Th2-dominant state by depleting intracellular glutathione. Mercury depletes glutathione, resulting in a Th2-dominant state. It is also known that several nutrients and hormones, such as plant sterols / sterolin, melatonin, probiotics, progesterone, and the minerals selenium and zinc, affect the Th1 / Th2 balance. Th1 / Th2-based immunotherapies, such as T cell receptor (TCR) peptides and interleukin-4 (IL-4) injections, are known (Non-Patent Document 1).

[0003] The Th1 / Th2 balance is known to play an important role in allergic diseases (Non-Patent Document 2), and Th2 cytokines such as interleukin (IL)-4 and IL-13 are known to promote class switching to IgG4. On the other hand, Th cells other than Th2, such as regulatory T cells and follicular helper T cells, are also known to be involved in IgG4-related diseases (Non-Patent Document 3).

[0004] Kidd, "Th1 / Th2 balance: the hypothesis, its limitations, and implications for health and disease," Galli and Tsai, Altern Med Rev. 2003 Aug;8(3):223-46. Proceedings of the 126th Japanese Medical Association Symposium, Tomohiro Yoshimoto, Cytokines and Allergic Diseases - IL-18 and Allergy, https: / / jams.med.or.jp / event / doc / 126028. pdfMoriyama and Nakajima “Th1 / Th2 immune balance and other helper subsets in IgG4-related disease”, Current Topics in Microbiology and Immunology (pp. 75-83). https: / / doi. org / 10.1007 / 82_2016_40

[0005] In one aspect, the present disclosure aims to provide a method and composition for adjusting the Th2-dominant immune balance. Another aspect of the present disclosure aims to provide a method and composition for improving the intestinal flora. Another aspect of the present disclosure aims to provide a method and composition for simultaneously suppressing a Th2 response and improving the intestinal flora.

[0006] The inventors of the present disclosure have conducted extensive research into the effects of bryostatin and have unexpectedly discovered that administration of bryostatin suppresses Th2 responses and improves the intestinal flora.

[0007] In one aspect, the present disclosure provides compositions containing bryostatin for use in suppressing Th2 responses and / or improving the gut microbiota.

[0008] In one aspect, the disclosure provides methods for suppressing Th2 responses and / or improving gut flora using bryostatin.

[0009] In one aspect, the present disclosure more specifically provides the following: [Item 1] A composition for use in suppressing a Th2 response, comprising bryostatin as an active ingredient. [Item 2] The composition of Item 1, wherein the suppression of a Th2 response includes prevention or treatment of a condition, disorder, or disease in which suppression of a Th2 response is beneficial. [Item 3] The composition of Item 1, wherein the condition, disorder, or disease in which suppression of a Th2 response is beneficial is selected from the group consisting of an eosinophil-associated disease, an allergic disease caused by an IgE antibody response, an IgG4-associated disease, a fibrotic disease, cancer, and an autoimmune disease. [Item 4] The composition of Item 1, wherein the condition, disorder, or disease for which suppression of a Th2 response is beneficial is selected from the group consisting of Mikulicz's disease, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, pancreatic cancer, liver cancer, breast cancer, type 1 diabetes, bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, food allergy, anaphylaxis, hay fever, drug allergy, urticaria including chronic idiopathic urticaria (CSU), eosinophilic asthma (bronchial asthma), eosinophilic sinusitis (ECRS), eosinophilic gastrointestinal disease (EGID), eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EGE), hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), and drug-induced hypersensitivity syndrome (DRESS syndrome). [Item 5] The composition of Item 1, wherein the condition, disorder, or disease for which suppression of the Th2 response is beneficial is an eosinophil-associated disease selected from the group consisting of eosinophilic asthma (bronchial asthma), eosinophilic sinusitis (ECRS), eosinophilic gastrointestinal disease (EGID), eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EGE), urticaria, hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), drug-induced hypersensitivity syndrome (DRESS syndrome), atopic dermatitis, allergic rhinitis, allergic conjunctivitis, and food allergy. [Item 6] A composition for use in improving the intestinal flora, comprising bryostatin as an active ingredient. [Item 7] The composition of Item 6, wherein the improvement of the intestinal flora includes prevention or treatment of a condition, disorder, or disease for which improvement of the intestinal flora is beneficial.[Item 8] The composition of Item 6, wherein the condition, disorder, or disease for which improvement of the intestinal microbiota is beneficial is selected from the group consisting of intestinal microbial dysbiosis, inflammatory bowel disease (IBD), metabolic disease, digestive disease, allergic disease, cardiovascular disease, renal disease, and autoimmune disease. [Item 9] The composition of Item 1 or 6, for oral, nasal, or enteral administration. [Item 10] A method for suppressing a Th2 response in a subject in need thereof, comprising administering bryostatin to the subject. [Item 11] The method of Item 10, wherein the suppression of a Th2 response comprises prevention or treatment of a condition, disorder, or disease for which suppression of a Th2 response is beneficial. [Item 12] The method of Item 10, wherein the condition, disorder, or disease for which suppression of a Th2 response is beneficial is selected from the group consisting of eosinophil-associated diseases, allergic diseases caused by IgE antibody responses, IgG4-associated diseases, fibrotic diseases, cancer, and autoimmune diseases. [Item 13] The method of Item 10, wherein the condition, disorder, or disease for which suppression of a Th2 response is beneficial is selected from the group consisting of Mikulicz's disease, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, pancreatic cancer, liver cancer, breast cancer, type 1 diabetes, bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, food allergy, anaphylaxis, hay fever, drug allergy, urticaria including chronic idiopathic urticaria (CSU), eosinophilic asthma (bronchial asthma), eosinophilic sinusitis (ECRS), eosinophilic gastrointestinal disease (EGID), eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EGE), hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), drug-induced hypersensitivity syndrome (DRESS syndrome), and food allergy. [Item 14] The method of Item 10, wherein the condition, disorder, or disease for which suppression of a Th2 response is beneficial is an eosinophil-associated disease selected from the group consisting of eosinophilic asthma (bronchial asthma), eosinophilic sinusitis (ECRS), eosinophilic gastrointestinal disease (EGID), eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EGE), urticaria including chronic idiopathic urticaria (CSU), hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), drug-induced hypersensitivity syndrome (DRESS syndrome), atopic dermatitis, allergic rhinitis, allergic conjunctivitis, and food allergy.[Item 15] A method for improving the intestinal flora in a subject in need thereof, comprising a step of administering bryostatin to the subject. [Item 16] The method according to Item 15, wherein the improvement of the intestinal flora includes prevention or treatment of a condition, disorder, or disease for which improvement of the intestinal flora is beneficial. [Item 17] The method according to Item 16, wherein the condition, disorder, or disease for which improvement of the intestinal flora is beneficial is selected from the group consisting of intestinal microbial dysbiosis, inflammatory bowel disease (IBD), metabolic diseases, digestive diseases, allergic diseases, cardiovascular diseases, renal diseases, and autoimmune diseases. [Item 17] The method according to Item 10 or 16, wherein the compound is administered orally, nasally, or enterally.

[0010] The present disclosure has an effect of providing a method and composition for adjusting a Th2-dominant immune balance. Also, in one aspect, the present disclosure has an effect of providing a method and composition for improving the intestinal flora. Also, in one aspect, the present disclosure has an effect of providing a method and composition for simultaneously suppressing a Th2 response and improving the intestinal flora.

[0011]

[0023] Figure 1 is a diagram showing the scheme of a test investigating the effect of Bryostatin 1 in a cedar pollen allergy model. Figure 2 is a diagram showing the results of a test investigating the effect of Bryostatin 1 in a cedar pollen allergy model. Figure 3 is a diagram showing the results of a test investigating the effect of Bryostatin 1 administration in a cedar pollen allergy model. Figure 4 is a diagram showing the results of a test investigating the effect of Bryostatin 1 administration in a cedar pollen allergy model. Figure 5 is a diagram showing the production of Th2 cytokines by mediastinal lymph node cells and the effect of Bryostatin 1 in a cedar pollen allergy model. Figure 6 is a diagram showing the scheme of a test evaluating the effect of Bryostatin 1 on Th2-related cytokine production in a cedar pollen allergy model. Figure 7 is a diagram showing the results of a test evaluating the effect of Bryostatin 1 on Th2-related cytokine production in a cedar pollen allergy model.

[0033] Figure 8 is a diagram showing the effect of Bryostatin 1 on the intestinal flora in a cedar pollen allergy model. Figure 9 is a diagram showing the effect of Bryostatin 1 on the intestinal flora in a cedar pollen allergy model. Figure 10 is a diagram showing the effect of Bryostatin 1 on the intestinal flora in a cedar pollen allergy model. Figure 11 is a diagram showing a test scheme investigating the effect of Bryostatin 1 in a food allergy model. Figure 12 is a diagram showing the results of a test investigating the effect of Bryostatin 1 in a food allergy model. Figure 13 is a diagram showing the results of a test investigating the effect of Bryostatin 1 in a food allergy model. Figure 14 is a diagram showing the results of a test investigating the effect of Bryostatin 1 in a food allergy model. Figure 15 is a diagram showing a test scheme evaluating the effect of Bryostatin 1 on Th2-related cytokine production in a food allergy model. Figure 16 shows the results of a test evaluating the effect of Bryostatin 1 on Th2-associated cytokine production in a food allergy model. Figure 17 shows the effect of Bryostatin 1 on the intestinal bacterial flora in a food allergy model.FIG. 18 is a diagram showing the effect of Bryostatin 1 on the intestinal microbiota in a food allergy model. FIG. 19 is a diagram showing the effect of Bryostatin 1 on the intestinal microbiota in a food allergy model. FIG. 20 is a diagram showing the results of evaluating the direct effect of Bryostatin 1 on T cell differentiation. FIG. 21 is a diagram showing the results of evaluating the direct effect of Bryostatin 1 on T cell differentiation. FIG. 22 is a diagram showing the results of evaluating the direct effect of Bryostatin 1 on T cell differentiation. FIG. 23 is a diagram showing the results of investigating the involvement of Bryostatin 1 in the regulation of Th2-associated cytokines and GATA3 expression. FIG. 24A is a diagram showing the results of investigating the involvement of IL-10 expression regulation in Bryostatin 1 suppressing the production of Th2-associated cytokines. FIG. 24B is a diagram showing the results of investigating the involvement of Lag3 and c-Maf expression regulation in Bryostatin 1 suppressing the production of Th2-associated cytokines. FIG. 25 is a diagram showing an outline of the mechanism by which Bryostatin 1 controls T cell differentiation and suppresses Th2 responses and eosinophil activity.

[0012] The inventors of the present disclosure unexpectedly discovered that administration of bryostatin suppresses Th2 responses and improves the intestinal microbiota. In particular, they found that bryostatin directly acts on T cells to suppress the production of Th2-associated cytokines and improves the bacterial diversity of the intestinal microbiota. During T cell differentiation, naive T cells differentiate into not only Th2 cells but also suppressor T cells. Suppressor T cells are known to suppress the activity of Th2 cells. Furthermore, while Th2 cells activate eosinophils through Th2-associated cytokines, suppressor T cells act antagonistically to suppress eosinophil activity. The inventors of the present disclosure discovered that bryostatin suppresses the differentiation of naive T cells into Th2 cells and simultaneously promotes the differentiation of naive T cells into suppressor T cells during T cell differentiation. Specifically, the inventors of the present disclosure have discovered that bryostatin simultaneously regulates two mechanisms, namely, the inhibition of differentiation into Th2 cells and the promotion of differentiation into regulatory T cells, thereby potently suppressing Th2 responses. Furthermore, they have also discovered that simultaneously regulating mechanisms that act antagonistically against eosinophils potently suppresses eosinophils. Based on this discovery, the inventors of the present disclosure have discovered that compositions containing bryostatin as an active ingredient can be used to suppress Th2 responses and / or improve intestinal flora, or to treat conditions, disorders, and / or diseases in which the inhibition of Th2 responses and / or the improvement of intestinal flora are beneficial.

[0013] (Definitions) In this specification, when multiple ranges of numerical values ​​are shown, the same applies to ranges formed by any combination of the lower and upper limits of those multiple ranges.

[0014] As used herein, "prevention" of a condition, disorder, or disease has its usual meaning in the art, such as preventing the occurrence of a condition, disorder, or disease, reducing the risk of the occurrence of a condition, disorder, or disease, etc.

[0015] As used herein, "treatment" of a condition, disorder, or disease has its usual meaning in the art, and means, for example, preventing the onset of a condition, disorder, or disease; reducing the risk of onset of a condition, disorder, or disease; inhibiting the progression of a condition, disorder, or disease that has already occurred; delaying the progression of a condition, disorder, or disease that has already occurred; eliminating a condition, disorder, or disease that has already occurred; ameliorating the symptoms of a condition, disorder, or disease that has already occurred; and the like.

[0016] 1. Compositions containing bryostatin for use in suppressing Th2 responses and / or improving intestinal flora In one aspect, the present disclosure provides compositions containing bryostatin as an active ingredient for use in suppressing Th2 responses and / or improving intestinal flora.

[0017] Bryostatins are naturally occurring macrocyclic compounds originally isolated from bryozoans. Currently, approximately 20 naturally occurring bryostatins are known, which share three six-membered rings designated A, B, and C and differ primarily in the nature of the substituents at C7 and C20. In one embodiment, the bryostatins of the present disclosure include bryostatin or its derivatives, pharmaceutically acceptable salts, and enantiomers thereof.

[0018] In one embodiment, the bryostatin of the present disclosure is bryostatin 1, represented by formula (I):

[0019] In one embodiment, the bryostatin of the present disclosure is bryostatin 1, represented by formula (II):

[0020] The bryostatins of the present disclosure include structures corresponding to the bryostatin-1 described above, as well as derivatives and prodrugs thereof, in which one or more carbon or oxygen atoms in the bryostatin backbone structure have been substituted, and which are expected to retain or enhance biological activity.

[0021] In one aspect, derivatives of bryostatin of the present disclosure are represented by the following formula, where the group indicated by "*" is substituted:

[0022] Examples of the substituent include, but are not limited to, a hydrogen atom, a halogen atom (fluorine, chlorine, bromine, iodine), a hydroxyl group, a substituted or unsubstituted saturated or unsaturated hydrocarbon group, and the like.

[0023] The term "substituted or unsubstituted saturated or unsaturated hydrocarbon group" refers to a group having a chain or cyclic hydrocarbon skeleton composed of carbon and hydrogen, in which the hydrogen may be replaced by another functional group (e.g., halogen, hydroxy, amino, oxy, alkoxy, alkyl).

[0024] The term "substituted or unsubstituted saturated or unsaturated hydrocarbon group" includes, but is not limited to, substituted or unsubstituted alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkenoxy groups, alkynoxy groups, hydroxyl group-containing alkyl groups, hydroxyl group-containing alkynyl groups, hydroxyl group-containing alkoxy groups, cycloalkyl groups, aryl groups, heteroaryl groups, and heterocyclic groups.

[0025] The term "alkyl group" refers to a saturated hydrocarbon group having a straight or branched chain and having 1 to 8 carbon atoms.

[0026] The term "alkenyl group" refers to a straight or branched chain unsaturated hydrocarbon group having one to eight carbon atoms and one or more carbon-carbon double bonds. Examples of alkenyl groups include CH ３ -CH ２ -CH ２ -CH=CH-CH=CH-CH ２ -, CH ３ -CH ２ -CH ２ -CH=CH-CH=CH-CH(OH)-, CH ３ -CH ２ -CH ２ -CH=CH-CH=CH-C(O)-, CH ３ -CH=CH-CH ２ -CH ２ -CH=CH-CH ２ -, CH ３-CH=CH-CH ２ -CH ２ -CH=CH-CH(OH)-, CH ３ -CH=CH-CH ２ -CH ２ It means —CH═CH—C(O)—, etc.

[0027] The term "alkynyl group" refers to a straight or branched chain unsaturated hydrocarbon group having one to eight carbon atoms and one or more carbon-carbon triple bonds. Examples of alkynyl groups include CH ３ -CH ２ -CH ２ -C≡C-C≡C-CH ２ -, CH ３ -CH ２ -CH ２ -C≡C-C≡C-CH(OH)-, CH ３ -CH ２ -CH ２ -C≡C-C≡C-C(O)-, CH ３ -C≡C-CH ２ -CH ２ -C≡C-CH ２ -, CH ３ -C≡C-CH ２ -CH ２ -C≡C-CH(OH)-, CH ３ -C≡C-CH ２ -CH ２ It means —C≡C—C(O)—, etc.

[0028] The "alkoxy group" is a group bonded to the alkyl group via an oxygen atom (-O-), and examples thereof include methoxy, ethoxy, and CH ３ OC(O)-, CH ３ It means OC(O)C- and the like.

[0029] The term "alkenoxy group" refers to a group bonded to the alkenyl group via an oxygen atom (-O-), and includes, for example, CH2=CH-O-, CH2=CH-CH2-O-, CH3-CH=CH-O-, CH2=C(CH3)-O-, CH2=CH-CH=CH-O-, etc.

[0030] The "alkynoxy group" is a group bonded to the alkynyl group via an oxygen atom (-O-), and examples thereof include HC≡C-O-, HC≡C-CH2-O-, CH3-C≡C-CH2-O-, and CH3CH2-C≡C-CH 2- O- means etc.

[0031] The term "hydroxyl group-containing alkyl group" refers to a group having one or more hydroxyl groups (-OH) in the alkyl chain, and includes hydroxymethyl, hydroxyethyl, hydroxypropyl, and the like.

[0032] The term "cycloalkyl group" refers to a cyclic saturated hydrocarbon group having 3 to 12 carbon atoms, and includes, for example, cyclopropyl, cyclopentyl, cyclohexyl, and the like.

[0033] The term "aryl group" refers to a group having an aromatic hydrocarbon ring containing 6 to 10 carbon atoms, and includes phenyl, naphthyl, and the like.

[0034] The terms "heteroaryl group" and "heterocyclic group" refer to groups containing an aromatic or non-aromatic heterocyclic structure, each containing a non-carbon atom such as oxygen, nitrogen, or sulfur within the ring. These include monocyclic rings such as pyridyl, thiazolyl, pyrazole, imidazole, and morpholine, as well as fused polycyclic rings such as benzimidazole and benzothiazole.

[0035] The bryostatins of the present disclosure may have multiple asymmetric centers. In preferred embodiments, they contain 6 to 12 asymmetric centers, resulting in a variety of stereoisomers. Accordingly, the present invention includes these enantiomers, diastereomers, racemates, and mixtures thereof.

[0036] Bryostatin 1 and derivatives of bryostatin 1 are described, for example, in US Pat. No. 4,560,774, which is incorporated herein by reference.

[0037] The bryostatins of the present disclosure include analogs of bryostatin, commonly referred to as bryologs. Bryologs are structural analogs of bryostatin. While bryostatin has two pyran rings and one six-membered acetal ring, most bryologs replace one of the pyrans of bryostatin with a second six-membered acetal ring. This modification reduces the stability of bryostatin relative to bryostatin, for example, in both strongly acidic and strongly basic conditions, but is less significant at physiological pH. Bryologs also have lower molecular weights (ranging from approximately 600 to 755) than bryostatin, which has a molecular weight of 988. Examples of bryologs suitable for use in the present invention include, but are not limited to, derivatives of bryostatin disclosed, for example, in U.S. Pat. Nos. 6,624,189, 7,256,286, and 8,497,385, the disclosures of which are incorporated herein by reference.

[0038] More specifically, bryologs include compounds represented by the following Formula III: and pharmaceutically acceptable salts thereof. Here, R ２０ is H, OH, or -T-U-V-R'; T is selected from -O-, -S-, -N(H)-, or -N(Me)-; U is absent, -C(O)-, -C(S)-, -S(O)-, or -S(O)-; ２ V is absent or selected from —O—, —S—, —N(H)—, or —N(Me)—, provided that when U is absent, V is also absent; R ２１ is =CR ａ R ｂ or R ２１ is the independent part R ｃ and R ｄ where: R ａ and R ｂ are independently H, CO ２ R', CONRcRd, or R', where Rc and Rd are independently H, alkyl, alkenyl, or alkynyl, or (CH ２ ) ｎ CO ２R′ (n is 1, 2 or 3), R ２６ is H, OH, or R'; R' (at each occurrence) is independently selected from the group of H, alkyl, alkenyl, or alkynyl, or aryl, heteroaryl, aralkyl, or heteroaralkyl; L is a linear or branched, straight-chain, cyclic, or polycyclic moiety preferably comprising a continuous chain of 6 to 14 chain atoms, which substantially maintains the relative distance between the C1 and C17 atoms and the orientation of the C1C2 and C16C17 bonds of native bryostatin; and Z is -O- or -N(H)-.

[0039] In this disclosure, pharmaceutically acceptable salts encompassed by the term "bryostatin" include salts with pharmaceutically acceptable acids or bases, such as inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, hydroiodic acid, and nitric acid, and organic acids such as citric acid, fumaric acid, maleic acid, malic acid, mandelic acid, ascorbic acid, oxalic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, cyclohexylsulfamic acid, or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metals (e.g., sodium or potassium), alkaline earth metals (e.g., calcium or magnesium), hydroxides, and organic bases (e.g., alkylamines, arylalkylamines, and heterocyclic amines).

[0040] The term "bryostatin" includes a prodrug, which refers to a modified form of bryostatin that has a lower therapeutic activity and that is capable of being converted to the active bryostatin form in vivo. Such prodrugs are known in the art and include, for example, acetyl esters, alanine esters, cyclocarbonates, monophosphate esters, ethers, carbamates, and the like of bryostatin (see Org Lett. 2006 Apr 27;8(9):1893-6. doi: 10.1021 / ol060457z., WO2018067382A1). These documents are incorporated herein by reference.

[0041] (Form and Use of Composition) The form and method of use of the composition of the present disclosure are not particularly limited. The composition of the present disclosure can be used as a medicine, as well as in the food and feed fields. Therefore, the composition of the present disclosure can be a pharmaceutical composition, a food composition, or a feed composition. The food composition includes not only general foods, but also foods for specified health uses, nutritional supplements, functional foods, foods for the sick, etc.

[0042] In one aspect, suppressing a Th2 response and / or improving the gut flora includes preventing or treating a condition, disorder or disease in which suppressing a Th2 response and / or improving the gut flora is beneficial.

[0043] In one embodiment, the suppression of Th2 responses in the compositions of the present disclosure comprises suppressing cytokine production by Th2 cells. Cytokines primarily produced by Th2 cells include IL-4, IL-5, IL-9, IL-10, and IL-13. In one embodiment, the suppression of Th2 responses comprises suppressing blood levels of IL-4, IL-5, IL-9, IL-10, or IL-13.

[0044] In one embodiment, the suppression of Th2 responses in the composition of the present disclosure includes suppression of the differentiation of naive T cells into Th2 cells. In one embodiment, the suppression of Th2 responses in the composition of the present disclosure includes promotion of the differentiation of naive T cells into suppressor T cells. In one embodiment, the suppression of Th2 responses in the composition of the present disclosure includes an increase in IL-10-producing suppressor T cells. In one embodiment, the suppression of Th2 responses in the composition of the present disclosure includes suppression of IgE antibody production through the suppression of differentiation into Th2 cells and the suppression of their activity. In one embodiment, the suppression of Th2 responses in the composition of the present disclosure includes suppression of the increase in eosinophils or suppression of their activity.

[0045] The compositions of the present disclosure directly affect the regulatory mechanism of T cell differentiation, suppressing differentiation into Th2 cells and promoting differentiation into inhibitory T cells that act antagonistically against Th2 cells, thereby enabling the prevention or treatment of conditions, disorders, or diseases for which suppression of Th2 responses is beneficial. Therefore, in preventing or treating these conditions, disorders, or diseases, they provide superior effects compared to regulating certain downstream responses of the Th2 response. For example, they provide a more fundamental immunomodulatory effect than simply acting on the class switching of antibodies produced by activated B cells, thereby increasing the production of IgA antibodies and suppressing the production of other antibodies, such as IgE antibodies. Furthermore, the compositions of the present disclosure provide superior preventive or therapeutic effects for diseases that have previously been prevented or treated by downstream responses of the Th2 response, such as suppressing the production of IgE antibodies through class switching control. Furthermore, the compositions of the present disclosure can provide preventive or therapeutic effects for conditions, disorders, or diseases for which sufficient preventive or therapeutic effects cannot be achieved by regulating downstream responses of the Th2 response. Thus, in one aspect, the compositions of the present disclosure are applied to the prevention or treatment of conditions, disorders or diseases in which suppression of a Th2 response is beneficial.

[0046] In one embodiment, the condition, disorder, or disease in which suppression of the Th2 response is beneficial is an IgG4-associated disease (e.g., Mikulicz's disease) (see, e.g., Arthritis Rheum. 2012 Jan;64(1):254-63. doi:10.1002 / art.33320), a fibrotic disease (e.g., pulmonary fibrosis, liver fibrosis, and kidney fibrosis) (see, e.g., Nat Rev Immunol. 2018 Jan;18(1):62-76. doi:10.1038 / nri.2017.90), a cancer (e.g., pancreatic cancer, liver cancer, breast cancer) (Clin Exp Immunol. 2014 Nov;178(2):201-11. doi:10.1111 / cei.12409, etc.), autoimmune diseases (e.g., multiple sclerosis, type 1 diabetes) (J Exp Med. 1997 Jul 21;186(2):307-12. doi:10.1084 / jem.186.2.307, etc.), and allergic diseases (e.g., bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, food allergies, anaphylaxis, hay fever, drug allergies, urticaria) (Clin Exp Immunol. 2014 Nov;178(2):201-11. doi:10.1111 / cei.12409, Japanese Society of Allergology website https: / / www. jsa-pr. jp / html / sickness. html).

[0047] Upon recognizing an allergen, Th2 cells activate B cells and promote the production of IgE antibodies. In one aspect, the composition of the present disclosure suppresses B cell activation and IgE antibody production by suppressing the Th2 response, and is particularly suitable for use in conditions, disorders, or diseases in which suppression of IgE antibody production is beneficial.

[0048] Non-limiting examples of conditions, disorders, or diseases for which suppression of IgE antibody production is beneficial include food allergies, allergic asthma, chronic idiopathic urticaria (CSU), and atopic dermatitis caused by IgE antibody responses (N Engl J Med 2024;390:889-899, DOI: 10.1056 / NEJMoa2312382, Annals of Allergy, Asthma & Immunology, Volume 127, Issue 2, 223-231, J Allergy Clin Immunol. 2016 Jun;137(6):1742-1750.e4. doi: 10.1016 / j. jaci. 2015.12.1342, G Ital Dermatol Venereol. 2019 Aug; 154(4):480-487. doi: 10.23736 / S0392-0488.19.06302-8. ).

[0049] In one aspect, the compositions of the present disclosure are particularly suitable for use in conditions, disorders, or diseases in which eosinophil suppression is beneficial, including eosinophil-associated diseases characterized by abnormal increase, activation, and tissue infiltration of eosinophils.

[0050] The eosinophil-related diseases include eosinophilic inflammatory diseases, diseases associated with eosinophilia, and eosinophil-mediated diseases. Specific, non-limiting examples of these diseases include eosinophilic asthma (bronchial asthma), eosinophilic sinusitis (ECRS), eosinophilic gastrointestinal disease (EGID), eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EGE), atopic dermatitis, allergic rhinitis, allergic conjunctivitis, and urticaria (including chronic and idiopathic). Eosinophilia includes, but is not limited to, hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), and drug-induced hypersensitivity syndrome (DRESS syndrome) (Semin Immunopathol. 2021 Apr 23;43(3):459-475. doi: 10.1007 / s00281-021-00849-w, Mayo Clin Proc. 2021 Oct;96(10):2694-2707. doi: 10.1016 / j.mayocp.2021.04.025, Allergol Int. 2019 Oct;68(4):403-412. doi: 10.1016 / j. alit. 2019.07.002. , Allergo J Int 33, 1-8 (2024). https: / / doi. org / 10.1007 / s40629-023-00265-6, Journal of Allergy and Clinical Immunology, Volume 113, Issue 1, 30 - 37, Curr Hematol Malig Rep. 2018 Jun;13(3):191-201. doi: 10.1007 / s11899-018-0448-8, Front Immunol. 2023 Apr 28;14:1134178. doi: 10.3389 / fimmu. 2023.1134178).

[0051] In one embodiment, the improvement of the intestinal microbiota in the composition of the present disclosure includes increasing the bacterial flora diversity, preferably α-diversity, in the gastrointestinal contents or excrement of the subject organism, and in another embodiment, the improvement of the intestinal microbiota includes increasing the amount of short-chain fatty acids in the gastrointestinal contents or excrement of the subject organism.

[0052] In one aspect, improving the gut microbiota in the compositions of the present disclosure includes preventing or treating a condition, disorder, or disease in which improving the gut microbiota is beneficial.

[0053] In one aspect, the condition, disorder, or disease for which improvement of the gut microbiota is beneficial may be selected from the group consisting of dysbiosis, inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis), metabolic disease (e.g., type 2 diabetes, obesity), digestive disease (e.g., irritable bowel syndrome, colorectal cancer), allergic disease, cardiovascular disease, renal disease, and autoimmune disease (e.g., Parkinson's disease, multiple sclerosis) (Inflamm Bowel Dis. 2016 May;22(5):1137-50. doi:10.1097 / MIB.0000000000000750, Sci Rep. 2019 Sep 9;9(1):12918. doi:10.1038 / s41598-019-49452-y, Healthline article, What Causes Dysbiosis and How Is It Treated? , https: / / www. healthline. com / health / digestive-health / dysbiosis#outlook, Microorganisms. 2022 Mar 7;10(3):578. doi:10.3390 / microorganisms10030578, Nature Reviews Immunology 2017, volume 17, pages 219-232. https: / / doi. org / 10.1038 / nri. 2017.7, Microbial Ecology in Health and Disease 2015, Volume 26, Article: 26191. https: / / doi. org / 10.3402 / mehd. v26.26191, Ann Nutr Metab (2013) 63 (Suppl. 2): 28-40. https: / / doi. org / 10.1159 / 000354902, Gut. 2018 Jun;67(6):1024-1032. doi:10.1136 / gutjnl-2017-314281, Infrastructure Bowel Diseases, Volume 22, Issue 5, 1 May 2016, Pages 1137-1150, https: / / doi. org / 10.1097 / MIB. 0000000000000750, Med Microbiol Immunol. 2017 Apr;206(2):83-91. doi:10.1007 / s00430-017-0496-z. PLoS One. 2017 Mar 22;12(3):e0174099. doi:10.1371 / journal. bone. 0174099 etc.). ;

[0054] In a preferred embodiment of the present disclosure, the composition of the present disclosure is applied for use in the prevention or treatment of conditions, disorders, or diseases in which both suppression of Th2 responses and improvement of intestinal flora are beneficial. Because the composition of the present disclosure has the effect of suppressing Th2 responses and improving intestinal flora, it has particularly excellent effects in the prevention or treatment of the above conditions, disorders, or diseases. In one embodiment, the conditions, disorders, or diseases in which both suppression of Th2 responses and improvement of intestinal flora are beneficial include cancer, autoimmune diseases, and allergies.

[0055] In one aspect, when the composition of the present disclosure is provided as a pharmaceutical composition, the composition may be provided for use in the prevention or treatment of conditions, disorders, or diseases in which suppression of the Th2 response and / or improvement of the intestinal flora is beneficial.

[0056] In one aspect, when the composition of the present disclosure is provided as a food composition or feed composition, the composition may be provided with labeling related to the suppression of the Th2 response and / or improvement of the intestinal flora, or the prevention or treatment of a condition, disorder, or disease in which the suppression of the Th2 response and / or improvement of the intestinal flora is beneficial. Such labeling includes, for example, labeling indicating that the food or feed is intended for a subject having a condition, disorder, or disease in which the suppression of the Th2 response and / or improvement of the intestinal flora is beneficial, or ... for whom the suppression of the Th2 response and / or improvement of the intestinal flora is beneficial.

[0057] The compositions of the present disclosure may include an effective amount of bryostatin.

[0058] The term "effective amount" as used herein refers to an amount that can exert the effect of suppressing Th2 response and / or improving the intestinal flora in the subject organism.

[0059] For example, when the subject is a mouse, bryostatin can be administered in an amount of 1 to 1000 ng per administration, such as 1 to 1000 ng, 2 to 1000 ng, 3 to 1000 ng, 4 to 1000 ng, 5 to 1000 ng, 6 to 1000 ng, 7 to 1000 ng, 8 to 1000 ng, 9 to 1000 ng, 10 to 1000 ng, 20 to 1000 ng, 30 to 1000 ng, 40 to 1000 ng, 50 to 1000 ng, 60 to 1000 ng, 70 to 1000 ng, 80 to 1000 ng, 90 to 1000 ng, 100 to 1000 ng, 110 to 1000 ng, 120 to 1000 ng, 130 to 1000 ng, 140 to 1000 ng, 150 to 1000 ng, 160 to 1000 ng, 170 to 1000 ng, 180 to 1000 ng, 190 to 2000 ng, 210 to 2100 ng, 220 to 2200 ng, 230 to 2300 ng, 240 to 2400 ng, 250 to 2500 ng, 260 to 2600 ng, 270 to 2700 ng, 280 to 2800 ng, 290 to 2900 ng, 300 to 3000 ng, 310 to 3 000ng, 90-1000ng, 100-1000ng, 110-1000ng, 120-1000ng, 130-1000ng, 140-1000ng, 150-1000ng, 160-1000ng, 1 70-1000ng, 180-1000ng, 190-1000ng, 200-1000ng, 250-1000ng, 300-1000ng, 350-1000ng, 400-1000ng, 500-1000 ng, 600-1000ng, 700-1000ng, 800-1000ng, 900-1000ng, 1-900ng, 1-800ng, 1-700ng, 1-600ng, 1-500ng, 1-400ng , 1-300ng, 1-250ng, 1-200ng, 1-150ng, 1-140ng, 1-130ng, 1-120ng, 1-110ng, 1-100ng, 1-90ng, 1-80ng, 1-70ng, 1 The dosage may be set to 60 ng, 1 to 50 ng, 1 to 40 ng, 1 to 30 ng, 1 to 20 ng, 1 to 10 ng, 1 to 9 ng, 1 to 8 ng, 1 to 7 ng, 1 to 6 ng, 1 to 5 ng, 1 to 4 ng, 1 to 3 ng, 1 to 2 ng, 10 to 900 ng, 20 to 800 ng, 30 to 700 ng, 40 to 600 ng, 50 to 500 ng, 60 to 400 ng, 70 to 350 ng, 80 to 300 ng, 90 to 250 ng, 100 to 200 ng, etc. When the target organism is a non-mouse, the dosage for the target organism can be determined in accordance with conventional methods based on the dosage for mice described above.

[0060] The dosage of the composition of the present disclosure is not particularly limited and can be appropriately selected depending on the dosage form, the age and body weight of the subject, the degree of desired effect, etc. The frequency of administration of the pharmaceutical composition can be, for example, 1 to 3 times a day, preferably once a day.

[0061] The compositions of the present disclosure may contain bryostatin together with a pharmaceutically acceptable carrier or additive. For example, in the case of a pharmaceutical composition, the pharmaceutically acceptable carrier or additive means any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavoring agent, or sweetener, and any known pharmaceutically acceptable carrier or additive may be used.

[0062] In one embodiment, the composition of the present disclosure may contain ingredients that are beneficial to the growth, survival, and health maintenance of microorganisms that are beneficial to the subject of administration. In one embodiment, the composition of the present disclosure may contain food ingredients, such as indigestible oligosaccharides and dietary fiber, that are generally called prebiotics, which reach the large intestine without being broken down or absorbed in the stomach or small intestine and serve as food for microorganisms that live in the large intestine.

[0063] In one embodiment, the compositions of the present disclosure are not used as adjuvants. In one embodiment, the compositions of the present disclosure are not used in combination with antigenic molecules.

[0064] The organisms to which the composition of the present disclosure is administered are not particularly limited, and examples include mammals such as humans, primates, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, and pigs, and birds such as chickens.

[0065] The administration method is not particularly limited, but is preferably administered directly to mucosal tissue or the digestive tract, and examples of such administration methods include oral administration, nasal administration, transmucosal administration, and enteral administration.

[0066] Enteral administration is not limited to administration via the anus, but also includes administration via a tube or the like inserted into the digestive tract from outside the individual, such as a gastrostomy. The location into which the digestive tract is inserted is not limited to the intestine, but includes the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), large intestine (including the cecum, colon, rectum, etc.), etc.

[0067] The composition according to the present disclosure may be administered in the above amount once a day or in divided doses. Furthermore, the administration interval may be daily, every other day, weekly, biweekly, every 2-3 weeks, monthly, bimonthly, or every 2-3 months, as long as it has a preventive or therapeutic effect against the above diseases.

[0068] When the composition of the present disclosure is provided as a food composition, its specific form is not particularly limited.For example, the composition of the present disclosure can be provided as a food itself or as a food additive to be incorporated into a food composition.More specifically, the composition can be provided in the form of beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, milk drinks, etc.; frozen desserts such as ice cream, ice sorbet, shaved ice, etc.; confectioneries such as candy, candy, gum, chocolate, tablet candy, snacks, biscuits, jelly, jam, cream, baked goods, etc.; noodles such as soba, udon, harusame, Chinese noodles, instant noodles, etc.; processed seafood and livestock foods such as kamaboko, ham, sausage, etc.; dairy products such as processed milk and fermented milk, oils and fats and oil-based foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing, etc.; seasonings such as sauces, sauces, etc.; soups, stews, salads, side dishes, sprinkles, pickles, bread, cereals, etc. In the case of foods for specified health uses, nutritional supplements, functional foods, etc., they may be provided in the form of powder, granules, capsules, lozenges, tablets, syrup, etc.

[0069] When the composition of the present disclosure is provided as a feed composition, its specific form is not particularly limited. For example, as long as the effects of the feed composition according to the present disclosure are not impaired, the feed composition may be prepared by mixing the composition with a normal feed or, if necessary, mixing it with components that can be incorporated into a normal feed, or the feed composition itself may be used as feed.

[0070] 2. Method for Suppressing Th2 Response and / or Improving Intestinal Microbiota Using Bryostatin In one aspect, the present disclosure provides a method for suppressing a Th2 response and / or improving intestinal microbiota in a subject in need thereof, the method comprising administering bryostatin to the subject.

[0071] In one embodiment, the suppression of a Th2 response in the method of the present disclosure comprises suppressing cytokine production by Th2 cells. Cytokines primarily produced by Th2 cells include IL-4, IL-5, IL-9, IL-10, and IL-13. In one embodiment, the suppression of a Th2 response comprises suppressing blood levels of IL-4, IL-5, IL-9, IL-10, or IL-13.

[0072] In one embodiment, the suppression of a Th2 response in the method of the present disclosure comprises suppressing the differentiation of naive T cells into Th2 cells. In one embodiment, the suppression of a Th2 response in the method of the present disclosure comprises promoting the differentiation of naive T cells into suppressor T cells. In one embodiment, the suppression of a Th2 response in the method of the present disclosure comprises increasing IL-10-producing suppressor T cells. In one embodiment, the suppression of a Th2 response in the composition of the present disclosure comprises suppressing IgE antibody production through the suppression of differentiation into Th2 cells and the suppression of their activity. In one embodiment, the suppression of a Th2 response in the method of the present disclosure comprises suppressing the increase of eosinophils or suppressing their activity.

[0073] The method of the present disclosure directly affects the regulatory mechanism of T cell differentiation, suppressing differentiation into Th2 cells and promoting differentiation into inhibitory T cells that act antagonistically against Th2 cells, thereby preventing or treating conditions, disorders, or diseases for which suppression of Th2 responses is beneficial. Therefore, in preventing or treating these conditions, disorders, or diseases, it provides a more effective effect than regulating certain downstream reactions of the Th2 response. For example, it provides a more fundamental immunomodulatory effect than simply acting on the class switching of antibodies produced by activated B cells, thereby increasing the production of IgA antibodies and suppressing the production of other antibodies, such as IgE antibodies. Furthermore, the method of the present disclosure provides a more effective preventive or therapeutic effect for diseases that have previously been prevented or treated by downstream reactions of the Th2 response, such as suppressing the production of IgE antibodies through class switching control. Furthermore, the method of the present disclosure can provide a preventive or therapeutic effect for conditions, disorders, or diseases for which control of downstream reactions of the Th2 response is insufficient to provide a sufficient preventive or therapeutic effect. In one aspect, the method of the present disclosure is applied to the prevention or treatment of conditions, disorders, or diseases for which suppression of a Th2 response is beneficial. Upon recognizing an allergen, Th2 cells activate B cells and promote the production of IgE antibodies. In one aspect, the method of the present disclosure suppresses B cell activation and IgE antibody production by suppressing a Th2 response, and is particularly suitable for use in conditions, disorders, or diseases for which suppression of IgE antibody production is beneficial.

[0074] In one embodiment, the condition, disorder, or disease in which suppression of the Th2 response is beneficial is an IgG4-associated disease (e.g., Mikulicz's disease) (see, e.g., Arthritis Rheum. 2012 Jan;64(1):254-63. doi:10.1002 / art.33320), a fibrotic disease (e.g., pulmonary fibrosis, liver fibrosis, and kidney fibrosis) (see, e.g., Nat Rev Immunol. 2018 Jan;18(1):62-76. doi:10.1038 / nri.2017.90), a cancer (e.g., pancreatic cancer, liver cancer, breast cancer) (Clin Exp Immunol. 2014 Nov;178(2):201-11. doi:10.1111 / cei.12409, etc.), autoimmune diseases (e.g., multiple sclerosis, type 1 diabetes) (J Exp Med. 1997 Jul 21;186(2):307-12. doi:10.1084 / jem.186.2.307, etc.), and allergic diseases (e.g., bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, food allergies, anaphylaxis, hay fever, drug allergies, urticaria) (Clin Exp Immunol. 2014 Nov;178(2):201-11. doi:10.1111 / cei.12409, Japanese Society of Allergology website https: / / www. jsa-pr. jp / html / sickness. html).

[0075] In one aspect, the method of the present disclosure suppresses B cell activation and IgE antibody production by suppressing the Th2 response, and is particularly suitable for use in conditions, disorders, or diseases in which suppression of IgE antibody production is beneficial.

[0076] Non-limiting examples of conditions, disorders, or diseases for which suppression of IgE antibody production is beneficial include food allergies, allergic asthma, chronic idiopathic urticaria (CSU), and atopic dermatitis caused by IgE antibody responses (N Engl J Med 2024;390:889-899, DOI: 10.1056 / NEJMoa2312382, Annals of Allergy, Asthma & Immunology, Volume 127, Issue 2, 223-231, J Allergy Clin Immunol. 2016 Jun;137(6):1742-1750.e4. doi: 10.1016 / j. jaci. 2015.12.1342, G Ital Dermatol Venereol. 2019 Aug; 154(4):480-487. doi: 10.23736 / S0392-0488.19.06302-8. ).

[0077] In one aspect, the method of the present disclosure is particularly suitable for use in conditions, disorders, or diseases in which eosinophil inhibition is beneficial, including eosinophil-associated diseases in which abnormal eosinophil increase, activation, and tissue infiltration are observed. In one aspect, the method of the present disclosure is particularly suitable for use in conditions, disorders, or diseases in which eosinophil inhibition is beneficial, including eosinophil-associated diseases in which abnormal eosinophil increase, activation, and tissue infiltration are observed.

[0078] The eosinophil-related diseases include eosinophilic inflammatory diseases, diseases associated with eosinophilia, and eosinophil-mediated diseases. Specific, non-limiting examples of these diseases include eosinophilic asthma (bronchial asthma), eosinophilic sinusitis (ECRS), eosinophilic gastrointestinal disease (EGID), eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EGE), atopic dermatitis, allergic rhinitis, allergic conjunctivitis, and urticaria (including chronic and idiopathic). Eosinophilia includes, but is not limited to, hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), and drug-induced hypersensitivity syndrome (DRESS syndrome) (Semin Immunopathol. 2021 Apr 23;43(3):459-475. doi: 10.1007 / s00281-021-00849-w, Mayo Clin Proc. 2021 Oct;96(10):2694-2707. doi: 10.1016 / j.mayocp.2021.04.025, Allergol Int. 2019 Oct;68(4):403-412. doi: 10.1016 / j. alit. 2019.07.002. , Allergo J Int 33, 1-8 (2024). https: / / doi. org / 10.1007 / s40629-023-00265-6, Journal of Allergy and Clinical Immunology, Volume 113, Issue 1, 30-37, Curr Hematol Malig Rep. 2018 Jun;13(3):191-201. doi: 10.1007 / s11899-018-0448-8, Front Immunol. 2023 Apr 28;14:1134178. doi: 10.3389 / fimmu. 2023.1134178).

[0079] In one embodiment, improving the intestinal microbiota in the method of the present disclosure comprises increasing the bacterial flora diversity, preferably α-diversity, in the gastrointestinal contents or excrement of the subject organism, and in another embodiment, improving the intestinal microbiota comprises increasing the amount of short-chain fatty acids in the gastrointestinal contents or excrement of the subject organism.

[0080] In one aspect, improving the gut microbiota in the methods of the present disclosure includes preventing or treating a condition, disorder, or disease in which improving the gut microbiota would be beneficial.

[0081] In one aspect, the condition, disorder, or disease for which improvement of the gut microbiota is beneficial may be selected from the group consisting of dysbiosis, inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis), metabolic disease (e.g., type 2 diabetes, obesity), digestive disease (e.g., irritable bowel syndrome, colorectal cancer), allergic disease, cardiovascular disease, renal disease, and autoimmune disease (e.g., Parkinson's disease, multiple sclerosis) (Inflamm Bowel Dis. 2016 May;22(5):1137-50. doi:10.1097 / MIB.0000000000000750, Sci Rep. 2019 Sep 9;9(1):12918. doi:10.1038 / s41598-019-49452-y, Healthline article, What Causes Dysbiosis and How Is It Treated? , https: / / www. healthline. com / health / digestive-health / dysbiosis#outlook, Microorganisms. 2022 Mar 7;10(3):578. doi:10.3390 / microorganisms10030578, Nature Reviews Immunology 2017, volume 17, pages 219-232. https: / / doi. org / 10.1038 / nri. 2017.7, Microbial Ecology in Health and Disease 2015, Volume 26, Article: 26191. https: / / doi. org / 10.3402 / mehd. v26.26191, Ann Nutr Metab (2013) 63 (Suppl. 2): 28-40. https: / / doi. org / 10.1159 / 000354902, Gut. 2018 Jun;67(6):1024-1032. doi:10.1136 / gutjnl-2017-314281, Infrastructure Bowel Diseases, Volume 22, Issue 5, 1 May 2016, Pages 1137-1150, https: / / doi. org / 10.1097 / MIB. 0000000000000750, Med Microbiol Immunol. 2017 Apr;206(2):83-91. doi:10.1007 / s00430-017-0496-z. PLoS One. 2017 Mar 22;12(3):e0174099. doi:10.1371 / journal. bone. 0174099 etc.). ;

[0082] In a preferred embodiment of the present disclosure, the method of the present disclosure is applied to use in the prevention or treatment of conditions, disorders, or diseases in which both suppression of Th2 responses and improvement of intestinal flora are beneficial. Because the method of the present disclosure has the effect of suppressing Th2 responses and improving intestinal flora, it is particularly effective in the prevention or treatment of the above conditions, disorders, or diseases. In one embodiment, the conditions, disorders, or diseases in which both suppression of Th2 responses and improvement of intestinal flora are beneficial include cancer, autoimmune diseases, and allergies.

[0083] (Bryostatin) The bryostatin used in the method of the present disclosure includes bryostatin or a derivative thereof. More specifically, the bryostatin described in (Bryostatin) in "1. Composition containing bryostatin for use in suppressing Th2 response and / or improving intestinal flora" above can be used.

[0084] (Target of administration)

[0085] The target organisms in the methods of the present disclosure are not particularly limited, but include, for example, mammals such as humans, primates, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, and pigs, and birds such as chickens.

[0086] (Mode of Administration) In the method of the present disclosure, bryostatin can be administered in the form of various compositions. More specifically, the compositions described in (Form and Use of Composition) of "1. Compositions Containing Bryostatin for Use in Suppressing Th2 Responses and / or Improving Intestinal Microflora" above can be used as these compositions. The dosage and administration method of the composition of the present disclosure can be appropriately determined taking into consideration the condition of the subject, the degree of effect exerted by administration, the dosage form, the administration method, the subject, the gender of the subject, etc.

[0087] The administration method in the method of the present disclosure is not particularly limited, but direct administration to mucosal tissue or the digestive tract is preferred, and examples of such administration methods include oral administration, nasal administration, transmucosal administration, and enteral administration.

[0088] Enteral administration is not limited to administration via the anus, but also includes administration via a tube or the like inserted into the digestive tract from outside the individual, such as a gastrostomy. The location into which the digestive tract is inserted is not limited to the intestine, but includes the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), large intestine (including the cecum, colon, rectum, etc.), etc.

[0089] The administration in the method of the present disclosure may be once a day or divided into several doses. Furthermore, the administration interval may be daily, every other day, weekly, biweekly, every 2-3 weeks, monthly, bimonthly, or every 2-3 months, as long as it has a preventive or therapeutic effect against the above-mentioned disease.

[0090] In one embodiment, the method of the present disclosure may further administer to the subject a component beneficial to the growth, survival, and health maintenance of microorganisms beneficial to the subject. In one embodiment, the method of the present disclosure includes administering to the subject food components, such as indigestible oligosaccharides and dietary fiber, generally referred to as prebiotics, which reach the large intestine without being broken down or absorbed in the stomach or small intestine and serve as food for microorganisms living in the large intestine.

[0091] In one embodiment, the bryostatin is not used as an adjuvant in the methods of the present disclosure. In one embodiment, the antigenic molecule is not used in combination in the methods of the present disclosure. In one embodiment, the bryostatin is administered 24 hours or later, 36 hours or later, 48 hours or later, 3 days or later, 4 days or later, 5 days or later, 6 days or later, or 7 days or later, before or after the antigenic molecule is administered to the subject.

[0092] In one aspect, improving the intestinal microbiota of a subject organism in the method of the present disclosure comprises increasing the diversity of the microbiota in the gastrointestinal contents or excrement of the subject organism. Also, in one aspect, improving the intestinal microbiota of a subject organism comprises increasing the amount of short-chain fatty acids in the gastrointestinal contents or excrement of the subject organism.

[0093] The present invention will be specifically explained below by showing examples, but the present invention is not limited by these examples.

[0094] (Materials and Methods) (1) Creation of a Cedar Pollen Allergy Model Mouse Eight-week-old BALB / c mice (purchased from Japan CLEA) were intraperitoneally administered 150 μg of cedar pollen antigen (Cosmobio) / 20 mg of potassium aluminum sulfate (Nacalai Tesque) suspended in PBS (Nacalai Tesque). Two weeks later, 25 μg of cedar pollen antigen was administered intranasally once a week for three weeks. In the third week, the cedar pollen antigen was administered intranasally for three consecutive days.

[0095] (2) Administration of Bryostatin 1 to cedar pollen immunized mouse model Two weeks after immunization with cedar pollen antigen, Bryostatin 1 (Enzo Life Science) was administered intraperitoneally (300 ng, 150 ng, 75 ng), orally (300 ng, 150 ng, 75 ng), or intranasally (40 ng, 20 ng, 10 ng) three times at 5-day intervals.

[0096] (3) Evaluation of Symptoms of Cedar Pollen Allergy Model Mice On the final day of cedar pollen antigen administration, the nose-scratching behavior and the number of sneezes of the mice were counted for 10 minutes.

[0097] (4) Collection of Samples from Cedar Pollinosis Model Mice Blood samples were collected from the eye of mice with cedar pollinosis induced under isoflurane anesthesia. The collected blood samples were left on ice for 15 minutes and then centrifuged at 2000 rpm for 15 minutes. The supernatant was collected and used as a serum sample for ELISA. After collecting the blood samples, the mice were euthanized by cervical dislocation. After spraying with 70% ethanol, an incision was made from the abdomen to the throat of the mouse to aseptically expose the bronchi. An incision was made in the bronchi with scissors, and 600 μL of PBS was injected into the alveoli. The PBS injected into the alveoli was collected in an Eppendorf tube and centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was used as alveolar lavage fluid for ELISA. The pellet after centrifugation was used for flow cytometric analysis of alveolar-infiltrating immune cells. Mediastinal and nasopharyngeal-associated lymph nodes were collected and gently crushed with two sterile glass slides in a culture dish containing 5 ml of cell culture medium (RPMI 1640 supplemented with 10% FCS (Nichirei), 0.05 μM 2-mercaptoethanol (Nacalai Tesque), and 80 μg / ml gentamicin (Nacalai Tesque)) to obtain single-cell suspensions of mediastinal and nasopharyngeal-associated lymph nodes. Mouse feces were collected and weighed, then suspended in a 9-fold volume of PBS and centrifuged at 50 g for 15 minutes at 4°C. After centrifugation, the supernatant was collected in a separate Eppendorf tube. The collected supernatant was centrifuged at 8,000 g for 5 minutes at 4°C to collect bacterial cells.

[0098] (5) Evaluation of alveolar-infiltrating immune cells by flow cytometry Cells collected from alveolar lavage fluid were counted under a microscope using trypan blue (Nacalai Tesque) and then suspended in 100 μL of FACS buffer (PBS supplemented with 10% FCS (Nichirei), 0.05 μM 2-mercaptoethanol (Nacalai Tesque), 80 μg / ml gentamicin (Nacalai Tesque), and 5 μM EDTA). To the cell suspension, PerCP-Cy5.5-CD45 (BioLegend) (1 μg / mL), FITC-CD3ε (BioLegend) (1 μg / mL), FITC-B220 (BioLegend) (1 μg / mL), PE-SiglecF (BioLegend) (1 μg / mL), Pacific Blue-CD11c (BioLegend) (1 μg / mL), and PEcy7-Ly6G (BioLegend) (1 μg / mL) were added, and the mixture was incubated on ice for 20 minutes in the dark. FACS buffer was added to the antibody reaction solution, and the mixture was centrifuged at 1,500 rpm for 5 minutes at 4 ° C. The supernatant was then removed. 400 μL of FACS buffer and 0.25 μg of propidium iodide were added to the cells, and then the cell suspension was measured using a Sony Cell Analyzer SA3800 (Sony).

[0099] (6) Measurement of total IgE antibody titer in serum and alveolar lavage fluid by ELISA. The capture antibody for ELISA was each isotype antibody (unlabeled, described below) diluted to 2 μg / ml in 0.05 M NaCl. ２ CO ３The antibody was diluted in 1x PBS and added to an ELISA plate (C96 MAXISORP NUNC-Immunoplate, Thermo Scientific) at 50 μl / well and coated overnight at 4°C. The following day, the coated ELISA plate was washed three times with 1x PBS. After washing, 50 μl of 1x PBS supplemented with 1% BSA was added to each well and blocked at room temperature for 1 hour. During the blocking reaction, serial dilutions of the serum, alveolar lavage fluid samples, and standards collected in (3) above were prepared using 1x PBS supplemented with 1% BSA. After blocking, the diluted samples were added to the plate at 50 μl / well and incubated at room temperature for 1 hour. The plate was then washed three times with 1x PBS solution supplemented with 0.05% Tween 20. Next, 0.5 μg / ml of secondary antibody (alkaline phosphatase labeled) recognizing each isotype was added to each well at 50 μl / well, and the plate was incubated at room temperature for 1 hour. After washing three times with 1×PBS solution containing 0.05% Tween 20, a substrate solution (Phosphatase substrate) (Sigma) was added, and the plate was incubated at room temperature in the dark for color development. The absorbance (OD ４０５ｎｍ ) were measured. The absolute antibody titers were calculated by comparison with standard isotype antibodies and graphed. The antibodies used are listed below. Goat anti-mouse IgE UNLB (SouthernBiotech) Goat anti-mouse IgE AP (SouthernBiotech) Purified mouse IgE kappa isotype control (BD Pharmingen)

[0100] (7) Evaluation of Th2-related cytokine production in mediastinal lymph node and nasopharyngeal-associated lymph node cells. Mediastinal lymph node and nasopharyngeal-associated lymph node-derived cells were cultured at 1 x 10 ６ The cells were diluted with cell culture medium to a concentration of 1 / ml and added to a 24-well culture dish. 25 μg / mL of cedar pollen antigen and 0.8 nM of Bryostatin 1 were added to each well as indicated in the results. The cultured cells were incubated at 37°C in 5% CO ２After the culture, the culture supernatant was collected, and the concentrations of each cytokine were measured using a Mouse IL-4 ELISA Kit (Proteintech), a Mouse IL-5 ELISA Kit (Proteintech), and a Mouse IL-13 ELISA Kit (Proteintech).

[0101] (8) Extraction of Bacterial DNA DNA lysis buffer for DNA extraction was prepared by adding Tris-HCl (pH 8.0, final concentration 50 mM), NaCl (final concentration 300 mM), EDTA (final concentration 1 mM), and 0.5% SDS to ultrapure water. The collected bacterial cells were suspended in 500 μl of DNA lysis buffer. After suspension, the cells were transferred to a 2.0 ml tube (TM-625S, TOMY) containing glass beads (GB-01, 0.1Φ, TOMY) and subjected to bead disruption at 3,500 rpm for 1 minute using a bead cell disrupter (MS-100, TOMY). Proteinase K (final concentration 0.5 mg / ml, Nacalai Tesque) was added to the bead-disrupted bacterial solution, vortexed, and allowed to stand overnight at 55°C to completely lyse the bacterial cells. The temperature was returned to room temperature from 55°C, and 500 μl of phenol-chloroform-isoamyl alcohol (Nacalai Tesque) was added to the bacterial cell lysate, mixed by vortexing, and centrifuged at 13,000 rpm for 5 minutes at 4°C. The upper layer was collected in another 1.5 ml tube. An equal volume of chloroform (Nacalai Tesque) was added, mixed by vortexing, and centrifuged at 13,000 rpm for 5 minutes at 4°C. The upper layer was again collected in another 1.5 ml tube, and equal volumes of isopropanol (Nacalai Tesque) and glycogen (final concentration 0.04 mg / ml, Nacalai Tesque) were added and mixed by inversion. The DNA solution was mixed by inversion and allowed to stand at -20°C for 20 minutes, then centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was removed. 500 μl of 70% ethanol was added to the precipitated DNA, and the mixture was centrifuged at 13,000 rpm for 5 minutes at 4°C, and the supernatant was removed. The precipitated DNA was air-dried, and then Ultra Pure Distilled Water (Invitrogen) was added to dissolve the DNA.

[0102] (9) 16S ribosomal RNA (rRNA) gene amplicon sequence analysis of bacterial DNA. Bacterial DNA was subjected to polymerase chain reaction (PCR) targeting the V3-V4 region of the variable region of the 16S rRNA gene. Primers were prepared for next-generation sequencing using Miseq, and index sequences were added to distinguish the samples being analyzed. The composition of the PCR reaction solution is shown in Table 1, and the PCR reaction is shown in Table 2. After the PCR reaction, 10x Loading Buffer (TAKARA) was added, and agarose electrophoresis was performed. The gel used to run the PCR samples was a 1.5% agarose gel (VWR Life Science) supplemented with LED Stain G (LABTAS+). 100 bp DNA Ladder (TAKARA) was used as a DNA size marker. Electrophoresis was performed at 100V for 30 minutes. After electrophoresis, approximately 500 bp of PCR product was excised and purified using a Fast Gene Gel Extraction Kit (Nippon Genetics). A portion of the purified PCR product was subjected to electrophoresis again to confirm whether it was purified. Sequence analysis was performed at the request of Myskin Inc. using Miseq Reagent Kit V3 (Illumina). The sequence data obtained by sequence analysis was subjected to bacterial flora analysis (phylogenetic analysis, alpha diversity analysis, beta diversity analysis) using Qiime2 software (ver. 2020-2). SILVA and Greengenes databases were used. For α diversity analysis, the Shannon index was calculated, and for β diversity analysis, principal coordinate analysis (PCoA) based on Jaccard and unweighted uniFrac distances was performed.

[0103] (10) Creation of a Food Allergy Model Mouse Eight-week-old BALB / c mice (purchased from Japan CLEA) were intraperitoneally administered 1 mg of ovalbumin (OVA) (Sigma) and 20 mg of potassium aluminum sulfate suspended in PBS. The mice were weighed at this time. Two weeks later, 200 mg of OVA was orally administered once a week for two weeks.

[0104] (11) Administration of Bryostatin 1 to Food Allergy Model Mice Two weeks after immunization with OVA, 150 ng of Bryostatin 1 was orally administered twice at an interval of 5 days to the mice.

[0105] (12) Evaluation of symptoms in food allergy model mice. On the final day of OVA administration, the mice were weighed to evaluate the weight loss rate due to food allergy. In addition, the fecal properties after OVA administration were evaluated, and diarrhea symptoms were scored from 0 to 5.

[0106] (13) Collection of Samples from Food Allergy Model Mice. Blood samples were collected from the eye of mice with induced food allergies under isoflurane anesthesia. The collected blood samples were left on ice for 15 minutes and then centrifuged at 2,000 rpm for 15 minutes. The supernatant was collected and used as a serum sample for ELISA. After collecting the blood samples, the mice were euthanized by cervical dislocation. After spraying with 70% ethanol, the abdomen was opened and the mesenteric lymph nodes were collected. The collected mesenteric lymph nodes were gently crushed with two sterile glass slides in a culture dish containing 5 ml of cell culture medium to obtain a single-cell suspension of mesenteric lymph nodes. Mouse feces were collected and weighed, then suspended in 9 volumes of PBS and centrifuged at 50 g for 15 minutes at 4°C. After centrifugation, the supernatant was collected in a separate Eppendorf tube. Bacterial cells were collected by centrifugation at 8,000 g for 5 minutes at 4°C. The supernatant after centrifugation was collected in another Eppendorf tube and used for ELISA.

[0107] (14) Measurement of total IgE and IgA antibody titers by ELISA The IgE and IgA antibody titers were measured for the serum and single-cell suspension of mesenteric lymph nodes collected in (13) above using the same method as in (6). The antibodies used are listed below. Goat anti-mouse IgE UNLB (SouthernBiotech) Goat anti-mouse IgE AP (SouthernBiotech) Purified mouse IgE kappa isotype control (BD Pharmingen) Goat anti-mouse IgA UNLB (SouthernBiotech) Goat anti-mouse IgA AP (SouthernBiotech) Purified mouse IgA kappa (Immunology Consultants Laboratory)

[0108] (15) Measurement of OVA-specific IgE / IgA antibody titers by ELISA OVA was diluted with 0.05 M NaCl to a concentration of 75 μg / ml. ２ CO ３The antibody was diluted in 1x PBS and added to an ELISA plate (C96 MAXISORP NUNC-Immunoplate, Thermo Scientific) at 50 μl / well, followed by overnight coating at 4°C. The following day, the coated ELISA plate was washed three times with 1x PBS. After washing, 50 μl of 1x PBS supplemented with 1% BSA was added to each well, and blocking was performed at room temperature for 1 hour. During the blocking reaction, a dilution series of the serum and feces collected in (10) above was prepared using 1x PBS supplemented with 1% BSA. After blocking, the diluted samples were added to the plate at 50 μl / well, and the plate was allowed to react at room temperature for 1 hour. The plate was then washed three times with 1x PBS solution supplemented with 0.05% Tween 20. Next, 0.5 μg / ml of secondary antibody (alkaline phosphatase labeled) recognizing each isotype was added to 50 μl / well and allowed to react for 1 hour at room temperature. After washing three times with 1x PBS solution containing 0.05% Tween 20, a substrate solution (Phosphatase substrate) was added, and color was developed at room temperature in the dark, and absorbance (OD405 nm) was measured using a plate reader. The antibody titer was plotted as a relative value using absorbance (OD405 nm) as an index. The antibodies used are listed below. Goat anti-mouse IgE AP (SouthernBiotech) Goat anti-mouse IgA AP (SouthernBiotech)

[0109] (16) Measurement of Th2-related cytokine production in mesenteric lymph node cells. 1 x 10 cells derived from mesenteric lymph nodes were cultured in a 1000 ml culture medium. ６ The cells were diluted with the above cell culture medium to a concentration of 1 / ml and added to a 24-well culture dish. 25 μg / mL of OVA and 0.8 nM of Bryostatin 1 were added to each well as indicated in the results. The cultured cells were incubated at 37°C in 5% CO ２After the incubation, the culture supernatant was collected, and the concentrations of each cytokine were measured using a Mouse IL-4 ELISA Kit (Proteintech), a Mouse IL-5 ELISA Kit (Proteintech), and a Mouse IL-13 ELISA Kit (Proteintech).

[0110] (17) Evaluation of intestinal flora in food allergy model mice The intestinal flora in food allergy mice was analyzed using the same method as in (8)-(9) above.

[0111] (18) Mouse CD4 from the spleen ＋ Isolation of T Cells After euthanasia, mice were opened and the spleen was removed. The spleen was mashed using a glass slide and suspended in RPMI 1640. The cells were collected through a cell strainer into a tube containing 10 ml of RPMI 1640. The cells were centrifuged at 1500 rpm for 5 minutes and the supernatant was removed. The cells were suspended in 10 ml of RPMI 1640 and centrifuged again at 1500 rpm for 5 minutes and the supernatant was removed. The spleen cells were suspended in 1 ml of cell culture medium, and dead cells were stained using 0.5% trypan blue staining solution to calculate the number of viable cells. Based on the results of viable cell count, CD4+ T Cell Isolation Kit for mouse (Miltenyi) was used to isolate CD4+ T cells. ＋ T cells were isolated.

[0112] (19) Mouse CD4 ＋ T cell culture: Anti-mouse CD3ε antibody (BioLegend) was coated onto a 24-well plate at room temperature overnight. ＋ 1 x 10 T cells ６The cell culture medium was adjusted to give a cell density of 1000 cells / ml, and the cells were seeded. Reagents required for differentiation into various T cells were added under the following conditions. Th0: anti-mouse CD28 antibody (Biolegend) 3μg / mL, anti-mouse IFN-γ antibody (Biolegend) 3μg / mL, anti-mouse IL-12 antibody (Biolegend) 3μg / mL, anti-mouse IL-4 antibody (Biolegend) 3μg / mL, recombinant mouse IL-2 (Biolegend) 20ng / mL Th1: anti-mouse CD28 antibody 3μg / mL, anti-mouse IL-4 antibody 3μg / mL, recombinant mouse IL-2 20ng / mL, recombinant mouse IL-12 (Biolegend) 10ng / mL Th2: anti-mouse CD28 antibody 3μg / mL, anti-mouse IFN-γ antibody 3μg / mL, anti-mouse IL-12 antibody 3μg / mL, recombinant mouse IL-2 20ng / mL, recombinant mouse IL-4 (Biolegend) 20ng / mL Th17:anti-mouse CD28 The media contained: antibody 3 μg / mL, anti-mouse IFN-γ antibody 3 μg / mL, anti-mouse IL-12 antibody 3 μg / mL, anti-mouse IL-4 antibody 3 μg / mL, recombinant mouse IL-2 20 ng / mL, recombinant mouse TGF-β (Biolegend) 10 ng / mL, recombinant mouse IL-6 (Biolegend) 20 ng / mL. Bryostatin 1 was added at a concentration of 0.8 nM. Three days after the start of culture, the cells were passaged onto a 6-well plate. Cells were collected on day 4 for Th1 and Th17, and on day 6 for Th2.

[0113] (20) Evaluation of T cell differentiation by flow cytometry. The collected cells were suspended in 100 μL of FACS buffer. To the cell suspension, PerCP-Cy5.5 anti-CD3ε antibody (BioLegend) 1 μg / mL, PEcy7 anti-CD4 antibody (BioLegend) 1 μg / mL, and Fixable Viability Dye eFluor® 660 (eBioscience) 0.5 μL were added and incubated on ice in the dark for 20 minutes. FACS buffer was added to the antibody reaction solution, and the mixture was centrifuged at 1,500 rpm for 5 minutes at 4°C, and the supernatant was removed. The cells were permeabilized using BD Cytofix / Cytoperm® Fixation / Permeabilization Kit (BD). After cell permeabilization, PerCP-Cy5.5 anti-IL-13 antibody (BioLegend) 1 μg / mL, FITC anti-IL-4 antibody (BioLegend) 1 μg / mL, FITC anti-IL17A antibody (BioLegend) 1μg / mL, PE anti-IL-10 antibody (BioLegend) 1μg / mL, Pacific Blue anti-IFNγ antibody (BioLegend) 1 μg / mL, BV421 anti-IL-5 antibody (eBioscience) 1 μg / mL on ice The reaction was allowed to proceed for 30 minutes. FACS buffer was added to the antibody reaction solution, and the mixture was centrifuged at 1,500 rpm for 5 minutes at 4°C, and the supernatant was removed. 400 μL of FACS buffer was added to the cells, and the cell suspension was measured using a Sony Cell Analyzer SA3800.

[0114] (21) Mouse CD4 ＋ RNA was purified from the collected cells using RNA Premium (Fast Gene). Finally, the RNA was eluted with 20 μL of RNase-free water, and the concentration was measured using BioDrop (BERTHOLD).

[0115] (22) Synthesis of complementary DNA (cDNA) GoScript ＴＭReverse transcription was performed using Reverse Transcriptase (Promega). ＴＭ Gospecific Mix was prepared by mixing 4 μl of Reaction Buffer + Random Primer Mixture, 2 μl of GoScript™ Enzyme Mixture, and 4 μl of Nuclease-Free Water. 10 μl of Gospecific Mix was mixed with 10 μl of RNA solution adjusted to 5 μg / 10 μl, and the mixture was incubated at 25°C for 10 minutes, 42°C for 60 minutes, and 85°C for 5 minutes. The synthesized cDNA was stored at -20°C.

[0116] (23) Analysis of Gene Expression by Quantitative PCR. Evaluation was performed using the synthesized cDNA and primers for the genes listed in Table 3. The PCR reaction solution was prepared by mixing 10 μL of KAPA SYBR® FAST qPCR Kit Master Mix (2x) (KAPABIOSYNTHESIS® System), 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), 0.5 μL of 3-fold diluted cDNA, and 9.1 μL of ultrapure water per well. Three wells were prepared per sample and analyzed. The PCR conditions were denaturation at 98°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds, for a total of 70 cycles (LightCycler® 480, Roche). The data after quantitative PCR were corrected by the expression level of the housekeeping gene β-actin. To obtain an accurate Cp value, three wells per sample were analyzed, and after confirming that the Cp value had an error of 1.0 or less, the average value of the three wells was calculated as the Cp value.

[0117]

[0118] Example 1 Effect of bryostatin in a cedar pollen allergy model As described in (1) to (9) of the above (Materials and Methods), the effect of bryostatin in a cedar pollen allergy model was tested. Bryostatin 1 was used in this test. The test scheme is shown in Figure 1.

[0119] In a cedar pollen allergy model, administration of Bryostatin 1 significantly suppressed allergic symptoms, such as the number of sneezing and nose-scratching movements. Oral or nasal administration demonstrated particularly effective suppression of allergic symptoms (Figure 2). Analysis of alveolar-infiltrating immune cells in bronchoalveolar lavage fluid (BAF) revealed significant decreases in the numbers of eosinophils and lymphocytes (Figure 3). A significant decrease in total IgE antibody titers was also observed in BAF, as well as in serum (Figure 4). Furthermore, a significant suppression of Th2-related cytokine production, such as IL-4, IL-5, and IL-13, was observed in the culture supernatant of mediastinal lymph node cells (Figure 5).

[0120] Next, we evaluated whether bryostatin directly affects the production of Th2-associated cytokines by acting on T cells in mediastinal lymph nodes and nasopharyngeal-associated lymph nodes (MALSNs) (Materials and Methods (7)). The experimental scheme is shown in Figure 6. As a result, we observed a significant suppression of Th2-associated cytokine production, such as IL-4, IL-5, and IL-13, in both mediastinal lymph node (mLN) and nasopharyngeal-associated lymph node (NALT) cells, through bryostatin's action on T cells (Figure 7).

[0121] Furthermore, the intestinal microbiota of the test mice was analyzed (Materials and Methods (8)-(9)). The results showed that bacterial diversity in the intestinal microbiota was reduced in subjects immunized with cedar pollen, and that administration of Bryostatin 1 suppressed this decrease in diversity and increased bacterial diversity in the intestinal microbiota (Figures 8, 9, and 10). A significant increase in bacterial diversity was observed, particularly in α-diversity (Figure 9). The list of bacterial names shown in Figure 9 is reproduced below. Note that "Unassigned;__;__;__;__" indicates that the corresponding bacteria could not be identified. The results of this example demonstrate that in a cedar pollen allergy model, Bryostatin 1 acts directly on T cells to suppress the production of Th2-related cytokines and improves the diversity of the intestinal microbiota, thereby ameliorating allergic symptoms.

[0122] Example 2 Effect of Bryostatin in a Food Allergy Model As described in (10) to (16) of the above (Materials and Methods), the effect of bryostatin in a food allergy model was tested. Bryostatin 1 was used in this test. The test scheme is shown in Figure 11.

[0123] The results of the study confirmed that administration of Bryostatin 1 significantly suppressed allergic symptoms, such as weight loss and diarrhea, in a food allergy model (Figure 12). Furthermore, analysis of serum and single-cell suspensions of mesenteric lymph nodes from the control mice revealed suppression of serum total IgE and OVA-specific IgE levels, and increased total and OVA-specific IgA production in mesenteric lymph node cells (Figure 13). Furthermore, evaluation of Th2-associated cytokine production in mesenteric lymph node cells revealed a significant suppression of Th2-associated cytokine production, including IL-4, IL-5, and IL-13 (Figure 14). Next, we evaluated whether Bryostatin directly affects the production of Th2-associated cytokines by acting on mesenteric lymph node T cells (Materials and Methods (16)). The experimental scheme is shown in Figure 15. As a result, it was observed that bryostatin acted on T cells in mesenteric lymph node cells, resulting in a marked suppression of the production of Th2-associated cytokines such as IL-4, IL-5, and IL-13 (FIG. 16).

[0124] Furthermore, the intestinal microbiota of the test mice was analyzed (Materials and Methods (17)). The results showed that bacterial diversity in the intestinal microbiota was reduced in subjects who received OVA, and that administration of Bryostatin 1 suppressed this decrease in diversity and increased bacterial diversity in the intestinal microbiota (Figures 17 and 18). A significant increase in bacterial diversity was observed, particularly in α-diversity (Figure 18). The list of bacterial names shown in Figure 18 is reproduced below. Note that "Unassigned;__;__;__;__" indicates that the corresponding bacteria could not be identified. The results of this example demonstrate that in a food allergy model, Bryostatin 1 acts directly on T cells to suppress the production of Th2-associated cytokines and improves the diversity of the intestinal microbiota, thereby ameliorating allergic symptoms.

[0125] Example 3: Evaluation of the Effect of Bryostatin on T Cell Differentiation As described in (18) to (23) in the Materials and Methods section above, the direct effect of bryostatin on T cell differentiation was evaluated. Bryostatin 1 was used in this study. Naive T cells were treated with bryostatin or vehicle (control), and the expression of IL-4, IL-13, IL-5, IL-10, IFN-γ, and IL-17 in CD4+ T cells was examined. Bryostatin treatment reduced CD4+ T cells expressing Th2-associated cytokines IL-4, IL-13, and IL-5, as well as IL-17, while increasing CD4+ T cells expressing IL-10 (Figures 20 to 22). These results indicate that bryostatin suppresses the differentiation of naive T cells into Th2 and Th17 cells and preferentially promotes their differentiation into suppressor T cells (Figure 25). Further investigation into the mechanism of this reaction revealed that bryostatin induces the expression of c-Maf and LAG-3, which are key regulators of inhibitory molecules ( Front. Immunol., 10 March 2022, Sec. B Cell Biology Volume 13, 2022, https: / / doi.org / 10.3389 / fimmu.2022.818814, Int. J. Mol. Sci. 2021, 22(10), 5282; https: / / doi.org / 10.3390 / ijms22105282 ), and through these, induces the expression of inhibitory molecules ( Figures 23-24A, B ). These results indicate that bryostatin acts directly on T cell differentiation, suppressing differentiation into Th2 cells, and also promotes differentiation into suppressor T cells by acting on key regulatory factors of inhibitory molecules. Suppressor T cells have the function of suppressing Th2 cells. Furthermore, while Th2 cells activate eosinophils through Th2-associated cytokines, suppressor T cells act antagonistically to suppress eosinophil activity. These results indicate that bryostatin simultaneously regulates two mechanisms: suppression of differentiation into Th2 cells and promotion of differentiation into suppressor T cells, thereby potently suppressing Th2 responses.It has also been shown that simultaneous regulation of these two antagonistic mechanisms on eosinophils leads to potent suppression of eosinophils (Fig. 25).

[0126] The results of Examples 1 to 3 show that bryostatin acts directly on T cells to significantly suppress Th2 responses and improve the intestinal flora, and that bryostatin is highly effective in preventing or treating conditions, disorders, or diseases in which suppression of Th2 responses is beneficial, as well as conditions, disorders, or diseases in which improvement of the intestinal flora is beneficial.

[0127] While preferred embodiments of the present invention are described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only, and that various modifications, changes, and substitutions may be made by those skilled in the art without departing from the invention. It should be understood that various alternative embodiments of the invention described herein may be used in practicing the invention. Furthermore, the contents of all publications, including patents and patent applications, referenced herein should be construed as incorporated by reference as if expressly set forth herein.

Claims

1. A composition for use in suppressing a Th2 response, comprising bryostatin as an active ingredient.

2. The composition of claim 1, wherein the suppression of a Th2 response includes the prevention or treatment of a condition, disorder, or disease in which suppression of a Th2 response is beneficial.

3. The composition of claim 1, wherein the condition, disorder, or disease in which suppression of the Th2 response is beneficial is selected from the group consisting of eosinophil-related diseases, allergic diseases caused by IgE antibody responses, IgG4-related diseases, fibrotic diseases, cancer, and autoimmune diseases.

4. The composition of claim 1, wherein the condition, disorder, or disease in which suppression of a Th2 response is beneficial is selected from the group consisting of Mikulicz's disease, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, pancreatic cancer, liver cancer, breast cancer, type 1 diabetes, bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, food allergy, anaphylaxis, hay fever, drug allergy, urticaria including chronic idiopathic urticaria (CSU), eosinophilic asthma (bronchial asthma), eosinophilic sinusitis (ECRS), eosinophilic gastrointestinal disease (EGID), eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EGE), hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), and drug-induced hypersensitivity syndrome (DRESS syndrome).

5. The composition of claim 1, wherein the condition, disorder, or disease in which suppression of a Th2 response is beneficial is an eosinophil-associated disease selected from the group consisting of eosinophilic asthma (bronchial asthma), eosinophilic sinusitis (ECRS), eosinophilic gastrointestinal disease (EGID), eosinophilic esophagitis (EoE), eosinophilic gastroenteritis (EGE), urticaria including chronic idiopathic urticaria (CSU), hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), drug-induced hypersensitivity syndrome (DRESS syndrome), atopic dermatitis, allergic rhinitis, allergic conjunctivitis, and food allergy.

6. A composition for use in improving the intestinal flora, comprising bryostatin as an active ingredient.

7. The composition of claim 5, wherein improving the intestinal flora includes preventing or treating a condition, disorder, or disease in which improving the intestinal flora is beneficial.

8. The composition of claim 6, wherein the condition, disorder, or disease for which improvement of the intestinal microbiota is beneficial is selected from the group consisting of intestinal microbial symbiotic imbalance (dysbiosis), inflammatory bowel disease (IBD), metabolic disease, digestive disease, allergic disease, cardiovascular disease, renal disease, and autoimmune disease.

9. A composition according to claim 1 or 6 for oral, nasal or enteral administration.

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