Immunosuppressive composition containing 3,6-anhydro-L-galactose (L-AHG)

Abstract

The present invention relates to immunosuppressive compositions containing 3,6-anhydro-L-galactose (L-AHG) as an active ingredient, as well as pharmaceutical compositions, functional health foods, and functional cosmetic compositions containing the active ingredient for the prevention or treatment of immune-mediated diseases. The immunosuppressive compositions, pharmaceutical compositions, functional health foods, and functional cosmetic compositions containing L-AHG of the present invention have the excellent effect of selectively and effectively suppressing the activation, proliferation, and differentiation of CD4 T cells, the main targets of immunosuppressants, and are therefore extremely useful inventions in the pharmaceutical, food, and cosmetic industries.

Description

[Technical Field]

[0001] The present invention relates to an immunosuppressive composition containing 3,6-anhydro-L-galactose (L-AHG) as an active ingredient, a pharmaceutical composition containing the active ingredient for the prevention or treatment of immune diseases, a functional health food, and a functional cosmetic composition. [Background technology]

[0002] The immune system can sometimes act inappropriately, typically by overreacting and causing a hypersensitivity reaction, or by failing to distinguish between self and non-self molecules and inducing an immune response against self molecules, resulting in autoimmune diseases. Furthermore, the immune rejection of an allograft transplanted for therapeutic purposes by the recipient's immune system can be considered an inappropriate immune response from the perspective of graft maintenance and survival.

[0003] Allergic or type 1 hypersensitivity reactions in humans are primarily mediated by immunoglobulin E (IgE) antibodies. When specific antigens bind to IgE present on the surface of mast cells and basophils in the body, these cells release mediators that cause hypersensitivity and inflammation. The main symptoms of hypersensitivity reactions include sneezing, wheezing, asthma, atopic dermatitis, and skin rashes.

[0004] Autoimmunity, in which the immune system loses the ability to distinguish between self and non-self molecules and attacks the host immunologically, causes a variety of chronic debilitating diseases, including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, psoriasis, Hashimoto's thyroiditis, and acute inflammatory bowel disease, which has recently become a problem among young people.

[0005] When the human immune system comes into contact with foreign cells and tissues, it responds strongly to eliminate these foreign elements. However, in some cases, even if the patient's immune system recognizes them as foreign and initiates an immune response to reject them, transplantation of cells, tissues, or organs from another donor may be the only treatment for the patient's disease. For example, it is estimated that more than 100,000 people require kidney transplantation treatment each year in the United States alone. However, if the immune system does not recognize the transplanted cells, tissues, or organs as self, it will attack and eliminate them through an immune rejection response. Therefore, immune rejection is a significant obstacle to this life-saving transplant therapy. An additional risk in transplants is that the transplanted immune cells may recognize the new host as non-self and react against it. This reaction, called graft-versus-host disease, can be fatal.

[0006] Therefore, immunosuppressive drugs are used to regulate inappropriate immune responses, such as hypersensitivity reactions, autoimmune diseases, allograft rejection, and graft-versus-host disease, induced by the human immune system. Currently used immunosuppressants include corticosteroids (prednisone), antiproliferative agents (mycophenolate mofetil (CellCept®)), calcineurin inhibitors (tacrolimus (Envarsus XR® or Protopic®), cyclosporine (Gengraf®, Neoral®, or Sandimmune®)), rapamycin (mTOR) inhibitors (sirolimus (Rapamun®)), and polyclonal and monoclonal antibodies targeting immune cells. Currently used immunosuppressants are associated with side effects, such as damage to other organs, such as renal dysfunction, and are not satisfactory in terms of efficacy. Therefore, there is a need for the development of safe, target-specific immunosuppressants that minimize drug side effects.

[0007] Until recently, with the advancement of biotechnology and an increased understanding of the human immune system, various types of immunosuppressants have been used as single or combined therapies to regulate inappropriate immune responses in patients with hypersensitivity reactions, autoimmune diseases, and organ transplants, including stem cell or bone marrow transplants, for whom administration of immunosuppressants is clinically indicated, depending on the patient's condition and symptoms.

[0008] The typical target of immunosuppressants used in patients to artificially suppress the body's immune function is T cells, and in fact, the main mechanism of action is suppression of the function of CD4+ T (Th) cells. This is because cytokines derived from effector Th cells are important not only in cellular immune responses, in which T cells play a major role, but also in humoral immune responses, in which B cell-derived antibody molecules play a major role. In particular, inhibiting T cell activation could be an important therapeutic option for Sjögren's syndrome, Behçet's disease, systemic sclerosis, polymyositis, and dermatomyositis, in which cytotoxic CD4+ T cells have been reported to play an important causative role.

[0009] Therefore, immunosuppressive effects can be fully achieved by appropriately blocking the activation, proliferation, and differentiation of resting Th cells into effector Th cells. The importance of Th cells in inducing and regulating immune responses is well-documented by the phenomenon of immunodeficiency in patients infected with human immunodeficiency virus (HIV) and suffering from Th cell dysfunction.

[0010] In order to improve the side effects, potential risks, or complications caused by the use of immunosuppressants developed to date, there is a continuing need to develop new immunosuppressants that can specifically suppress only inappropriate immune responses. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention has been made to solve the above-mentioned problems of the prior art, and its purpose is to provide a novel immunosuppressive composition, a pharmaceutical composition for the prevention or treatment of immune diseases, a functional health food, and a functional cosmetic composition that can effectively suppress the activation, proliferation, and differentiation of CD4 T cells, which are the main targets of immunosuppressants. [Means for solving the problem]

[0012] To achieve the above object, the present invention provides an immunosuppressive composition containing 3,6-anhydro-L-galactose as an active ingredient.

[0013] Preferably, the composition inhibits the activation, proliferation or differentiation of CD4 T cells.

[0014] Preferably, the composition blocks cell cycle progression in the G1 or S phase.

[0015] Preferably, the composition suppresses differentiation into Th1 cells or Th2 cells.

[0016] Preferably, the composition inhibits the production of interferon-γ, interleukin-2, interleukin-4 or interleukin-13.

[0017] The present invention also provides a pharmaceutical composition for preventing or treating immune diseases, which contains 3,6-anhydro-L-galactose as an active ingredient.

[0018] Preferably, the immune disease is selected from the group consisting of hypersensitivity immune diseases, autoimmune diseases, immune rejection, graft-versus-host disease, Sjogren's syndrome and Behcet's disease.

[0019] Preferably, said hypersensitivity immune disorder is selected from the group consisting of allergies, sneezing, wheezing, asthma, atopic dermatitis and urticaria (hives).

[0020] Preferably, the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, psoriasis, Hashimoto's thyroiditis, systemic lupus erythematosus and acute inflammatory bowel disease.

[0021] The present invention also provides a health functional food containing 3,6-anhydro-L-galactose for preventing or ameliorating immune-mediated diseases.

[0022] Preferably, the immune disease is selected from the group consisting of hypersensitivity immune diseases, autoimmune diseases, immune rejection, graft-versus-host disease, Sjogren's syndrome and Behcet's disease.

[0023] Preferably, said hypersensitivity immune disorder is selected from the group consisting of allergies, sneezing, wheezing, asthma, atopic dermatitis and urticaria (hives).

[0024] Preferably, the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, psoriasis, Hashimoto's thyroiditis, systemic lupus erythematosus and acute inflammatory bowel disease.

[0025] The present invention also provides a functional cosmetic composition for preventing or improving hypersensitive skin diseases, which contains 3,6-anhydro-L-galactose as an active ingredient.

[0026] The hypersensitive skin disease is preferably selected from the group consisting of photodermatitis, contact dermatitis and atopic dermatitis. [Effects of the Invention]

[0027] The immunosuppressive compositions, pharmaceutical compositions, health functional foods, and functional cosmetic compositions of the present invention, which contain 3,6-anhydro-L-galactose (L-AHG), have the excellent effect of selectively and effectively suppressing the activation, proliferation, and differentiation of CD4 T cells, which are the main targets of immunosuppressants. Therefore, these compositions are extremely useful in the pharmaceutical, food, and cosmetic industries. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows the distribution of CD4 (Th) cells and CD8 (Tc) cells in G0 T cells isolated from mouse spleens. [Figure 2] FIG. 1 shows the results of a [H]thymidine incorporation assay of the proliferative effects of 3,6-anhydro-L-galactose (L-AHG), neoagarobiose (NA2), neoagarotetraose (NA4), and neoagarohexaose (NA6), obtained by enzymatic hydrolysis of agarose, on T cells activated with immobilized anti-CD3 / anti-CD28. [Figure 3] FIG. 1 shows the results of L-AHG specifically blocking cell cycle progression in CD4 T cells. G0 T cells were treated with nocodazole 20 hours after activation. [Figure 4] FIG. 1 shows how L-AHG blocks cell cycle progression in nocodazole-naive CD4 T cells. [Figure 5] FIG. 1 shows the production of Th1-secreted cytokines (INF-γ, IL-2) and Th2-secreted cytokines (IL-4, IL-13) in T cells activated with immobilized anti-CD3 / anti-CD28 by treatment with L-AHG. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be described in detail below.

[0030] In this study, agarose was saccharified using a combined enzyme process using recombinant GH16B β-agarase, GH50A β-agarase, and GH117 α-neoagarobiose hydrolase derived from the agar-degrading bacterium Cellvibrio sp. KY-GH-1 (KCTC 13629BP), isolated from a non-marine freshwater environment. The resulting monomeric 3,6-anhydro-L-galactose and D-galactose products were fractionated by size-exclusion chromatography on a Sephadex G-10 column, resulting in purified L-AHG. To determine the immunosuppressive activity of purified L-AHG, we investigated the effects of L-AHG on the activation and proliferation of resting Th cells in a mouse model in which in vitro activation and cell cycle initiation of resting (G0) T cells were induced. As a result, it was confirmed that when resting Th cells are activated by simultaneous stimulation with immobilized anti-CD3 bound to the T cell receptor complex and immobilized anti-CD28 bound to the T cell costimulatory receptor, they leave the G0 phase and progress through the cell cycle in the order of G1 → S → G2 / M, and the presence of L-AHG can exert an immunosuppressive effect that reduces T cell proliferation by inhibiting the progression of activated Th cells through the G1 and S phases.

[0031] Therefore, the present invention provides an immunosuppressive composition containing 3,6-anhydro-L-galactose as an active ingredient.

[0032] The active ingredient can be obtained from acid hydrolysis or enzymatic degradation of agarose.

[0033] In a preferred embodiment, the active ingredient of the present invention can be obtained by subjecting agarose to acid hydrolysis.

[0034] In another preferred embodiment, the active ingredient of the present invention can be obtained by saccharifying agarose using an enzymatic process in which agarose is treated with a combination of recombinant GH16B β-agarase, GH50A β-agarase, and GH117 α-neoagarobiose hydrolase derived from the agar-degrading bacterium Cellvibrio sp. KY-GH-1 strain (KCTC 13629BP), and then fractionating the mixture of monomers, 3,6-anhydro-L-galactose and D-galactose, produced as the final degradation products, by size exclusion chromatography using a Sephadex G-10 column.

[0035] In yet another preferred embodiment, the active ingredient of the present invention can be obtained from the agarase-3,6-anhydro-L-galactosidase-arabinose isomerase enzyme conjugate product of agarose.

[0036] In addition to the several methods for obtaining L-AHG described above, the active ingredient of the present invention can also be obtained commercially, for example, from Toronto Research Chemicals, North York, Ontario, Canada.

[0037] Preferably, the composition inhibits the activation, proliferation or differentiation of CD4 T cells.

[0038] Preferably, the composition blocks cell cycle progression in the G1 or S phase.

[0039] Preferably, the composition suppresses differentiation into Th1 cells or Th2 cells.

[0040] Preferably, the composition inhibits the production of interferon-γ, interleukin-2, interleukin-4 or interleukin-13.

[0041] The present invention also provides a pharmaceutical composition for preventing or treating immune diseases, which contains 3,6-anhydro-L-galactose as an active ingredient.

[0042] Preferably, the immune disease is selected from the group consisting of hypersensitivity immune diseases, autoimmune diseases, immune rejection, graft-versus-host disease, Sjogren's syndrome and Behcet's disease.

[0043] Preferably, said hypersensitivity immune disorder is selected from the group consisting of allergies, sneezing, wheezing, asthma, atopic dermatitis and urticaria (hives).

[0044] Preferably, the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, psoriasis, Hashimoto's thyroiditis, systemic lupus erythematosus and acute inflammatory bowel disease.

[0045] The immunosuppressant compositions and pharmaceutical compositions containing the active ingredients of the present invention can be formulated into various forms, such as oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injections of sterile injection solutions, according to the usual methods, depending on the intended purpose of use, and can be administered orally or via various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

[0046] Such pharmaceutical compositions may further comprise a carrier, excipient, or diluent, and examples of suitable carriers, excipients, or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the pharmaceutical composition of the present invention may further comprise a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, an emulsifier, a preservative, etc.

[0047] In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are formulated by mixing the immunosuppressive composition and pharmaceutical composition with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0048] In a preferred embodiment, oral liquid preparations include suspensions, liquid preparations, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents.

[0049] In a preferred embodiment, parenteral administration preparations include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Injections may include conventional additives such as solubilizers, isotonicity adjusting agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

[0050] The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors including the type and severity of the patient's disease, the activity of the drug, its sensitivity, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors known in the medical field. The immunosuppressive composition and pharmaceutical composition of the present invention can be administered as a single therapeutic agent, in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking the above factors into consideration, it is important to administer an amount that provides maximum efficacy at the minimum dose without side effects, which can be easily determined by one skilled in the art.

[0051] In a preferred embodiment, the effective amount of the active ingredient in the immunosuppressive composition and pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, but generally ranges from 1 mg to 1,000 mg per kg of body weight, preferably 5 mg to 100 mg, and more preferably 10 mg to 20 mg, which may be administered daily or every other day, or in one to three divided doses per day. However, the dosage may increase or decrease depending on the route of administration, severity of the disease, sex, weight, age, etc., and the above dosage does not in any way limit the scope of the present invention.

[0052] The immunosuppressive and pharmaceutical compositions of the present invention can be administered to a subject by a variety of routes, including oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine, dura, or intracerebroventricular injection, although any mode of administration is contemplated.

[0053] In the present invention, "administration" means providing a predetermined substance to a patient by any appropriate method, and the immunosuppressive composition and pharmaceutical composition of the present invention can be administered orally or parenterally via any common route as long as it can reach the target tissue. Furthermore, the immunosuppressive composition and pharmaceutical composition of the present invention can be administered using any device that can deliver the active ingredient to the target cell.

[0054] In the present invention, the term "subject" is not particularly limited to, but includes, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, and preferably refers to mammals, more preferably humans.

[0055] The present invention also provides a health functional food containing 3,6-anhydro-L-galactose for preventing or ameliorating immune-mediated diseases.

[0056] Preferably, the immune disease is selected from the group consisting of hypersensitivity immune diseases, autoimmune diseases, immune rejection, graft-versus-host disease, Sjogren's syndrome and Behcet's disease.

[0057] Preferably, said hypersensitivity immune disorder is selected from the group consisting of allergies, sneezing, wheezing, asthma, atopic dermatitis and urticaria (hives).

[0058] Preferably, the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, psoriasis, Hashimoto's thyroiditis, systemic lupus erythematosus and acute inflammatory bowel disease.

[0059] The functional health food of the present invention can be used in a variety of foods and beverages that are effective in preventing or improving immune diseases. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, and dietary supplements, and can be used in the form of powder, granules, tablets, capsules, or beverages.

[0060] The active ingredient of the present invention can generally be added in an amount of 0.001 to 10% by weight based on the total weight of the food, and in the case of a health drink composition, it can be added in an amount of 0.001 to 10 g, preferably 0.01 to 1 g, based on 100 ml.

[0061] In addition to containing the above-mentioned compounds as essential ingredients in the indicated proportions, the health functional food of the present invention may also contain nutrient-acceptable food supplements, such as natural carbohydrates and various flavoring agents, as additional ingredients.

[0062] Examples of the natural carbohydrates include common sugars such as monosaccharides (glucose, fructose, etc.), disaccharides (maltose, sucrose, etc.) and polysaccharides (dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, erythritol, etc.

[0063] The flavoring agent may be natural flavoring agents (thaumatin, stevia extract (rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.)). The natural carbohydrate is generally used in an amount of about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the functional health food of the present invention.

[0064] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, minerals, flavorings such as synthetic flavorings and natural flavorings, coloring agents and enhancers, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc.

[0065] In addition, the health functional food of the present invention may also contain fruit pulp used in the production of natural fruit juice, fruit juice drinks, vegetable drinks, etc. These ingredients can be used alone or in combination. The proportion of these additives is generally selected from the range of 10 to about 50 parts by weight per 100 parts by weight of the active ingredient of the present invention.

[0066] The present invention also provides a functional cosmetic composition for preventing or improving hypersensitive skin diseases, which contains 3,6-anhydro-L-galactose as an active ingredient.

[0067] The hypersensitive skin disease is preferably selected from the group consisting of photodermatitis, contact dermatitis and atopic dermatitis.

[0068] The active ingredient of the cosmetic composition of the present invention may be contained in an amount of 0.001 to 1 wt %, preferably 0.01 to 0.1 wt %, and more preferably 0.05 to 0.5 wt %, based on the total weight of the composition. Within this content range, appropriate formulation stability can be ensured and the desired antioxidant effect can be expected.

[0069] In addition to the active ingredient of the present invention, the composition of the present invention may further contain known natural extracts or other ingredients for the effects of antioxidants, preventing skin aging, and promoting skin regeneration, as long as they do not inhibit the effective activity of the present invention.

[0070] Furthermore, the composition can be molded into a specific dosage form by incorporating ingredients commonly added to cosmetic compositions, such as adjuvants and carriers commonly used in the fields of cosmetics or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, moisturizing agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, etc.

[0071] The cosmetic composition of the present invention can be prepared in any formulation commonly used in the art, including, but not limited to, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, pack, soap, surfactant-containing cleanser, oil, spray, etc. More specifically, it can be prepared in the form of a softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, powder foundation, emulsion foundation, wax foundation, cleansing cream, cleansing foam, cleansing water, pack, spray, powder, compact, lip gloss, lipstick, eyeshadow, shampoo, rinse, etc.

[0072] When the formulation of the present invention is a paste, cream or gel, the carrier component may be an animal oil, a vegetable oil, a wax, a paraffin, a starch, tragacanth, a cellulose derivative, a polyethylene glycol, a silicone, a bentonite, silica, talc or zinc oxide.

[0073] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as a carrier component, and particularly when it is a spray, it may further contain a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

[0074] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan fatty acid esters.

[0075] When the dosage form of the present invention is a suspension, the carrier component may be a liquid diluent such as water, ethanol, or propylene glycol; a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester; microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant.

[0076] When the dosage form of the present invention is a surfactant-containing cleanser, the carrier component may be a fatty alcohol sulfate, a fatty alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyl taurate, a sarcosinate, a fatty acid amide ether sulfate, an alkylamidobetaine, a fatty alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a lanolin derivative, or an ethoxylated glycerol fatty acid ester.

[0077] The present invention also provides a reagent composition containing L-AHG for inhibiting the activation, proliferation, or differentiation of T cells.

[0078] Preferably, the T cells are CD4 T cells.

[0079] Said activation refers to the activation of resting T cells.

[0080] The term "proliferation" refers to an increase in the number of T cells as they progress through the cell cycle.

[0081] The differentiation means differentiation into Th1 cells and Th2 cells.

[0082] The composition can suppress the activation, proliferation and differentiation of T cells by applying it to T cells under ex vivo conditions, preferably in vitro conditions.

[0083] The present invention also provides a method for inhibiting the activation, proliferation, or differentiation of T cells, preferably CD4 T cells, which comprises treating T cells with the L-AHG of the present invention under ex vivo conditions.

[0084] The in vitro conditions refer to a state in which T cells are physically completely separated from the living body, and are preferably in vitro conditions.

[0085] The T cells can be cells isolated from the blood of a subject, cells subcultured therefrom, or a cell line.

[0086] The treatment can be carried out, for example, by adding L-AHG to a medium containing T cells in a cell culture dish, and the treatment concentration is preferably 1.0 to 1,000 μg / mL, and more preferably 12.5 to 400 μg / mL.

[0087] The inhibition of differentiation may be inhibition of differentiation into Th1 and Th2 cells, which may reduce the secretion of interferon-γ, interleukin-2, interleukin-4, or interleukin-13.

[0088] The present invention will be described in more detail below with reference to specific examples. The following examples are intended to illustrate preferred embodiments of the present invention, and the scope of the present invention should not be construed as being limited by the details described in the following examples. [Example]

[0089] 1. Materials and Methods 1.1.Animals Five- to six-week-old C57BL / 6J male mice were purchased from Orient Co. (322, Kalmaji-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do, Korea) and used in the experiments while maintained under specific pathogen-free conditions at the Kyungpook National University-Laboratory Animal Resources Center (680, Gukjeol-ro, Jung-gu, Daegu-si, Korea).

[0090] 1.2. Chemicals, Antibodies, Reagents and Media Mouse recombinant IL-2 (rIL-2) was purchased from Cambridge Bioscience (Cambridge, UK). Rabbit anti-mouse IgG was purchased from Jackson ImmunoResearch (West Grove, PA, USA), and anti-CD3 was purchased from BD Pharmingen (Sandiego, CA, USA). Goat anti-mouse IL-2 antibody to neutralize IL-2 activity was purchased from R&D Systems (Minneapolis, MN, USA), and rat IL-2R neutralizing antibody was purchased from Abcam (Cambridge, UK). The ECL Western blotting kit was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Immobilon-P membranes for Western analysis were purchased from EMD Millipore Corporation (Temecula, CA, USA). The microtubule depolymerizing agent nocodazole and all other electrophoresis reagents were purchased from Sigma Chemical (St. Louis, MO, USA).

[0091] To obtain the agarose hydrolysis products, neoagarotetraose (NA4) and neoagarohexaose (NA6), 9.0% agarose dissolved in water by heat treatment was hydrolyzed with GH16B β-agarase (1.6 μg / mL) under optimal reaction conditions (1 mM MnCl2, 10 mM TCEP, 50 mM Tris-HCl, pH 7.0, 50°C) for 14 h with continuous magnetic stirring to produce NA4 and NA6. The resulting NA4 / NA6 mixture was fractionated and purified by size-exclusion chromatography on a Sephadex G-15 column. To obtain neoagarobiose (NA2) from agarose, 9% agarose was first hydrolyzed with GH16B β-agarase at 50°C to generate neoagarooligosaccharides (NAOS) containing NA4 / NA6. The temperature was then lowered to 35°C, and GH50A β-agarase (20 μg / mL) was added. The reaction mixture was incubated for 14 hours to generate NA2. NA2 in the reaction product was fractionated and purified by size-exclusion chromatography using a Sephadex G-15 column. To obtain L-AHG, a monomer of agarose, 9% agarose was first hydrolyzed with GH16B β-agarase at 50°C to form NAOS, which contain NA4 and NA6. The temperature was then lowered to 35°C, and GH50A β-agarase (20 μg / mL) and GH117A α-NABH (60 μg / mL) were added simultaneously. The resulting mixture was then incubated for 14 hours, yielding a mixture of L-AHG and D-galactose monomers. To purify the L-AHG in this mixture, the mixture was fractionated by size-exclusion chromatography using a Sephadex G-10 column.

[0092] The medium used for activating and culturing T lymphocytes was RPMI 1640 containing 10% FBS (HyClone, Logan, Utah, USA), 20 mM HEPES, 50 μM β-mercaptoethanol, and 100 μg / mL gentamicin, designated as complete medium.

[0093] 1.3. Isolation, activation and differentiation of resting (G0) T cells 3Measurement of [H]thymidine incorporation Resting (G0) T lymphocytes were isolated from the spleens of 4- to 5-month-old C57BL / 6J male mice by negative selection using a T cell purification column kit (R&D Systems, Minneapolis, MN, USA). To activate G0 T cells, we modified the method described by Kim et al. In this case, we pretreated the surface of the plastic culture vessel with rabbit anti-hamster IgG antibody (20 μg / mL) to facilitate coating with anti-CD3 (2.0 μg / mL) and anti-CD28 (2.0 μg / mL).

[0094] To assess the proliferation of T cells activated by immobilized anti-CD3 / anti-CD28, 1 × 10 cells were added to each well of a 96-well plate coated with anti-CD3 (2.0 μg / mL) and anti-CD28 (2.0 μg / mL). 5 Activation was initiated by adding 100 G0 T cells and various concentrations of L-AHG together with 200 μL of RPMI 1640 complete medium and culturing them. 29 hours after activation, 0.25 μCi of [ 3 H]thymidine was added and cultured for an additional 12 hours, and the [ 3 The amount of [H]thymidine was quantified using a liquid scintillation counter.

[0095] To analyze the cell cycle of T cells activated by immobilized anti-CD3 / anti-CD28 or to analyze cytokine concentrations in the culture supernatant, 3 × 10 cells were placed in each well of a 35 mm plate coated with anti-CD3 (2.0 μg / mL) and anti-CD28 (2.0 μg / mL). 6 Activation of G0 T cells was initiated by adding 1000 cells and various concentrations of L-AHG to 2 ml of RPMI 1640 complete medium and culturing them. 30 hours after activation, the culture was stopped and the cells and culture supernatant were collected by centrifugation.

[0096] Under the same culture conditions, the first cell cycle progression initiated by activation of G0 T cells was arrested at prometaphase by nocodazole treatment. After activation for 20 hours, nocodazole was added at a concentration of 0.2 μg / mL and the cells were cultured for an additional 10 hours. After the culture was completed, the culture was centrifuged to collect the cells and culture supernatant.

[0097] 1.4. T cell surface staining using fluorescently labeled anti-CD4 / anti-CD8 antibodies and intracellular DNA staining using DAPI Resting (G0) T cells isolated from mouse spleen cells using a T cell purification column kit (R&D) and T cells obtained by activating G0 T cells for a certain period with immobilized anti-CD3 / anti-CD28 were stained with anti-CD3, anti-CD4, anti-CD8, and DAPI. Approximately 5 × 10 5 The cells were suspended in 100 μL of 1X PBS / 1% FBS solution, and 0.5 μL each of anti-CD3, anti-CD4, and anti-CD8 antibodies were added. The cells were incubated at 4°C for 15 minutes and then washed twice with 1X PBS / 1% FBS. The cells were then fixed with 1% paraformaldehyde for 15 minutes at 4°C. The fixed cells were then washed twice with 1X PBS and stained with 10 μM DAPI for 15 minutes. The stained cells were analyzed using a flow cytometer (Attune NxT flow cytometer, Thermo Fisher Scientific, Waltham, MA, USA) to determine the ratios of CD3 T cells, CD4 T cells, and CD8 T cells, as well as their cell cycle distribution, based on the increase in blue fluorescence due to the amount of DAPI bound to intracellular DNA.

[0098] 1.5. Enzyme-linked immunosorbent assay (ELISA) T cell culture supernatants were collected and analyzed for IFN-γ, IL-2, IL-4, and IL-13 concentrations using an ELISA kit (Mouse Uncoated Elisa Kit, Invitrogen) according to the manufacturer's instructions. Briefly, 100 μl of a diluted capture antibody solution was added to each well of a 96-well plate and incubated at 4°C for 16 hours, followed by three washes with washing buffer. 200 μl of blocking solution was added to each well, followed by blocking at 25°C for 1 hour and washing once with washing buffer. Next, 100 μl of appropriately diluted standard samples and culture supernatants were added to each well and incubated for 2 hours at 25°C. After washing three times with washing buffer, 100 μl of secondary antibody (detection antibody) solution was added to each well and incubated for 1 hour at 25°C. After washing three times with washing buffer, 100 μl of avidin-HRP solution was added to each well and incubated at 25°C for 30 minutes. After washing five times with washing buffer, 100 μl of substrate solution was added to each well and incubated at 25°C for 15 minutes in the dark. The reaction was terminated by adding 100 μl of stop solution to each well, and the absorbance was measured at wavelengths of 450 nm and 570 nm using a plate reader (Varioskan Lux Multimode Microplate Reader, Thermo Fisher Scientific). The absorbance measured at 450 nm minus the absorbance measured at 570 nm was compared with a standard curve to calculate cytokine concentrations in the culture supernatant. Serially diluted recombinant IFN-γ, IL-2, IL-4, and IL-13 were used as standards to generate standard curves.

[0099] 2.Results 2.1. Immobilized anti-CD3 / anti-CD28 stimulated G0 T cells [ 3 Effect of enzymatic hydrolysates of agarose on [H]thymidine incorporation Resting (G0) lymphocytes were isolated from mouse spleens by Percoll gradient centrifugation. G0 T lymphocytes were isolated from G0 lymphocytes by negative selection using a T cell purification column kit (R&D Systems, Minneapolis, MN, USA). Surface staining of G0 T cells isolated using the T cell purification column with fluorescently labeled anti-CD3, anti-CD4, and anti-CD8 antibodies confirmed that 96% of the cells were CD3+ T cells (Figure 1). Furthermore, 50.6% of these CD3+ T cells were confirmed to be CD4+ T (Th) cells, and 43.8% were confirmed to be CD8+ T (Tc) cells.

[0100] Resting T cells were activated in 96-well plates by stimulation with immobilized anti-CD3 / CD28. NA4 and NA6, obtained by hydrolyzing agarose with GH16B β-agarase, NA2, obtained by hydrolyzing agarose with GH16B β-agarase followed by the addition of GH50B β-agarase, and L-AHG, obtained by hydrolyzing agarose with GH16B β-agarase followed by the simultaneous addition of GH50B β-agarase and GH117A α-NABH, were added at concentrations of 12.5–400 μg / mL. The effects of these agarose degradation products (L-AHG, NA2, NA4, and NA6) on cell cycle progression and proliferation upon activation of resting T cells were examined.

[0101] First, resting T cells (1 x 10) were cultured in a 96-well plate. 5 G0 T cells (per well) were activated by stimulation with immobilized anti-CD3 / anti-CD28 in the presence of various concentrations (0, 12.5, 25, 50, 100, 200, and 400 μg / mL) of L-AHG, NA2, NA4, or NA6. At 29 hours post-activation, each well received 0.25 μCi of [ 3 H]thymidine was added and the cells were further cultured for 12 hours. During this additional culture period, T cell proliferation occurred, and [ 3The amount of [H] thymidine was quantified using a liquid scintillation counter to determine the degree of T cell proliferation that occurred in each well.

[0102] As a result, as shown in Figure 2, 12.5 μg / ml of L-AHG had little effect on the proliferation of activated T cells, [ 3 The level of [H]thymidine incorporation was almost the same as that in the control group without L-AHG. On the other hand, in the presence of 25-400 μg / ml of L-AHG, [H]thymidine incorporation increased in a concentration-dependent manner. 3] In particular, in the presence of 100 μg / mL of L-AHG, [ 3 H]thymidine incorporation was reduced to approximately 22% of that in the control group without L-AHG, and at L-AHG concentrations of 200–400 μg / mL, [ 3] However, under the same conditions, agarose degradation products such as NA2, NA4, and NA6 significantly reduced the [H]thymidine incorporation of activated T cells at concentrations of 25–400 μg / mL. 3 3H]thymidine incorporation was not significantly affected.

[0103] Therefore, these results confirmed that, among the enzymatic hydrolysis products of agarose examined, only L-AHG has the physiological activity to significantly suppress the proliferation of activated T cells.

[0104] Recently, various physiological activities of agar hydrolysates (NA2, L-AHG, NAOS, and AOS) have been reported. These include antioxidant, prebiotic, antitumor, anti-inflammatory, antidiabetic, anti-obesity, skin moisturizing, and skin whitening activities. Furthermore, oral administration of NA4 to mouse models has been shown to protect against fatigue and liver damage induced by strenuous exercise by regulating the intestinal microbiota (gut flora, microbiota). However, the inhibitory effect of L-AHG on activated T cell proliferation is a novel physiological activity that has not been reported previously.

[0105] 2.2. Effect of L-AHG on cell cycle progression of G0 T cells stimulated with immobilized anti-CD3 / anti-CD28

[0106] T lymphocytes that make up the immune system are divided into CD4 T (Th) lymphocytes and CD8 T (T C ) lymphocytes and are broadly divided into two types. 3 We investigated whether the growth-suppressive activity of L-AHG, which was confirmed by [H]thymidine incorporation assay, through inhibition of DNA synthesis in activated T cells, occurs in both CD4+ Th cells and CD8+ Tc cells, or whether it is exclusive to either of these two types of T cells.

[0107] To this end, resting T cells were activated by stimulation with immobilized anti-CD3 / anti-CD28. At 20 h after activation, when they had exited G0 (resting phase), entered G1, and reached the onset of S phase, the microtubule-depolymerizing agent nocodazole (a cell cycle arresting agent that arrests eukaryotic cell cycle progression at prometaphase of mitosis) was added and cultured for an additional 10 h. This unique experimental method allowed for the initial cell cycle progression initiated by activation of resting T cells to progress through G1, S, and G2 phases, before being arrested at prometaphase of M due to microtubule damage induced by nocodazole. These cells were then harvested, stained with fluorescently labeled anti-CD4 and anti-CD8 antibodies, and the DNA fluorescent dye DAPI, and analyzed by flow cytometry. As a result, we sought to clarify how L-AHG affects the initial cell cycle progression (G1, S, and G2 / M phases) initiated by activation of resting CD4 T cells and resting CD8 T cells.

[0108] As shown in Figures 3A and 3B, flow cytometry analysis of stained cells revealed that when resting T cells were activated by stimulation with immobilized anti-CD28 / anti-CD28, 4.4%, 8.8%, and 77.9% of CD8 T cells were distributed in G1, S, and G2 / M phases, respectively, whereas 9.9%, 35.4%, and 49.9% of CD4 T cells were distributed in G1, S, and G2 / M phases, respectively. That is, 30 hours after activation, only about 13.2% of CD8 T cells remained in G1+S phase without entering G2 / M phase, whereas about 45.3% of CD4 T cells remained in G1+S phase. These results indicate that among resting T cells, CD8 T cells progress through the cell cycle relatively rapidly and smoothly after activation with immobilized anti-CD28 / anti-CD28 compared with CD4 T cells.

[0109] The initial cell cycle progression upon activation of resting CD4 T cells and CD8 T cells, as confirmed above, was significantly and differentially affected by the addition of L-AHG at concentrations of 25–100 μg / ml. Specifically, after activation with immobilized anti-CD3 / anti-CD28, the percentage of resting CD4 T cells remaining in G1 and S phases without entering G2 / M increased in an L-AHG concentration-dependent manner. In particular, with the addition of 100 μg / ml L-AHG, the percentage of cells in G1 phase was 35.4%, the percentage of cells in S phase was 54.6%, while the percentage of cells in G / M phase was only approximately 8.8%, demonstrating that L-AHG significantly inhibited cell cycle progression. On the other hand, cell cycle progression after activation of resting CD8 T cells was hardly affected by the addition of 25–50 μg / mL L-AHG. When 100 μg / mL L-AHG was added, the change in the proportion of G1-phase cells was negligible, but the proportion of S-phase cells increased to 46.6% and the proportion of G2 / M-phase cells was 41.8%.

[0110] These study results showed that the addition of 25-100 μg / mL L-AHG inhibited cell cycle progression upon activation of resting CD4 T cells, primarily in the G1 and S phases, in a concentration-dependent manner. In particular, the addition of 100 μg / mL L-AHG resulted in approximately 90.0% of cells remaining in the G1+S phase, unable to enter the G2 / M phase. Under the same conditions, the addition of 25-50 μg / mL L-AHG did not significantly affect cell cycle progression upon activation of resting CD8 T cells. The addition of 100 μg / mL L-AHG did not affect G1 phase progression, but did slightly inhibit S phase progression. Thus, although the presence of L-AHG (25-100 μg / mL) inhibited cell cycle progression upon activation of resting CD4 T cells and CD8 T cells in different ways, L-AHG did not induce apoptotic cell death (based on the percentage of apoptotic sub-G1 cells) in these cells.

[0111] On the other hand, when resting T cells were stimulated and activated with immobilized anti-CD3 / anti-CD28 under the same conditions and then cultured for 30 hours without the addition of nocodazole, the inhibitory effect of L-AHG on cell cycle progression associated with activation of resting CD4 T cells and CD8 T cells was not readily confirmed by flow cytometry (Figures 4A and 4B). This is likely because the cell cycle progression initiated by activation of resting CD4 T cells or CD8 T cells is inhibited in some cells by the presence of L-AHG, whereas the remaining cells are not inhibited by L-AHG and complete the initial cell cycle progression, then progress through the G1, S, and G2 phases again. Finally, cells whose cell cycle progression is inhibited by L-AHG coexist with cells that progress through the cell cycle even in the presence of L-AHG.

[0112] Taken together, these results suggest that the inhibitory effect of L-AHG on activated T cell proliferation is primarily directed at CD4 T cells rather than CD8 T cells, and is mediated by inhibition of cell cycle progression in the G1 and S phases.

[0113] 2.3. Effect of L-AHG on cytokine production in G0 T cells stimulated with immobilized anti-CD3 / anti-CD28 Unlike CD8 T cells, CD4 T cells differentiate into several functionally distinct effector CD4 T cell subtypes that mediate various immune responses. Known effector CD4 T cell subtypes include Th1, Th2, Th17, Tfh, and Treg cells. These subtypes can be distinguished from each other based on the characteristic combinations of cytokines they secrete.

[0114] Th1 cells produce IFN-γ and promote macrophage activation, allowing for more efficient destruction of microorganisms that can survive or multiply within the macrophage. Th2 cells produce cytokines such as IL-4, IL-5, and IL-13, which are involved in the recruitment and activation of eosinophils (IL-5) and mast cells / basophils (IL-4) and enhance barrier immunity at mucosal surfaces (IL-13). They contribute to the suppression of parasitic infections by promoting defense responses primarily via IgE. In particular, IL-4 and IL-13 are required for class switching of IgE-producing B cells. Thus, IgE produced by B cells with the help of Th2 cells can also cause hypersensitivity reactions such as allergies and asthma. Th17 cells secrete IL-17A, IL-17F, and IL-22, inducing local epithelial and stromal cells to produce chemokines that recruit neutrophils to the site of infection. This amplifies the neutrophil response, which eliminates pathogens such as extracellular bacteria and fungi. Furthermore, it activates epithelial cells in mucosal epithelia and skin to produce antimicrobial peptides that attack bacteria. Tfh cells are a subtype of effector CD4 T cells that assist B cells in lymphoid tissues. Tfh cells interact with naive B cells through mechanisms such as trafficking to B cell follicles and linked recognition of antigens, promoting germinal center responses and supporting B cell antibody production and class switching. Tfh cells produce large amounts of the cytokine IL-21, which supports B cell proliferation and differentiation into antibody-producing plasma cells. Treg cells limit immune responses by normally suppressing T cell activity and the activity of innate immune cells through the production of TGF-β and IL-10, helping to prevent the development of autoimmunity during immune responses.

[0115] The stability of effector CD4 T cell subtypes differs. While naive CD4 T cells have diverse potential, Th1 and Th2 cells exhibit a relatively stable basal state and are highly resistant to conversion to other subtypes. In contrast, iTreg and Th17 cells are less stable and can be converted to other subtypes by cytokines that primarily influence them. iTreg cells can be converted to Th17 cells in response to IL-6 and IL-1, and to Th1 cells in response to IL-12. Th17 cells can also be converted to Th1 cells in response to IL-12. It is understood that the conversion of iTreg cells to Th17 cells and Th17 cells to Th1 cells is unidirectional or irreversible.

[0116] Based on the differentiated immune-related functions of effector CD4 T cell subtypes, it is expected that the use of immunosuppressants that exert specific suppressive abilities targeting Th1 and Th2 effector CD4 T cell subtypes will be more effective in controlling inappropriate immune responses to required levels in patients with hypersensitivity reactions, autoimmune diseases, and organ transplant patients, including those receiving stem cell or bone marrow transplants.

[0117] L-AHG (25-100 μg / mL) significantly suppressed proliferation of activated CD4 T cells by inhibiting cell cycle progression, primarily in the G1 and S phases, in a concentration-dependent manner. Resting T cells were activated under the same conditions by stimulation with immobilized anti-CD3 / anti-CD28 antibodies 30 hours later, and the concentrations of Th1 signature cytokines (IFN-γ, IL-2) and Th2 signature cytokines (IL-4, IL-13) characteristically secreted by Th1 and Th2 cells, respectively, were measured by ELISA.

[0118] The results showed that the addition of L-AHG (25, 50, and 100 μg / mL) significantly reduced the levels of IL-2 and IFN-γ secreted by Th1 subtype cells, as well as IL-4 and IL-13 secreted by Th2 subtype cells, in a concentration-dependent manner (Figures 5A and 5B). Interestingly, the levels of Th2 signature cytokines were more significantly reduced than those of Th1 signature cytokines. In particular, in the presence of 25 μg / mL of L-AHG, the levels of IFN-γ were barely affected, whereas the levels of IL-4 were significantly reduced to 38.5% of the levels in the L-AHG-untreated control group.

[0119] In conclusion, when stimulation with immobilized anti-CD3 / anti-CD28 activates resting Th cells to initiate the cell cycle, leading to clonal expansion and differentiation into effector Th cell subtypes, the presence of L-AHG can suppress differentiation into Th1 and Th2 subtypes by inhibiting cell cycle progression of CD4 T cells. 、 This suggests that differentiation into Th2 subtype can be more strongly suppressed than that into Th1 subtype. [Accession number]

[0120] [Microorganism deposit] Depository institution: Korea Institute of Bioscience and Biotechnology (KCTC: Korean Collection for Type Cultures) Accession number: KCTC13629BP Date of acceptance: 20180827

Claims

1. An immunosuppressive composition containing 3,6-anhydro-L-galactose as an active ingredient.

2. The immunosuppressive composition according to claim 1, which suppresses the activation, proliferation or differentiation of CD4 T cells.

3. The immunosuppressive composition according to claim 2, characterized in that it blocks progression of the cell cycle in the G1 or S phase.

4. The immunosuppressive composition according to claim 1, characterized in that it suppresses differentiation into Th1 cells or Th2 cells.

5. The immunosuppressive composition according to claim 4, which suppresses the production of interferon-γ, interleukin-2, interleukin-4 or interleukin-13.

6. A pharmaceutical composition for preventing or treating immune diseases, which comprises 3,6-anhydro-L-galactose as an active ingredient.

7. 7. The pharmaceutical composition according to claim 6, wherein the immune disease is selected from the group consisting of hypersensitivity immune diseases, autoimmune diseases, immune rejection reactions, graft-versus-host disease, Sjogren's syndrome and Behcet's disease.

8. 8. The pharmaceutical composition according to claim 7, wherein the hypersensitivity immune disease is selected from the group consisting of allergies, sneezing, wheezing, asthma, atopic dermatitis and urticaria (hives).

9. 8. The pharmaceutical composition according to claim 7, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, psoriasis, Hashimoto's thyroiditis, systemic lupus erythematosus, and acute inflammatory bowel disease.

10. A functional health food containing 3,6-anhydro-L-galactose for preventing or improving immune diseases.

11. The health functional food according to claim 10, wherein the immune disease is selected from the group consisting of hypersensitivity immune disease, autoimmune disease, immune rejection, graft-versus-host disease, Sjogren's syndrome, and Behcet's disease.

12. The health functional food according to claim 11, wherein the hypersensitivity immune disease is selected from the group consisting of allergies, sneezing, wheezing, asthma, atopic dermatitis, and urticaria (hives).

13. The health functional food according to claim 11, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, psoriasis, Hashimoto's thyroiditis, systemic lupus erythematosus, and acute inflammatory bowel disease.

14. A functional cosmetic composition for preventing or improving hypersensitive skin diseases, which contains 3,6-anhydro-L-galactose as an active ingredient.

15. The functional cosmetic composition according to claim 14, wherein the hypersensitive skin disease is selected from the group consisting of photodermatitis, contact dermatitis, and atopic dermatitis.

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