KR102404844B1 - Composition for preventing or treating Sjogren's syndrome comprising sodium butyrate - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating Sjogren's syndrome comprising sodium butyrate or a pharmaceutically acceptable salt thereof as an active ingredient, wherein sodium butyrate inhibits HDAC8, thereby reducing the activity of Th17 cells It can be usefully used as a treatment for Sjogren's syndrome by increasing the activity of Treg cells.

Description

A composition for preventing or treating Sjogren's syndrome comprising sodium butyrate, comprising sodium butyrate

The present invention relates to a composition for preventing and treating Sjogren's syndrome, comprising sodium butyrate, and various applications thereof.

Sjogren's syndrome is a systemic chronic inflammatory disease that affects the lacrimal and salivary glands. Sjogren's syndrome is the most prevalent autoimmune disease affecting 1-3% of the world's population, with 4,000,000 patients in the United States. More than 90% of patients with Sjogren's syndrome are women, and the disease may be accompanied by a variety of other autoimmune phenomena. The physiological cause of Sjogren's syndrome is not precisely understood, but two distinct phenomena are believed to occur. There are two distinct periods: the initiation stage, when autoreactive lymphocyte activation is induced, and the effect of reducing the amount of tears and saliva, respectively, by infiltrating and accumulating pathogenic lymphocytes in the lacrimal and salivary glands.

In other words, it is divided into primary Sjogren's syndrome and secondary Sjogren's syndrome according to the stage of symptom progression. Primary Sjogren's syndrome mainly presents with dry eyes, oral cavity, and skin. As it progresses further, the eyes sting, tears do not come out, and there is a feeling of redness or fatigue. However, if this state is left as it is, it leads to damage to the cornea and conjunctiva, and the lack of saliva in the mouth causes a lot of tartar to occur, and tooth decay and periodontitis often occur. In addition, the secretion of sweat glands and sebaceous glands of the skin is also reduced, so that the skin becomes dry.

Secondary Sjogren's syndrome is also accompanied by rheumatoid arthritis, lupus, systemic sclerosis, and dermatomyositis. As the disease progresses, damage to the digestive endocrine system such as the respiratory system, stomach and intestines, and nervous system such as kidney and heart. In addition, it is known that about 5% of patients with Sjogren's syndrome develop lymphoma, and it is known that patients with Sjogren's syndrome have a mortality rate of about 2.7 times higher than that of the general public.

On the other hand, there is still no fundamental treatment for Sjögren's syndrome, and all it takes is ancillary drug treatment to alleviate the progression of symptoms in each affected area and prevent complications. An artificial salivary agent should be used, and pilocarpine may be administered. In addition, the dry skin should be sufficiently moisturized and non-steroidal anti-inflammatory drugs are used to relieve inflammation. Therefore, there is an urgent need to develop a novel therapeutic agent for Sjogren's syndrome that is more fundamental and effective.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating Sjogren's syndrome comprising sodium butyrate as an active ingredient.

Another object of the present invention is to provide a food composition for preventing and improving Sjogren's syndrome comprising sodium butyrate as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating Sjogren's syndrome comprising sodium butyrate as an active ingredient.

In addition, the present invention provides a food composition for preventing and improving Sjogren's syndrome comprising sodium butyrate as an active ingredient.

Sodium butyrate according to the present invention can be usefully used as a therapeutic agent for Sjogren's syndrome by inhibiting HDAC8 to reduce the activity of Th17 cells and increase the activity of Treg cells.

1 is after injection of sodium butyrate into an animal model of Sjogren's syndrome, salivation capacity (FIG. 1a, flow rate = saliva/weight value per minute), saliva volume (FIG. 1B), weight (FIG. 1C) and IgG antibody amount (FIG. 1d) is the measurement result.
2 is a result of confirming whether or not to maintain cell shape and infiltrate inflammatory cells in the lacrimal and salivary glands after sodium butyrate is injected into the Sjogren's syndrome animal model.
3 is after injection of sodium butyrate into an animal model of Sjogren's syndrome, the spleen (Spleen (SP)), salivary gland (SG), lacrimal gland (LG), the expression level of IL-10 was measured. Results (black marks = 19 weeks of age (19w); and red marks = 21 weeks of age (21w)).
Figure 4A is a confocal microscopy of Th17 cells (CD4:red; and IL-17:green) and Treg cells (CD4:red; Foxp3:green; and CD25:blue) cells in spleen tissue in an animal model of Sjogren's syndrome in which sodium butyrate is administered. This is the result confirmed through
FIG. 4B shows CD4 ⁺ CD62L ^high CD44 ^low -naive CD4 ⁺ T cells isolated from spleen tissue in a mouse model of Sjogren's syndrome and cultured with 100 μM or 500 μM sodium butyrate as Th17 differentiation conditions, and Th17 cells and Treg cells after 72 hours. This is the result confirmed through Flow Cytometry.
FIG. 4C shows that CD4 ⁺ CD62L ^high CD44 ^low -naive CD4 ⁺ T cells were isolated from the spleen tissue of a mouse model of Sjogren's syndrome and cultured with sodium butyrate under Th17 differentiation conditions. After 48 hours, the mRNA of Foxp3 and CTLA4, markers of Treg cells. It is the result of confirming the expression level through real-time PCR.
5A shows the results of culturing naive CD4 ⁺ T cells under the Th17 differentiation condition, and confirming the mRNA expression level according to the histone deacetylase (HDAC) subtype through real time-PCR 24 hours later.
Figure 5B shows EL4 cells (Mus musculus lymphoma, ATCC® TIB39™) were cultured with anti-CD3, IL-6 and sodium butyrate, followed by immunoprecipitation (IP) using an anti-ERRα (estrogen-related receptor alpha) antibody. This is the result of performing IB (immuniblotting) using an anti-ERRα antibody and an anti-acetyl lysine antibody.
5C is a result of culturing naive CD4 ⁺ T cells under Th17 differentiation conditions, and confirming the expression of CPT IA and NR1D1 through confocal microscopy 72 hours later.
5D shows the results of culturing naive CD4 ⁺ T cells under Th17 differentiation conditions, and confirming the mRNA expression levels of CPT IA and NR1D1 through real time-PCR 24 hours later.
Figure 5E shows the results of culturing naive CD4 ⁺ T cells in Th17 differentiation conditions with sodium butyrate, Etomoxir or SR8278 alone or with sodium butyrate, and analyzing Th17 cells and Treg cells through Flow Cytometry after 72 hours.

The present invention is characterized in that it was found that sodium butyrate represented by the following Chemical Formula 1 can be used as a therapeutic agent for Sjogren's syndrome. Sodium butyrate inhibits HDAC8, reduces the activity of Th17 cells, and increases the activity of Treg cells, so it can be used as a treatment for Sjogren's syndrome. The present invention was completed by revealing that it can be used as a therapeutic agent for Sjogren's syndrome by reducing the infiltration of and increasing the expression level of IL-10, an anti-inflammatory cytokine.

Accordingly, the present invention can provide a pharmaceutical composition for the prevention or treatment of Sjogren's syndrome comprising sodium butyrate represented by the following formula (1) as an active ingredient.

[Formula 1]

As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions or similar reactions such as gastrointestinal disorders, dizziness, etc., when administered to humans. In addition, the composition of the present invention may include a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like, and carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above.

That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and as a representative dosage form for parenteral administration, an isotonic aqueous solution or suspension is preferred as an injectable dosage form. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be dissolved in saline or buffer to be formulated for injection. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

The pharmaceutical composition formulated in the above manner may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, the term "effective amount" refers to an amount exhibiting a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected according to the route of administration, administration, age, sex and weight, individual differences and disease state. Preferably, the pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease, but preferably 1 to 10000 μg/kg body weight/day, more preferably 10 to 1000 mg/kg body weight It can be administered repeatedly several times a day at an effective dose of /day. In addition, the composition of the present invention may be administered in combination with a known compound having an effect of preventing, improving or treating immune diseases.

In addition, the present invention can provide a food composition for preventing or improving Sjogren's syndrome comprising sodium butyrate as an active ingredient, and the food composition according to the present invention is a food that can prevent or improve Sjogren's syndrome , For example, it can be easily utilized as a main raw material, auxiliary raw material, food additive, functional food or beverage of food.

As used herein, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state that can be eaten directly through a certain processing process, , refers to all foods, food additives, functional foods and beverages.

Foods to which the composition for food according to the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, and the like. In addition, in the present invention, food includes special nutritional food (eg, formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), breads, health supplements, seasonings Food (eg soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, sweets (eg snacks), candy, chocolate, gum, ice cream, dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, Pickled foods (various kimchi, pickles, etc.), beverages (eg fruit beverages, vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (eg ramen soup, etc.) are included, but not limited thereto. The food, beverage or food additive may be prepared by a conventional manufacturing method.

In addition, the term "functional food" refers to a food group or food composition that has added value to act and express the function of the food for a specific purpose by using physical, biochemical, bioengineering methods, etc. It refers to food that has been designed and processed to sufficiently express the body control functions related to and recovery, etc., and may specifically be a health functional food. The functional food may include a food supplementary additive that is pharmaceutically acceptable, and may further include an appropriate carrier, excipient and diluent commonly used in the manufacture of functional food.

In addition, in the present invention, the "beverage" refers to a generic term for drinking to quench thirst or enjoy taste, and includes functional beverages. The beverage is not particularly limited in other components other than including the composition for improving or preventing Sjogren's syndrome as an essential component in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional components like a conventional beverage. have.

Furthermore, in addition to those described above, the food containing the composition for food for improving or preventing Sjogren's syndrome of the present invention contains various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and natural flavoring agents, coloring agents and fillers. (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. may be contained. , The components may be used independently or in combination. In the food containing the composition for food of the present invention, the amount of the composition according to the present invention may include 0.001% to 90% by weight of the total food weight, preferably 0.1% to 40% by weight. In the case of beverage, it may be included in a ratio of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml, but in the case of long-term intake for health and hygiene purposes or for health control may be less than the above range, and since the active ingredient has no problem in terms of safety, it may be used in an amount greater than the above range, so it is not limited to the above range.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Salivation and maintenance capacity of butyrate in an animal model of Sjogren's syndrome

In order to confirm the effect of sodium butyrate for the treatment of Sjogren's syndrome, the present inventors injected sodium buryrate (Sigma-Aldrich) into a non-obese diabetic (NOD) mouse, an animal model of Sjogren's syndrome, and then Salivation ability and secretion maintenance capacity, which are indicators of the onset of Sjogren's syndrome, were investigated. A control (vehicle) or 1 g/kg of sodium butyrate was injected into NOD/ShiLtJ mice three times a week by intraperitoneal injection.

The amount of saliva was measured by collecting saliva after administering 0.1 mg of pilocarpine. As a result, in the group treated with sodium butyrate, compared to the control group (vehicle), it was confirmed that salivation capacity (FIG. 1a, measured by flow rate, which is a saliva/weight value per minute) and saliva volume (FIG. 1b, saliva per minute) were significantly increased. did It was confirmed that there was no significant difference in mouse weight (Fig. 1c).

In addition, the amount of IgG antibody (IgG2a) from mouse serum (23-week-old mouse) was measured by an ELISA method known in the art. As a result, it was confirmed that when sodium butyrate was injected, the amount of IgG antibody was reduced compared to the control ( FIG. 1d ).

Therefore, it was confirmed that sodium butyrate has an effect of increasing and maintaining salivation and thus can be used for the treatment of Sjogren's syndrome, a type of autoimmune disease.

Example 2. Effect of maintaining cell morphology and reducing infiltration of inflammatory cells in lacrimal and salivary glands by sodium butyrate in an animal model of Sjogren's syndrome

The present inventors, in order to confirm the effect of sodium butyrate for the treatment of Sjogren's syndrome, whether to maintain the shape of cells in the lacrimal and salivary glands of mice, which are animal models of Sjogren's syndrome, and infiltration of inflammatory cells An experiment was performed to confirm whether this was done through H&E staining method known in the art.

As a result, when sodium butyrate was injected, it was confirmed that the cells in the lacrimal and salivary glands were well maintained, and the infiltration of inflammatory cells was significantly reduced (FIG. 2). Moreover, it was confirmed that the infiltration of inflammatory cells was significantly reduced even in the spleen, an immune organ (FIG. 2).

Therefore, it was confirmed that sodium butyrate is effective in the treatment of Sjogren's syndrome by maintaining the cell morphology of the lacrimal and salivary glands and reducing the infiltration of inflammatory cells.

Example 3. Effect of increasing IL-10 expression in the spleen, salivary glands and lacrimal glands by sodium butyrate in an animal model of Sjogren's syndrome

The present inventors reported the expression level of IL-10, an anti-inflammatory cytokine, in the spleen (Spleen (SP)), salivary gland (SG), and lacrimal gland (LG) after sodium butyrate was injected into a Sjogren's syndrome mouse model. To compare the real-time PCR known in the art was performed.

As a result, when sodium butyrate was injected, it was confirmed that the IL-10 expression level was increased in the spleen (SP) and lacrimal gland (LG) compared to the control (vehicle) ( FIG. 3 ).

Therefore, it was confirmed that sodium butyrate is effective in the treatment of Sjogren's syndrome by increasing the expression level of IL-10, an anti-inflammatory cytokine, in the spleen and lacrimal gland.

Example 4. Effect of Sodium Butyrate on Th17/Treg Cell Modulation in Mouse T Cells

The present inventors performed confocal microscopy to identify Th17 cells (immunostained with CD4 and IL-17 antibodies) and Treg cells (immunostained with CD4, CD25, and Foxp3 antibodies) in the spleen tissue of a Sjogren's syndrome mouse model. As a result, it was confirmed that sodium butyrate decreased Th17 cells and increased Treg cells in the spleen tissue ( FIG. 4A ). In the ratio of Th17/Treg cells, it was confirmed that when sodium butyrate was injected, it was reduced by about 4 times compared to the control group.

In addition, the present inventors isolated CD4 ⁺ CD62L ^high CD44 ^low -naive CD4 ⁺ T cells from the spleen tissue of a mouse model of Sjogren's syndrome and incubated them with 100 μM or 500 μM sodium butyrate as a Th17 differentiation condition. After 72 hours, Th17 cells and Treg After immunostaining with antibodies to CD4, CD25 and Foxp3 to identify the cells, flow cytometry analysis was performed. As a result, when sodium butyrate was injected, it was confirmed that Th17 cells were decreased and Treg cells were increased compared to the control group (FIG. 4B).

In addition, the present inventors isolated CD4 ⁺ CD62L ^high CD44 ^low -naive CD4 ⁺ T cells from the spleen tissue of a mouse model of Sjogren's syndrome and incubated them with sodium butyrate under Th17 differentiation conditions. After 48 hours, Foxp3 and CTLA4, which are Treg cell markers, were Real-time PCR was performed to confirm the mRNA expression level of As a result, it was confirmed that when sodium butyrate was injected, the mRNA expression levels of Foxp3 and CTLA4, which are markers of Treg cells, were increased compared to the control group ( FIG. 4C ).

Therefore, it was confirmed that sodium butyrate is effective in the treatment of Sjogren's syndrome by decreasing Th17 cells and increasing Treg cells.

Example 5. Modulation effect of T cells through inhibition of HDAC8 (histone deacetylase 8) of sodium butyrate

The present inventors cultured naive CD4 ⁺ T cells under the Th17 differentiation condition, and after 24 hours, in order to check the mRNA expression level according to the HDAC (histone deacetylase) subtype in the cells cultured under the Th17 differentiation condition compared to the condition without stimulation. Real time-PCR was performed. As a result, it was confirmed that the mRNA expression level of HDAC8 (histone deacetylase 8) was significantly increased in cells cultured under the Th17 differentiation condition (FIG. 5A).

In addition, the present inventors incubated EL4 cells (Mus musculus lymphoma, ATCC® TIB39™) with anti-CD3, IL-6 and sodium butyrate, and then used an anti-ERRα (estrogen-related receptor alpha) antibody for immunoprecipitation (IP). ), and IB (immuniblotting) was performed using an anti-ERRa antibody and an anti-acetyl lysine antibody. As a result, it was confirmed that when treated with sodium butyrate, acetylated ERRα was increased compared to the control (FIG. 5B).

In addition, the present inventors cultured naive CD4 ⁺ T cells under Th17 differentiation conditions, and performed confocal microscopy to confirm the expression of CPT IA (Carnitine Palmitoyltransferase IA) and NR1D1 (Nuclear receptor subfamily 1 group D member 1) after 72 hours. did. As a result, it was confirmed that when sodium butyrate was treated, CPT IA was increased and NR1D1 was decreased ( FIG. 5C ).

In addition, the present inventors cultured naive CD4 ⁺ T cells under Th17 differentiation conditions, and performed real time-PCR to check the mRNA expression levels of CPT IA and NR1D1 after 24 hours. As a result, it was confirmed that when sodium butyrate was treated, CPT IA was increased and NR1D1 was decreased ( FIG. 5D ).

In addition, the present inventors incubated naive CD4 ⁺ T cells with sodium butyrate, Etomoxir (inhibitor of carnitine palmitoyltransferase-1) or SR8278 (antagonist of REV-ERBα) alone or with sodium butyrate under Th17 differentiation conditions, 72 hours later Th17 Flow Cytometry analysis was performed to identify cells and Treg cells. As a result, when treated with Etomoxir, Treg cells were reduced, and when treated with SR8278, it was confirmed that Th17 cells were reduced (FIG. 5E).

Therefore, it was confirmed that sodium butyrate is effective in the treatment of Sjogren's syndrome by inhibiting Th17 cells by inhibiting histone deacetyltransferase 8 (HDAC8) and increasing the activity of Treg cells.

Claims (4)

As a pharmaceutical composition for the prevention or treatment of Sjogren's syndrome comprising sodium butyrate represented by the following formula (1) as an active ingredient,
In the Sjogren's syndrome, by the expression of HDAC8 (histone deacetyltransferase 8), Th17 cells are increased, and the activity of Treg cells is decreased,
IgG antibody level is increased, IL-10 expression level is decreased,
acetylated ERRa is reduced and
The composition, wherein CPT IA is reduced and NR1D1 is increased:
[Formula 1]

.
delete delete As a food composition for preventing or improving Sjogren's syndrome comprising sodium butyrate represented by the following formula (1) as an active ingredient,
In the Sjogren's syndrome, by the expression of HDAC8 (histone deacetyltransferase 8), Th17 cells are increased, and the activity of Treg cells is decreased,
IgG antibody level is increased, IL-10 expression level is decreased,
acetylated ERRa is reduced and
The composition, wherein CPT IA is reduced and NR1D1 is increased:
[Formula 1]

.

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