KR102563307B1 - Composition for enhancing immune response comprising dcGMP - Google Patents

Abstract

In vitro ( It relates to a method for activating NK cells in vitro .
The present invention specifically confirmed that dcGMP has an immune enhancing activity by increasing NK cell killing ability and NK receptor activity and increasing IFN-γ expression. It can be usefully used in a wide range of fields such as food and cosmetics as well as the pharmaceutical field related to

Description

Composition for enhancing immune response comprising dcGMP as an active ingredient {Composition for enhancing immune response comprising dcGMP}

In vitro ( It relates to a method for activating NK cells in vitro .

The immune response is a self-defense mechanism to protect our body, including a process of removing or neutralizing various antigens introduced from the outside and unnecessary metabolites in the body, and plays a role in maintaining homeostasis and suppressing the occurrence of diseases. Immune responses in the body are largely divided into innate immunity, which is present from birth, and acquired immunity, which is acquired through adaptation to life.

Innate immunity, also called innate immunity, responds non-specifically to antigens and does not show any special memory function. This innate immunity actually defends against most infections. The innate immune system includes skin and mucous tissues that block the invasion of antigens, strong acidic stomach acid, and complement present in the blood. Cells include phagocytosis There are macrophages, polymorphonuclear leukocytes, and NK cells responsible for

Acquired immunity, also called adaptive immunity, remembers the first invaded antigen and reacts specifically when invaded again to effectively remove the antigen. Such acquired immunity is divided into humoral immunity and cellular immunity. In humoral immunity, after recognizing an antigen, B lymphocytes differentiate and secrete antibodies, and these antibodies mainly eliminate infected bacteria. Cellular immunity plays a role in T lymphocytes (T cells) derived from the thymus recognizing antigens and secreting lymphokine or directly killing infected cells. Such cellular immunity can mainly grow in viruses or cells. It performs the function of removing cells infected with bacteria.

Among the cells involved in the immune response, NK cells (natural killer cells) are an innate immune response that directly attacks host-infected cells or tumor cells, and an acquired immune response that activates cytotoxic T cells or B cells through cytokine secretion. (Advances in Immunology, 2014, 137-170). NK cells are activated by IFN or macrophage-derived cytokines, and activated NK cells secrete cytokines such as IFN-γ and TNF-α to promote apoptosis of tumor cells or to activate other immune cells. contribute The activity of NK cells is regulated by the signal transduction balance due to the presence of various activating and inhibitory receptors, and through this regulation, normal cells are not attacked and infected cells are eliminated (Cancer Immunol. Immunother. 2014; 63 (6): 571-580).

Regulation of NK cell activity is known to be associated with various diseases such as cancer or immune diseases, and the need for an efficient immune enhancement method using NK cells is emerging.

Under this background, the present inventors have completed the present invention by confirming that the activity of NK cells increases when NK cells are treated with dcGMP, the function of which is unknown, and by identifying the related mechanism of action.

An object of the present invention is to provide a composition for enhancing immunity comprising deoxy cyclic guanosine monophosphate (dcGMP).

Another object of the present invention is to provide a food composition for enhancing immunity comprising dcGMP as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating immune diseases containing dcGMP.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing dcGMP.

Another object of the present invention is to provide a method for activating NK cells in vitro comprising the step of treating NK cells with dcGMP.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be included in this invention.

One aspect of the present invention for achieving the above object provides a composition for enhancing immunity containing dcGMP (deoxy cyclic guanosine monophosphate) as an active ingredient.

"dcGMP (deoxy cyclic guanosine monophosphate)" of the present invention is a structure in which one molecule of nucleotide forms a ring, and may be referred to as "cyclic deoxyguanosine monophosphate", and the compound represented by the following formula (1) it means.

The molecular weight of the dcGMP is 351.2, the chemical formula is C ₁₀ H ₁₁ N ₅ O ₆ P·Na, and the CAS number is known as 93919-42-7. The nucleic acid derivative, dcGMP, is an analog of cGMP, and cGMP is an important physiologically active substance in cells and is well known to be involved in intercellular signal transduction pathways. However, the function of dcGMP has not been clarified. Thus, the immune enhancing activity of dcGMP was first identified by the present inventors.

The acquisition method of the dcGMP is not particularly limited, and may be chemically synthesized using a method known in the art or a commercially available material may be used. In addition, the dcGMP may exist in a solvated or non-solvated form, and may exist in a crystalline or amorphous form, and all such physical forms are included in the scope of the present invention.

"Immune enhancement" of the present invention is to enhance the body's ability to defend against antigens, which are external factors in the internal environment of the body. It can be distinguished, and the immunity includes both innate immunity and acquired immunity, and may increase innate immunity or innate immunity and acquired immunity together, but is not limited thereto.

NK cells (natural killer cells, natural killer cells) of the present invention are one of the immune cells and activate cytotoxic T cells or B cells through an innate immune response that directly attacks host-infected cells or tumor cells and secretion of cytokines Shiki is involved in the acquired immune response. NK cells are activated by cytokines, and activated NK cells secrete cytokines such as IFN-γ and TNF-α to promote apoptosis of tumor cells or to activate other immune cells.

The dcGMP of the present invention may increase the activity of NK cells. In the present invention, increasing the activity of NK cells may mean increasing the cytotoxicity (cytotoxicity) activity of NK cells, and "cytotoxic activity" or "killing ability" may be used interchangeably herein.

In a specific embodiment of the present invention, when NK cells were treated with dcGMP at a concentration of 20 μm or 50 μm, the cytotoxic activity of NK was significantly increased, and compared to similar nucleic acid derivatives, cAMP or cGMP, the activity was confirmed to be superior (Example 1).

The function of the NK cells is regulated by the interaction of cell surface receptors and corresponding target cell ligands without stimulation of specific antigens, and these receptors are largely divided into active receptors and inhibitory receptors (Clinical Immunology 2006; 118(1 ): 1-10). Natural cytotoxicity receptors (NCRs), NKG2D, and the like are well known as representative activating receptors that transmit activation signals to NK cells to regulate NK cell functions. Among these NCRs, NKp30, NKp44, and NKp46 have been identified. NKp30 and NKp44 are also found in T cells, but NKp46 is known to be specifically expressed in NK cells. It has been found that NK cells with high NCRs expression have higher cytotoxicity than cells with low NCRs expression, so the degree of NK cell activation can be confirmed according to the level of NCRs expression (Annual review of immunology 2001; 19: 197 -223).

In the present invention, the increase in NK cell activity may be achieved through an increase in the expression of an NK-activating receptor, and the NK-activating receptor may be one or more selected from the group consisting of NKp30, NKp44, and NKG2D, but is not limited thereto.

In a specific embodiment of the present invention, as a result of treating NK cells with 50 μm of dcGMP, it was confirmed that the expression of NKp30, NKp44, and NKG2D increased compared to cAMP or cGMP (Example 2), dcGMP of the present invention activates NK It can be seen that the activity of NK cells is increased through the increase of receptor expression.

NK cells secrete various cytokines and chemokines and play an important role in activating acquired immune responses through direct interaction with antigen-presenting cells. In particular, IFN-γ, a cytokine secreted from activated NK cells, activates and matures monocytes and dendritic cells to increase the innate immune response to pathogens and is known to play an important role in the early stage of the Th1 immune response (Nature Immunology 2008; 9(5): 503-10).

The dcGMP of the present invention may exhibit immune enhancing activity by increasing the secretion of IFN-γ in NK cells, but is not limited thereto.

In a specific embodiment of the present invention, as a result of treating NK cells with 20 μm of dcGMP, it was confirmed that the expression of IFN-γ increased (Example 3).

Studies have reported that adenosine A2B receptors are highly expressed in various immune cells, including NK cells, and studies on cancer treatment and immune response enhancement targeting adenosine receptors are being actively conducted.

In this regard, in a specific embodiment of the present invention, in order to reveal the mechanism of action of NK cell activation, as a result of measuring the mRNA expression level of the adenosine receptor after dcGMP treatment of NK cells, it was confirmed that the expression of the A2B receptor increased, p38 and phosphorylation of Erk were significantly increased (Example 4). That is, the dcGMP of the present invention may activate the A2B receptor of NK cells to increase NK cell activity, but is not limited thereto.

The composition of the present application may be a pharmaceutical composition, a food composition, or a health functional food composition, but is not limited thereto. The composition may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier and provided as pharmaceuticals, foods, and health functional foods.

Another aspect of the present invention provides a food composition for enhancing immunity comprising dcGMP as an active ingredient.

Description of "dcGMP" and "immunity enhancement" of the present invention is as described above.

The term "food" of the present invention refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, It includes all foods in the usual sense, such as vitamin complexes and health functional foods, and is not limited thereto as long as it can include the dcGMP of the present invention. In addition, the food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, and food additives, and the type of food composition is known in the art. It can be prepared in various forms according to conventional methods known to. When preparing the food composition, it may be prepared by adding raw materials and ingredients commonly added in the art, and the type is not particularly limited. For example, as in conventional foods, various herbal extracts, food-acceptable food supplements, or natural carbohydrates may be included as additional components, but are not limited thereto. The mixing amount of the active ingredient can be suitably determined depending on the purpose of use.

In a specific aspect, the food composition of the present invention may be a health functional food.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating immune diseases comprising dcGMP as an active ingredient.

Description of the "dcGMP" of the present invention is as described above.

The immune diseases of the present invention collectively refer to diseases caused by an abnormal immune system function, and if a preventive or therapeutic effect can be obtained through an increase in NK cell activity, it may be included in the scope of the immune diseases of the present invention. NK cells secrete various cytokines and chemokines and activate acquired immune responses through direct interaction with antigen-presenting cells. Since it increases the response and plays an important role in the early stage of the Th1 immune response, it is obvious that the prevention or treatment of immune diseases can be obtained by increasing the activity of NK cells.

Specifically, the immune disease may be an autoimmune disease, a viral infection disease, or an inflammatory disease. Specifically, the autoimmune diseases include Crohn's disease, erythema, atopic dermatitis, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, thyroid function Hypothyroidism and hyperthyroidism, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis , psoriasis, vitiligo, systemic scleroderma, asthma or ulcerative colitis, but not limited thereto.

In the present invention, "prevention" refers to all activities that suppress or delay the occurrence of immune diseases using a composition containing the dcGMP as an active ingredient. The term "treatment" of the present invention refers to all activities of controlling or improving the symptoms of an immune disease using a composition containing the dcGMP as an active ingredient.

The "pharmaceutical composition" of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent. Such pharmaceutically acceptable carriers, excipients, or diluents may be non-naturally occurring. Specifically, the composition is formulated in the form of oral formulations such as powders, granules, tablets, solutions, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can be used In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient in the dcGMP, for example, starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition may be appropriately administered to a subject according to a conventional method, administration route, and dosage used in the art according to purpose or necessity. Examples of routes of administration include oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, an appropriate dosage and frequency of administration may be selected according to a method known in the art, and the amount and frequency of administration of the pharmaceutical composition of the present invention actually administered are the type of symptom to be treated, the route of administration, gender, health condition, It can be appropriately determined by various factors such as diet, the age and weight of the individual, and the severity of the disease.

As the immune enhancing activity of dcGMP through NK cell activation was confirmed in the present invention, a pharmaceutical composition containing dcGMP as an active ingredient can be usefully used for preventing or treating immune diseases.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising dcGMP as an active ingredient.

Descriptions of "dcGMP, "prevention or treatment", and "pharmaceutical composition" of the present invention are as described above.

Cancer of the present invention refers to a disease caused by excessive proliferation of cells when the normal cell death balance is broken. These abnormal hyperproliferating cells, in some cases, invade surrounding tissues and organs to form masses, destroying or transforming normal structures in the body, and this condition is called cancer.

The types of cancer include cerebrospinal tumor, brain tumor, head and neck cancer, lung cancer, breast cancer, thymoma, neutrophil tumor, esophageal cancer, colon cancer, liver cancer, stomach cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovary cancer, cervical cancer, endometrial cancer, colorectal cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, and skin cancer.

Since NK cells can distinguish between cancer cells and normal cells through various immune receptors present on their surface, they exhibit selective cytotoxicity against cancer cells. Many studies have identified that NK cells play a key role in eliminating cancer cells, and it has been reported that cancer patients have defects in the number or activity of NK cells (Hanyang Med Rev 2013: 59-64). Therefore, the increase in activity of NK cells can lead to the prevention or treatment of cancer through the elimination of cancer cells.

In a specific embodiment of the present invention, the NK cell activating effect of dcGMP was confirmed, suggesting that a pharmaceutical composition containing dcGMP as an active ingredient can be usefully used for preventing or treating cancer.

Another aspect of the present invention provides a method for activating NK cells in vitro , comprising the step of treating NK cells with dcGMP.

Description of the "dcGMP" and "NK cells" of the present invention is as described above.

In the present invention, the treatment of dcGMP may be performed according to a conventional method in the art, and the treatment concentration of dcGMP may be 1 to 100 μm, 1 to 90 μm, 1 to 80 μm, 1 to 70 μm, and 1 to 60 μm, but is limited thereto. It doesn't work.

" In vitro " of the present invention refers to an experimental process performed in a controllable environment such as a test tube rather than inside a living organism. In the environment inside the test tube, various variables can be easily controlled, and the results can be efficiently confirmed.

In a specific embodiment of the present invention, the dcGMP was confirmed to increase the cytotoxic activity of NK cells by treating NK cells, increase the expression of NK-activating receptors, and increase the amount of IFN-γ secretion, which is ( in vitro ) suggests that dcGMP can activate NK cells.

The present invention specifically confirmed that dcGMP has an immune enhancing activity by increasing NK cell killing ability and NK receptor activity and increasing IFN-γ expression. It can be usefully used in a wide range of fields such as food and cosmetics as well as the pharmaceutical field related to

1 is a diagram of analyzing the cytotoxic activity of NK cells according to dcGMP treatment.
Figure 2 is a diagram comparing the cytotoxic activity of NK cells according to cAMP, cGMP, and dcGMP treatment.
3 is a diagram confirming the effect of cAMP, cGMP, and dcGMP on NK receptor expression, FIG. 3a is a graph comparing MFI values of FACS analysis, and FIG. 3b is FACS analysis data.
Figure 4 is a diagram confirming the effect of cAMP, cGMP, and dcGMP on the expression of IFN-γ, Figure 4a is a graph comparing MFI values of FACS analysis, Figure 4b is FACS analysis data.
5 is a diagram confirming the effect of dcGMP on A2B receptor and A2A receptor expression.
Figure 6 is a diagram confirming the expression levels of sub-markers of the A2B receptor, Figure 6a is western blot data of p38 and Erk, and Figure 6b is a graph quantifying the western blot results.

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

Example 1. Confirmation of the effect of dcGMP on the activity of NK cells

NK92 cells were subjected to IL-2 starvation for 24 hours. Thereafter, cell seeding was performed in 6 wells at 2×10 ⁵ /ml, and cAMP, cGMP, and dcGMP were treated with 20 μM and 50 μM, cultured for 24 hours, and experiments were performed.

NK cell activity assay was evaluated using the calcein-AM release assay. K562 target cells were labeled with calcein for 1 hour, and the labeled target cells (1×10 ⁴ cells) and diluted NK92 cells according to the number ratio were cultured in a 96 well round bottom plate for 4 hours. “Maximum release” was tested by adding 4% Triton X-100 to target cells, and “minimum release” by adding culture medium to target cells. Calcein released into the supernatant was measured using a multi-mode microplate reader. The activity analysis of NK cells was calculated and expressed as calcein release %=(sample release−spontaneous release)/(maximum release−spontaneous release)×100.

As a result, it was confirmed that the apoptotic ability of NK cells was increased compared to the control group when dcGMP was treated with 20 μm and 50 μm (FIG. 1). In addition, as a result of comparing the apoptosis of NK cells treated with cAMP, cGMP, and dcGMP, respectively, dcGMP showed the highest apoptosis (FIG. 2). That is, it was found that dcGMP markedly increased the activity of NK cells.

Example 2. Confirmation of the effect of dcGMP on NK receptor expression

NK92 cells stimulated with cAMP, cGMP, and dcGMP for 24 hours were analyzed by staining with appropriate antibodies. For surface staining, PE-anti-NKp30, PE-anti-NKp44, and APC-anti-NKG2D were used. The data of the samples were obtained in Canto II and analysis was expressed as mean fluorescence intensity (MFI) values using the software FlowJo.

Various activating or inhibitory receptors are involved in the activity of NK cells. As a result of treating NK92 cells with 50 μM of dcGMP, it was confirmed that the expression of NKp30, NKp44, and NKG2D, which are activating receptors, increased compared to cAMP or cGMP (FIG. 3).

Example 3. Confirmation of the effect of dcGMP on the expression of IFN-γ

Expression of IFN-γ was measured in the same manner as in Example 2 above. IFN-γ is a cytokine secreted by NK cells and plays an important role in increasing the innate immune response and activating the acquired immune response. As a result of treating NK92 cells with 20 μM of dcGMP, it was confirmed that the expression of IFN-γ, an immunoactive factor, increased (FIG. 4).

Example 4. Analysis of the mechanism of action of NK cell activation

In order to elucidate the mechanism of activation of NK cells, the amount of mRNA expression of adenosine receptors expressed in NK cells was measured. As a result of treating NK cells with dcGMP, it was confirmed that the expression of the A2B receptor among receptors expressed in NK cells increased, but the expression of the A2A receptor decreased (FIG. 5).

In this regard, Western blotting was performed to confirm the expression levels of p38 and Erk, submarkers of the A2B receptor. Specifically, NK92 cells stimulated with dcGMP for 24 hours were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor. Then, the same amount of protein was separated by 10% SDS PAGE and transferred to a polyvinylidene difluoride membrane. Blocking was performed with 5% skim milk for 1 hour, and the primary antibody was incubated overnight. As the primary antibodies used at this time, extracellular signal-regulated kinase (ERK), phosphorylated (p-)ERK, p38, and p-p38 were used. Then, the membrane was washed with PBST and incubated with the corresponding HRP-conjugated secondary rabbit antibody for 1 hour at room temperature. The intensity of each band was obtained using the program CSAnalyzer 4, and the phosphorylation form was quantified as the total form.

As a result of confirming the expression levels of p38 and Erk, which are submarkers of the A2B receptor, it was confirmed that the phosphorylation of p38 and Erk significantly increased when treated with dcGMP. Through this, in the present invention, it was found that dcGMP activates the A2B receptor of NK cells to increase NK cell activity (FIG. 6).

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts rather than the detailed description above.

Claims (10)

A food composition for enhancing immunity containing dcGMP (deoxy cyclic guanosine monophosphate) as an active ingredient,
The immune enhancement is to increase the activity of NK cells, immune enhancement food composition.
delete The food composition for enhancing immunity according to claim 1, wherein the increase in activity of NK cells increases the expression of NK activating receptors.
The food composition for enhancing immunity according to claim 3, wherein the NK-activating receptor is at least one selected from the group consisting of NKp30, NKp44 and NKG2D.
The food composition for enhancing immunity according to claim 1, wherein the dcGMP increases the secretion of IFN-γ in NK cells.
delete delete delete delete A method of activating NK cells in vitro , comprising the step of treating NK cells with dcGMP.