KR20090009540A - Composition for immunological enhancement comprising thymosin beta 4 - Google Patents

Abstract

A composition comprising thymosin beta 4 is provided to increase immunity by inducing the proliferation and activation of the natural killer cell, so that the composition is useful as non-specific immunity reinforcing agent. The composition for reinforcing the immunity comprises thymosin beta 4 capable of inducing the proliferation of the natural killer cell as an effective ingredient, and further comprises carrier, excipient and diluent such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. The non-specific immunity reinforcing agent comprises the immunity reinforcing composition comprising thymosin beta 4.

Description

Composition for immunological enhancement comprising thymosin beta 4

The present invention relates to a composition for enhancing immunity, and more particularly, to a composition for immunostimulation containing thymosin beta4 as an active ingredient.

Immunity is largely divided into innate immunity that has been born since birth and acquired immunity that is acquired by adapting to life. Innate immunity, also known as 'natural immunity', is a nonspecific reaction to antigens and does not have a special memory function. Innate immune systems include skin, mucous tissues, strong acidic acids, and complement that are present in the blood to block antigen invasion. Cells include macrophage and polymorpho nuclear leukocytes, which are responsible for phagocytosis, and natural killer (NK) cells that can kill infected cells. In fact, most infections are protected by this innate immunity.

In particular, the natural killer (NK) cells have a function of directly attacking and killing cancer cells or virus-infected cells through various mechanisms, and account for only 5 to 10% of all lymphocytes, but abnormalities have been detected in normal cells. In addition to having a strong function of removing the cells by defending for the first time, it also plays a very important role in regulating the function of other immune cells. Since the number and function of natural killer cells is extremely reduced in cancer patients, increasing the number and function of natural killer cells can be an effective immunotherapy method by remarkably increasing the immunity of cancer patients. The immunotherapy is a method of treating diseases related to immune function by differentiating or multiplying immune-related cells obtained from a patient and injecting them into the patient. Immunotherapy has been evaluated as a safe and powerful treatment with few side effects because it regulates the patient's immune function to heal the disease on its own and has proven its excellence in the toxicity test of immunotherapy.

Therefore, the necessity of more effectively proliferating natural killer cells and using them in the immunotherapy has been constantly required.

On the other hand, thymosin is a small protein originally discovered from the thymus but now widely found in many tissues and cells. It is classified into three classes of thymosin (thymosin alpha, thymosin beta, and thymosin gamma) based on the isoelectric point. Is known to exist.

In particular, thymosin beta 4 and thymosin alpha 1 is a protein that is in the spotlight because of its great clinical potential. The thymosin beta 4 was first discovered in 1981 and composed of 43 amino acids and had a molecular weight of about 4.9 kDa. The thymosin alpha 1 was found in 1972 and composed of 28 amino acids and had a molecular weight of about 3.1 kDa. Haritos et al ., FEBS Lett , 218 (1): 107-112, 1987).

Although thymosin beta 4 and thymosin alpha 1 have very small molecular weights, they have different molecular structures and have very low homology of 25%, thus playing different roles in terms of biological function (Koutrafouri et. al ., Biochim Biophys Acta , 1568 (1): 60-66, 2001). Specifically, thymosin alpha1, along with factors such as interferon and interleukin, is known to have a function of promoting the activity of natural killer cells to induce chemotherapy more effectively (Jean Marx et al ., Science , 316 (5825). : 682-683, 2007) Thymosin beta4, which has a completely different structure and function, is not known for its ability to induce the activity and proliferation of natural killer cells.

Accordingly, the present inventors completed the present invention by revealing that thymosin beta4 promotes the proliferation and activity of natural killer cells while studying a substance that effectively induces the proliferation of natural killer cells without side effects and economical. It was.

Therefore, an object of the present invention to provide a composition for immuno-enhancement containing thymosin beta4 (thymosin beta4) as an active ingredient.

Another object of the present invention to provide a non-specific immunopotentiator containing the composition as an active ingredient.

For the above purpose, the present invention provides a composition for immuno-enhancing containing thymosin beta4 (thymosin beta4) as an active ingredient. Specifically, the present invention provides an immune enhancing composition comprising thymosin beta 4 as an active ingredient to induce proliferation of natural killer cells.

For another object of the present invention provides a non-specific immunopotentiator containing the composition as an active ingredient.

Hereinafter, the present invention will be described in detail.

Thymosin beta 4 (thymosin beta 4) was first discovered as an extract of the thymus, consisting of 43 amino acids, a relatively small molecular weight of 5 KDa, and is present in almost all cells except red blood cells. The thymosin beta4 is a protein that originally regulates actin, which binds to G-actin, inhibits the synthesis of polymers of actin, induces differentiation and migration of endothelial cells, and induces neovascularization. It is reported to have an excellent effect on the regeneration of cardiomyocytes. However, the effect of thymosin beta4 on the activity or proliferation of natural killer cells is unknown.

Thus, the present inventors found that after thymosin beta 4 was treated with natural killer cell lines, the concentration of natural killer cell lines was compared with that of the control group, which was significantly increased compared to the control group (Example 1).

In addition, the inventors of the present invention treated the thymosin beta 4 in the natural killer cell line and analyzed the expression level of the CD activator, a natural killer cell activity marker, and found that it was significantly increased compared to the control group (Example 2).

In addition, the present inventors found that the treatment of thymosin beta 4 in natural killer cell lines and then the number of natural killer cells using a hemocytometer shelf increased significantly compared to the control group (Example 3).

Therefore, since thymosin beta 4 has an activity of inducing proliferation and activity of natural killer cells, it can be used in an immune enhancing composition containing the same as an active ingredient. The immunopotentiating composition is not limited thereto, but refers to a pharmaceutical composition or a composition for food addition.

The pharmaceutical composition containing thymosin beta 4 of the present invention as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in pharmaceutical compositions in addition to thymosin beta 4 (Remington's Pharmaceutical Science, Mack Publishig Co. , Easton PA). In addition, the composition for immuno-enhancement comprising thymosin beta 4 according to the present invention, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It can be formulated and used in the form of sterile injectable solutions.

Carriers, excipients and diluents that may be included in the pharmaceutical composition containing thymosin beta 4 include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used can be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, and sucrose in the polygamma glutamic acid. (sucrose), lactose (lactose), gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. May be included. Formulations for non-oral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous preparation and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, lauridge, glycerogelatin and the like can be used. The immunopotentiator of the present invention is not limited to a specific form so long as it can be administered to humans or animals, but may be a dosage form supported by a carrier. As the carrier, at least one of solids, semiconductors, liquid diluents, fillers, and other formulation auxiliaries is used at a ratio of 0.1 to 99.5%.

When the pharmaceutical composition containing thymosin beta4 is formulated, the preparation may be administered orally or parenterally. Parenteral administration can be local, subcutaneous, intramuscular, arterial or intravenous, and rectal administration. Suitable dosage forms for the route of administration may be prepared using conventional technical means. Oral administration can be carried out using solid or liquid unit dosage forms such as bulk powders, powders, granules, tablets, capsules, syrups and suspensions and the like. Unit dosage forms of oral administration may be microencapsulated if necessary. By further coating such formulations or incorporating the active ingredient in polymers or waxes, the active period can be extended and sustained release can be obtained.

The amount of the pharmaceutical composition containing thymosin beta 4 may vary depending on the age, sex, and weight of the patient, but is generally in an amount of 5 to 500 mg / kg, preferably in an amount of 100 to 250 mg / kg. It can be administered divided into 1 to 3 times a day, the dosage may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The composition for food addition containing thymosin beta 4 of the present invention as an active ingredient may be added to foods or beverages for the purpose of immune enhancement. At this time, the amount of thymosin beta4 in the food or beverage may generally be added in the range of 0.01 to 30% by weight of the total food weight, 0.01 to 10 g, preferably 0.1 to 2 g based on 100 ml of the beverage It can be added in a ratio and may be in powder, granule, tablet, capsule or beverage form.

The food to which the composition of the present invention may be added is not limited thereto, but various foods, meats, beverages, chocolates, snacks, confections, pizzas, ramen noodles, other noodles, gums, ice creams, alcoholic beverages, vitamin complexes, and health supplements. Foods;

When the composition of the present invention is used in a beverage, in addition to containing the thymosin beta 4 as an essential ingredient, there is no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, as in general beverages. . Examples of such natural carbohydrates include conventional sugars such as monosaccharides (glucose, fructose, etc.), disaccharides (maltose, sucrose, etc.) and polysaccharides (dextrins, cyclodextrins, etc.) and xylitol, sorbitol, erythritol And sugar alcohols. As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extract (rebaudioside A, glycyrzin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention, in addition to the various nutrients, vitamins, minerals (electrolytes), minerals, synthetic flavors and natural flavors. Carbonates used in agents, colorants, neutralizers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonated drinks And fruit flesh and grains for the production of natural fruit juice and fruit juice beverages, cereal beverages and vegetable beverages, etc. These ingredients may be used independently or in combination . The proportion of the additive is not so critical but is generally selected in the range of about 0.01 to 20 parts by weight per 100 parts by weight of the total composition of the present invention.

In addition, the immunopotentiating composition may be used as a nonspecific immunopotentiator for inducing proliferation of natural killer cells.

The non-specific adjuvant of the present invention can be used without limitation so long as it can be administered to humans or animals, it is preferable to be administered in a carrier supported form. As the carrier, at least one of solids, semiconductors, liquid diluents, fillers, and other formulation auxiliaries is used in a ratio of 0.1 to 99.55. The therapeutic agent can be administered orally or parenterally. Parenteral administration can be local, subcutaneous, intramuscular, arterial or intravenous, and rectal administration in a tissue. Suitable dosage forms for the route of administration may be prepared using conventional technical means. Oral administration can be carried out using solid or liquid unit dosage forms such as bulk powders, powders, granules, tablets, capsules, syrups and suspensions and the like. Unit dosage forms of oral administration may be microencapsulated as necessary. By further coating such formulations or incorporating the active ingredient in polymers or waxes, the active period can be extended and sustained release can be obtained. The dosage of the therapeutic agent may be preferably determined in consideration of the age, weight, route of administration, disease, the extent and other factors of the patient.

Therefore, the composition containing thymosin beta 4 of the present invention can be usefully used as an immune enhancing composition by inducing proliferation and activity of natural killer cells and can be used as a non-specific immunopotentiator.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

Thymosin Beta 4 ( thymosin beta  4) natural killing NK ) Cell aggregation enhancement effect

<1-1> Thymosin Verify aggregation promotion effect of beta-fourth of natural killer (NK) cells

1 with 2 mM L-glutamine, 0.2 mM i -inositol, 0.02 mM folic acid, 0.1 mM 2-mercaptoethanol (Sigma, USA), 15% fetal calf serum (Gibco, USA) Disperse the natural killer cells in 1% MEM vitamin medium (Gibco, USA) in an amount of 1 × 10 ⁵ / ml and treat thymosin beta 4 at a concentration of 50 ng / ml, followed by an optical microscope (Olympus, Japan) The photo taken with the control is shown in Figure 1 compared with the control.

As shown in FIG. 1, when the thymosin beta4 was treated, it was confirmed that the aggregation of natural killer cells was significantly increased as compared to the control group, indicating that thymosin beta4 could induce proliferation of natural killer cells.

<1-2> Thymosin Confirmation of Aggregation Enhancement Effect of Natural Killer ( NK ) Cells at Different Concentrations of Beta 4

Photograph taken with an optical microscope (Olympus, Japan) 24 hours after treatment with thymosin beta4 at concentrations of 10, 50 and 100 ng / ml in medium in which natural killer cells were dispensed as in Example <1-1> 2 is shown in comparison with the control.

As shown in FIG. 2, when thymosin beta 4 was treated, the concentration of natural killer cells was remarkably increased as compared to the control group. Particularly, when thymosin beta 4 was treated, natural killer cell aggregation was increased. It was confirmed that the greatest increase. Therefore, thymosin beta4 was found to induce proliferation of natural killer cells.

< Example  2>

Thymosin Beta 4 ( thymosin beta  4) cell membrane proteins of natural killer cells ( CD  56) Effect on expression

Antibodies that specifically bind to CD 56 after treatment with thymosin beta4 at concentrations of 10, 50 and 100 ng / ml in cultured medium to which natural killer cells were dispensed as in Example <1-1> and after 24 hours , USA) is shown in FIG. 3 by the expression of CD 56 protein by indirect immunofluorescent staining (Indirect FACS staining) compared to the control. In addition, the results of analyzing the M.F.I (Mean Fluorescence Intensity) value of Figure 3 using the CELLQuest program (Becton Dickinson, USA) is shown in Figure 4.

As shown in FIG. 3, the treatment of thymosin beta4 at all concentrations increased the expression of CD 56 protein compared to the control. In addition, as shown in FIG. 4, it was confirmed that the expression of CD 56 protein was increased in all groups treated with thymosin beta 4 compared to the control group, and especially when treated at a concentration of 50 ng / ml, the expression of CD 56 protein was significantly increased. It was confirmed. Therefore, thymosin beta4 may be used as an effective ingredient in the composition for enhancing immune cells by promoting the expression of cell membrane proteins of natural killer cells.

< Example  3>

Thymosin Beta 4 ( thymosin beta  4) natural killing NK ) Cell proliferation induction activity measurement

<3-1> Thymosin Confirmation of beta4 's natural killer cell proliferation induction activity

The number of spontaneous killer cells was treated with thymosin beta4 at a concentration of 10 ng / ml in the medium to which spontaneous killer cells were dispensed as in Example <1-1>, using a hematocutometer after 24 and 72 hours. Are measured and shown in FIG. 5 in comparison with the control.

As shown in Figure 5 when treated with thymosin beta 4 significantly increased the number of natural killer cells compared to the control thymosin beta 4 has the activity of inducing the proliferation of natural killer cells used as an effective ingredient in the composition for enhancing immune Could be found.

<3-2> Thymosin Confirmation of Induction Activity of Natural Killer Cells by Concentration of Beta 4

Treatment of thymosin beta4 at concentrations of 10, 50, and 100 ng / ml in the medium to which natural killer cells were dispensed as in Example <1-1> using a hematocutometer after 24, 48 and 72 hours The number of natural killer cells was measured and shown in FIG. 6 in comparison with the control group.

As shown in FIG. 6, the number of natural killer cells was significantly increased in all cases treated with thymosin beta 4 compared to the control group, and in particular, when treated with thymosin beta 4 at a concentration of 50 ng / ml, about 2 times more than the control group. Increased. Therefore, since thymosin beta4 effectively induces natural killer cell proliferation, it can be seen that it can be used as an effective ingredient in an immune enhancing composition.

As described above, the thymosin beta4-containing composition of the present invention can be usefully used as an immune enhancing composition by inducing proliferation and activity of natural killer cells and can be used as a non-specific immunopotentiator.

1 is a photograph showing the natural killer cell line aggregation after treatment with thymosin beta 4 natural killer cell line compared to the control group.

Figure 2 is a picture showing the concentration of natural killer cell line after treatment with thymosin beta4 in natural killer cell line concentration compared to the control.

3 is a graph showing the expression of cell membrane protein (CD 56) of natural killer cells after treatment with thymosin beta 4 in natural killer cell lines compared with the control group.

Figure 4 is a bar graph showing the expression of the cell membrane protein (CD 56) of natural killer cells after treatment with thymosin beta 4 by natural concentration in the cell line.

FIG. 5 is a graph showing the proliferation of natural killer cells after treatment with thymosin beta 4 in natural killer cell lines.

6 is a graph showing the proliferation of natural killer cells after treatment with thymosin beta 4 by natural concentration in the cell line, compared with the control group.

Claims (4)

Immunity-enhancing composition containing thymosin beta 4 as an active ingredient. The composition for immunopotentiation according to claim 1, which induces proliferation of natural killer cells. A nonspecific immunopotentiator comprising the composition of claim 1 as an active ingredient. 4. The nonspecific immunopotentiator according to claim 3, which induces proliferation of natural killer cells.

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