Use of amino acid chelated zinc in immunomodulation and treating autoimmune diseases

Abstract

Use of amino acid chelated zinc in preparing a composition for immunomodulation and / or inhibition of dendritic cell maturation, particularly a pharmaceutical composition for treating allergic diseases, treating autoimmune diseases, and / or preventing organ transplant rejection; and a combination for preparing tolerogenic dendritic cells, comprising (1) amino acid chelated zinc, and (2) a dendritic cell culture solution.

Description

Application of amino acid chelated zinc in immunomodulation and treatment of autoimmune diseases Technical Field

[0001] The present invention relates to the use of zinc amino acid chelate, and more particularly to the use of zinc amino acid chelate for immunomodulation of an individual, particularly providing benefits of suppressing the immune response of an individual in need (e.g., an autoimmune disease patient, an organ transplant patient). Background Art

[0002] According to statistics from the Autoimmune Association of America, more than 100 autoimmune diseases have been discovered, and in the United States, there are more than 50 million patients suffering from autoimmune diseases. In addition, due to the gradual aging of the social structure and changes in the lifestyle of modern people, the number of patients suffering from organ failure has increased year by year. Organ transplantation is currently the most effective method for clinical treatment of end-stage organ failure. The global organ transplantation market is expected to reach US$61.5 billion by 2027, and will increase at a compound annual growth rate of 9.9% during the forecast period. In order to prevent the recipient's immune system from rejecting the transplanted organ, the recipient usually needs to take immunosuppressants for a long time. Therefore, the industry is committed to developing drugs that can effectively regulate immunity (especially suppressing unwanted immune responses).

[0003] Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that serve as a bridge between the innate and adaptive immune systems. When the immune system recognizes foreign antigens, DCs mature, increasing the expression of costimulatory factors such as CD80, CD86, and CD40, as well as major histocompatibility complex class II (MHC II) on their surfaces. DCs also secrete proinflammatory cytokines to activate downstream T cells, leading to cytotoxicity and clearance of foreign antigens.

[0004] Recent studies have revealed that DCs (i.e., semi-mature DCs) with low co-stimulatory molecule and MHC II expression levels have the ability to induce immune tolerance, leading to T cell anergy, activation of regulatory T cells (Treg cells), and secretion of anti-inflammatory factors. These semi-mature DCs are also known as tolerant DCs. Due to their anti-inflammatory and immunosuppressive properties, tolerant DCs are considered potential cell therapies for allergic diseases, autoimmune diseases (such as type 1 diabetes, multiple sclerosis, and rheumatoid arthritis), and organ transplant rejection. Summary of the Invention

[0005] The inventors of this application have discovered that zinc amino acid chelate has excellent inhibitory effects on dendritic cell (DC) maturation. Compared to known active ingredients that inhibit DC maturation (e.g., zinc sulfate), zinc amino acid chelate can achieve the desired inhibitory effect at relatively low zinc concentrations. The inventors of this application have also discovered that administering zinc amino acid chelate to an individual suffering from an autoimmune disease can effectively alleviate symptoms associated with the autoimmune disease.

[0006] Therefore, one object of the present invention is to provide a use of zinc amino acid chelate in preparing a composition for immunomodulation. In some embodiments, the composition is a pharmaceutical composition or a food composition. In some embodiments, the pharmaceutical composition is used to treat allergic diseases, treat autoimmune diseases, and / or prevent organ transplant rejection.

[0007] In some embodiments of the use of the present invention, the composition is an aqueous composition and has a pH value less than 7.

[0008] Another object of the present invention is to provide a use of amino acid chelated zinc in preparing a composition, wherein the composition is used to inhibit dendritic cell maturation.

[0009] Another object of the present invention is to provide a combination comprising:

[0010] (1) amino acid chelated zinc; and

[0011] (2) A dendritic cell culture medium.

[0012] (3) In some embodiments of the combination according to the present invention, the combination further comprises dendritic cells.

[0013] In some embodiments of the combination according to the present invention, the content of the amino acid chelated zinc is 1 mg to 25 mg of zinc per mL of the dendritic cell culture medium, based on the content of the dendritic cell culture medium.

[0014] Another object of the present invention is to provide a method for immunomodulation and / or inhibition of dendritic cell maturation, comprising administering an effective amount of the composition described above to a subject in need thereof.

[0015] In some embodiments of the immunomodulatory method according to the present invention, the method is used to treat allergic diseases, treat autoimmune diseases, and / or prevent organ transplant rejection.

[0016] Another object of the present invention is to provide a method for preparing tolerogenic dendritic cells, comprising contacting a dendritic cell with an effective amount of amino acid chelated zinc. In some embodiments, the contacting is performed by culturing the dendritic cell in a dendritic cell culture medium containing amino acid chelated zinc.

[0017] In some embodiments of the uses, combinations, and methods of the present invention, the molar ratio of zinc to amino acid in the amino acid chelated zinc is 1:1 to 1:10.

[0018] In some embodiments of the uses, combinations and methods of the present invention, the amino acid chelated zinc is glycine chelated zinc, alanine chelated zinc, valine chelated zinc, leucine chelated zinc and / or isoleucine chelated zinc.

[0019] In order to make the above-mentioned objectives, technical features and advantages of the present invention more obvious and easy to understand, some specific implementation plans and drawings are described in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 shows the results of detecting CD11c expression of isolated mouse bone marrow dendritic cells (BMDCs) using flow cytometry. 97.22% of the cells expressed CD11c, and only 2.78% of the cells did not express CD11c, indicating that the purity of the BMDCs was quite high.

[0021] Figures 2A to 2K are the results of using flow cytometry to detect the expression of CD80, CD83, CD86, CD40, CD11c and MHC II on the cell surface of BMDCs treated with different treatments, wherein Figure 2A shows the detection results of BMDCs without any treatment (i.e., immature DCs), Figure 2B shows the detection results of activated BMDCs (i.e., mature DCs), Figure 2C shows the detection results of BMDCs treated with zinc sulfate at a zinc concentration of 3.27 mg / mL, Figure 2D shows the detection results of BMDCs treated with zinc sulfate at a zinc concentration of 6.54 mg / mL, Figure 2E shows the detection results of BMDCs treated with zinc sulfate at a zinc concentration of 13.08 mg / mL, and Figure 2F shows the detection results of BMDCs treated with glycine chelated zinc (CMC-Zn) solution at a zinc concentration of 3.27 mg / mL. BMDC test results, Figure 2G is the BMDC test results treated with a CMC-Zn solution with a zinc concentration of 6.54 mg / mL, Figure 2H is the BMDC test results treated with a CMC-Zn solution with a zinc concentration of 13.08 mg / mL, Figure 2I is the BMDC test results treated with a modified CMC-Zn solution with a zinc concentration of 3.27 mg / mL, Figure 2J is the BMDC test results treated with a modified CMC-Zn solution with a zinc concentration of 6.54 mg / mL, and Figure 2K is the BMDC test results treated with a modified CMC-Zn solution with a zinc concentration of 13.08 mg / mL.

[0022] Figure 3 is a line graph showing the average weight percentage of the two paws of rats over time, wherein the control group is untreated healthy rats, the CIA group is rats with collagen-induced arthritis, the MTX group is arthritic rats administered with MTX from day 17, the CMC-Zn group is arthritic rats administered with CMC-Zn solution from day 17, and the modified CMC-Zn group is arthritic rats administered with modified CMC-Zn solution from day 17.

[0023] Figure 4 is a bar graph showing the average weight percentage of the two paws of rats on day 24. The treatment conditions of the rats in each group were the same as those in Figure 3. "*" indicates a significant difference compared with the control group, P < 0.05; "#" indicates a significant difference compared with the CIA group, P < 0.05. DETAILED DESCRIPTION

[0024] Some specific embodiments according to the present invention will be described below; however, without departing from the spirit of the present invention, the present invention can also be practiced in a variety of different forms of solutions, and the scope of protection of the present invention should not be interpreted as being limited to the specific embodiments described in the description.

[0025] Unless otherwise indicated, the use of "a," "an," "the," and similar terms in this specification (especially in the claims) should be understood to include both the singular and the plural. As used herein, an "individual" may be a human or a non-human mammal. Examples of non-human mammals include, but are not limited to, cattle, horses, sheep, pigs, donkeys, mules, dogs, cats, rabbits, mice (e.g., hamsters, guinea pigs, etc.), apes, monkeys, and gorillas.

[0026] Herein, the numerical range used (e.g., 5 to 100) should be understood to also include all rational numbers in the range and ranges consisting of any rational numbers in the range. Therefore, the numerical range used in this specification includes all possible combinations of numerical values ​​between the lowest value and the highest value listed.

[0027] As mentioned above, the inventors of this application have discovered that zinc amino acid chelates have excellent inhibitory effects on DC maturation and can alleviate symptoms when administered directly to individuals suffering from autoimmune diseases. Therefore, the present invention relates to the use of zinc amino acid chelates for immunomodulation and / or inhibition of DC maturation.

[0028] Amino acid chelated zinc

[0029] The amino acid chelated zinc used in the present invention can be a commercially available product or obtained through a synthetic method. Methods for synthesizing amino acid chelated zinc are known to those skilled in the art. For example, amino acid chelated zinc can be obtained by physical mixing and / or heating to produce a chelation reaction between the amino acid and zinc. In the amino acid chelated zinc used in the present invention, the molar ratio of zinc to amino acid can be 1:1 to 1:10, for example, a molar ratio of zinc to amino acid of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (zinc:amino acid). In some embodiments, the molar ratio of zinc to amino acid is 1:1 to 1:4.

[0030] There are no particular limitations on the amino acids used to prepare the zinc amino acid chelate used in the present invention, as long as they do not affect the intended efficacy of the present invention (i.e., immunomodulation and / or inhibition of DC maturation). For example, glycine, alanine, valine, leucine, and / or isoleucine can be used to prepare the zinc amino acid chelate, but the present invention is not limited thereto. The zinc amino acid chelate used in the present invention can be prepared using zinc-containing inorganic compounds as a zinc source. Examples of suitable zinc-containing inorganic compounds include, but are not limited to, zinc sulfate, zinc carbonate, zinc oxide, zinc chloride, and the like.

[0031] In some embodiments, the amino acid chelated zinc used in the present invention can be prepared by sequentially mixing an amino acid and a zinc source, refrigerating, and heating. The refrigeration step preferably comprises placing the mixture obtained in the mixing step at 0°C to 4°C for at least 8 hours, for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, or 48 hours, or a range between any two of the foregoing values. The heating step is preferably performed by heating the refrigerated mixture at 50°C to 100°C for 6 to 24 hours, for example, at 50°C, 52.5°C, 55°C, 57.5°C, 60°C, 62.5°C, 65°C, 67.5°C, 70°C, 72.5°C, 75°C, 77.5°C, 80°C, 82.5°C, 85°C, 87.5°C, 90°C, 92.5°C, 95°C, 97.5°C, or 100°C for 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, or 15 minutes. Hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, 14.5 hours, 15 hours, 15.5 hours, 16 hours, 16.5 hours, 17 hours, 17.5 hours, 18 hours, 18.5 hours, 19 hours, 19.5 hours, 20 hours, 20.5 hours, 21 hours, 21.5 hours, 22 hours, 22.5 hours, 23 hours, 23.5 hours, or 24 hours. Suitable heating temperatures and heating times may also be within a range consisting of any two of the above values. In some embodiments, a drying step (e.g., heat drying or freeze drying) may be further performed after the heating step to provide the amino acid chelated zinc in powder form.

[0032] Compositions for immunomodulation / inhibition of DC maturation

[0033] As mentioned above, amino acid chelated zinc has the effect of immunoregulation and / or inhibition of DC maturation, and can therefore be used to provide a composition for immunoregulation / inhibition of DC maturation.

[0034] Therefore, in one embodiment of the present invention, a composition for immunomodulation and / or inhibition of DC maturation is provided, comprising an effective amount of zinc amino acid chelate. In some embodiments of the composition provided by the present invention, the zinc amino acid chelate can be mixed with water (e.g., sterile water, purified water) or an aqueous solution to form an aqueous composition. Examples of such aqueous solutions include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and acidic aqueous solutions (e.g., aqueous sulfuric acid, aqueous hydrochloric acid, aqueous phosphoric acid, aqueous acetic acid, and aqueous citric acid). The acidic aqueous solution is preferably a food-grade acidic aqueous solution, such as food-grade aqueous sulfuric acid, food-grade aqueous hydrochloric acid, or food-grade aqueous acetic acid. In some embodiments, the zinc amino acid chelate is mixed with an acidic aqueous solution to provide an aqueous composition having a pH less than 7, such as a pH of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 6.9, or a range between any two of the foregoing values.

[0035] The pH of the composition can be adjusted as needed based on the actual use requirements of the composition (e.g., the subject to be administered, the method of administration, etc.). For example, when the composition is orally administered to humans or mice / rats, the composition can be an aqueous composition with a pH of 2; when the composition is orally administered to adult pigs, the composition can be an aqueous composition with a pH of 3.5; and when the composition is orally administered to suckling pigs, the composition can be an aqueous composition with a pH of 4.5.

[0036] The amount of zinc amino acid chelate in the composition provided herein can be determined based on the individual's daily zinc intake requirement. Generally, the recommended daily zinc intake for healthy adult women is 12 mg, and for healthy adult men is 15 mg, with an upper limit of 35 mg. Furthermore, the amount of zinc amino acid chelate in the composition can be adjusted as needed based on the desired administration frequency (e.g., once daily, several times daily, or once every few days).

[0037] In some embodiments of the composition provided by the present invention, the composition is a pharmaceutical composition, and the pharmaceutical composition can be used to treat allergic diseases, treat autoimmune diseases and / or prevent organ transplant rejection, or to alleviate the discomfort caused by the aforementioned diseases. Examples of allergic diseases include, but are not limited to, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, urticaria, allergic gastroenteritis, and asthma. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, and ankylosing spondylitis.

[0038] The pharmaceutical composition provided by the present invention can be used for systemic administration or local administration, and can be delivered by various drug delivery systems (drug delivery system, DDS), suitable drug delivery systems include but are not limited to oral drug delivery systems (oral drug delivery system) and injection drug delivery systems (injectable drug delivery system). For example, but not limited to, the pharmaceutical composition can be administered orally, intravenously (including drip infusion and rapid injection), intramuscularly, subcutaneously, intraarterially or intraperitoneally to an individual in need. In addition, the pharmaceutical composition provided by the present invention can be delivered in the form of liposomes (liposomes), microcapsules (microcapsules), nanoparticles (nanoparticles), etc., to achieve the effects of improving bioavailability, controlling drug release rate, accurately administering medicine for lesions, reducing drug side effects, etc.

[0039] The pharmaceutical compositions provided according to the present invention may be in any suitable form, without particular limitation, and may be presented in a suitable dosage form depending on the intended use. Examples include, but are not limited to, suspensions, emulsions, solutions (e.g., syrups, elixirs, tinctures, etc.), coated tablets, tablets, granules, powders, capsules, pellets, pills, and injections. Depending on the form and intended use, the pharmaceutical compositions may be provided with pharmaceutically acceptable excipients, which are well known to those skilled in the pharmaceutical art and include, but are not limited to, diluents, surfactants, glidants, disintegrants, binders, buffers, colorants, flavorings, antioxidants, preservatives, and film-forming agents.

[0040] In some embodiments of the composition provided herein, the composition can be a food composition, and the food composition can be used for immunomodulation. The food composition can be a beverage, a solid food, or a semi-solid food, and can be provided in the form of a health food, a dietary supplement, a functional food, a nutritional supplement, or a special nutritional food. In some embodiments, the food composition is a health food.

[0041] Depending on the intended use and needs, the food composition provided by the present invention may contain any suitable food additive. Examples of such food additives include, but are not limited to, preservatives, bactericides, antioxidants, bleaching agents, color preservatives, bulking agents, nutritional additives, colorants, flavorings (e.g., sweeteners), thickeners, binders, food industry chemicals, emulsifiers, and agents for quality improvement, brewing, and food manufacturing.

[0042] Methods for immunomodulation / inhibition of DC maturation

[0043] In another embodiment of the present invention, a method for immunomodulation and / or inhibition of DC maturation is provided, comprising administering an effective amount of the composition described above to a subject in need thereof.

[0044] In the method of immunomodulation and / or inhibition of DC maturation according to the present invention, the subject in need is a subject with immune imbalance (especially abnormal immune activation) (eg, a patient suffering from an autoimmune disease) or a subject who has received an organ transplant.

[0045] Combinations for preparing tolerogenic DCs

[0046] In another embodiment of the present invention, a combination is provided, comprising: (1) an amino acid chelated zinc; and (2) a dendritic cell culture medium, wherein the combination can be used to prepare tolerogenic DCs, and the amino acid chelated zinc is as defined above.

[0047] In the combinations provided herein, the dendritic cell culture medium is not particularly limited, as long as it can provide the nutrients required for culturing dendritic cells. In some embodiments, the dendritic cell culture medium is RPMI-1640 medium containing interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fetal bovine serum (FBS).

[0048] In some embodiments of the combination provided by the present invention, the combination further comprises dendritic cells, and the dendritic cells are preferably immature dendritic cells.

[0049] In some embodiments of the combination provided by the present invention, the content of the amino acid chelated zinc is 1 mg to 25 mg of zinc per ml of the dendritic cell culture fluid, based on the content of the dendritic cell culture fluid. For example, based on the content of the dendritic cell culture fluid, the content of the amino acid chelated zinc is 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg per ml of the dendritic cell culture fluid. g, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg, 17 mg, 17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21 mg, 21.5 mg, 22 mg, 22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg, or 25 mg, or a range consisting of any two of the above values.

[0050] The combination provided by the present invention can be a set or a composition. When the combination provided by the present invention is a set, the component (1) amino acid chelated zinc and the component (2) dendritic cell culture medium are typically packaged separately and stored in different containers (e.g., plastic bags, plastic bottles, glass bottles, ampoules), and can be shipped or sold separately, or can be combined and distributed and sold together as a set. The set may also include an instruction manual to facilitate the user to mix the components on-site for cell culture, processing, and administration according to the procedures and processes provided therein.

[0051] When component (1) and component (2) are packaged separately and stored in different containers, they can be stored under different environmental conditions. For example, but not limited to, the container containing component (1) can be stored at room temperature, 0°C to 7°C, or -20°C, and the container containing component (2) can be stored at 0°C to 7°C.

[0052] In addition, when the combination provided by the present invention is a composition, the component (1) amino acid chelated zinc and the component (2) dendritic cell culture medium are usually mixed and stored in the same container (e.g., plastic bag, plastic bottle, glass bottle, ampoule).

[0053] When the combination provided by the present invention further comprises dendritic cells, the dendritic cells can be stored in a cell storage container (e.g., a cryopreservation tube) known in the art. In some embodiments, the dendritic cells are mixed with a cell cryopreservation solution and placed in a cryopreservation tube for storage at -80°C or in liquid nitrogen. Such a cryopreservation solution is known to those skilled in the art, for example, a cell culture medium containing 5% to 10% by volume of dimethyl sulfoxide (DMSO) or a commercially available cell cryopreservation solution.

[0054] Method for preparing tolerogenic DCs

[0055] In another embodiment of the present invention, a method for preparing tolerogenic DCs is provided, comprising contacting a dendritic cell with an effective amount of amino acid-chelated zinc.

[0056] In some embodiments, the contacting is performed by culturing the dendritic cells in a dendritic cell culture medium containing amino acid chelated zinc, wherein the amino acid chelated zinc, the dendritic cells, and the dendritic cell culture medium are as defined above.

[0057] The present invention is further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of the present invention. The scope of the present invention is as set forth in the claims.

[0058] Example

[0059] The materials and instruments used in the examples herein are listed below:

[0060] (1) Zinc sulfate: purchased from SIGMA, product number: 1282101001.

[0061] (2) PBS: purchased from Corning, product number: 21-040-CM.

[0062] (3) Mice: purchased from Lesco Biotechnology Co., Ltd., strain: C57BL / 6.

[0063] (4) Lewis rats: purchased from Lesco Biotechnology Co., Ltd., 7 weeks old, female.

[0064] (5) RPMI-1640 culture medium: purchased from Thermo Fisher Scientific, product number: 11875093.

[0065] (6) RBC lysis buffer: purchased from SIGMA, product number: R7757.

[0066] (7) DC culture medium: RPMI-1640 medium containing 500 U / mL IL-4 (purchased from Thermo Fisher, product number: 214-14), 1000 U / mL GM-CSF (purchased from Thermo Fisher, product number: 315-03) and 10% volume of FBS (purchased from Thermo Fisher, product number: A3160402).

[0067] (8) Flow cytometer: CytoFLEX S, Beckman.

[0068] (9) Collagen: Immunization Grade Bovine Type II Collagen purchased from Chondrex.

[0069] (10) Freund's Incomplete Adjuvant (IFA): purchased from Sigma.

[0070] (11) Interferon-γ (IFN-γ): purchased from Thermo Fisher Scientific, product number: 315-05.

[0071] (12) R848 (resiquimod): purchased from MCE, product number: HY-13740.

[0072] (13) Polyinosinic-Polycytidylic acid (poly I:C): purchased from MCE, product number: HY-107202.

[0073] (14) Lipopolysaccharide (LPS): purchased from SIGMA, product number: L2630.

[0074] (15) Flow cytometry analysis reagents and antibodies: FcR blocking reagent (FcRBlocking Reagent; purchased from MACS, product number: 130-092-575), CD11c antibody (purchased from BioLegend, product number: 117309), CD40 antibody (purchased from BioLegend, product number: 124607), CD80 antibody (purchased from BioLegend, product number: 104705), CD83 antibody (purchased from BioLegend, product number: 121509), CD86 antibody (purchased from BioLegend, product number: 105031), MHC II antibody (purchased from BioLegend, product number: 107631), isotype control antibody (isotype control; purchased from BioLegend, product number: 400905, 400411, 400535).

[0075] (16) IL12p70 ELISA kit: purchased from Arigo, product number: ARG80201, including a 96-well plate pre-coated with IL12p70 antibody, IL12p70 standard, wash buffer, antibody conjugate, HRP-Streptavidin solution, TMB reagent, and stop solution. Before use, each reagent / solution was placed at room temperature for 20 minutes.

[0076] (17) Methotrexate (MTX): purchased from MCE Company, product number: HY-14519.

[0077] [Preparation Example]

[0078] A. Preparation of Amino Acid Chelated Zinc

[0079] The amino acid chelated zinc used in the examples was prepared as follows: food-grade zinc sulfate and food-grade glycine were sieved through a 30-mesh sieve and mixed in a molar ratio of 1:1 to 1:4; the resulting mixture was refrigerated at 0°C to 4°C for 12 to 24 hours; and the refrigerated mixture was heated at 80°C for 12 hours to obtain glycine chelated zinc powder.

[0080] The glycine chelated zinc powder was prepared using DC culture medium (Example 1, cell experiments) to prepare solutions with zinc concentrations of 13.08 mg / mL, 6.54 mg / mL, and 3.27 mg / mL, respectively. Alternatively, the glycine chelated zinc powder was dissolved in 2 mL of PBS (Example 2, animal experiments) to obtain a solution containing 0.075 mg of zinc (hereinafter referred to as "CMC-Zn"). Furthermore, the CMC-Zn solutions with different zinc concentrations were further adjusted to pH 2 using citric acid (hereinafter referred to as "modified CMC-Zn").

[0081] B. Preparation of Mouse Bone Marrow Dendritic Cells (BMDCs)

[0082] Mice were euthanized with carbon dioxide and immersed in 75% ethanol. After immersion for 3 minutes, the mice were removed from the alcohol, dried, and laid flat on a workbench. Next, the skin and muscle were carefully removed to completely remove the femur and tibia. A 100-micron cell sieve was placed in a 3-cm culture dish and 2 mL of RPMI-1640 medium was added. The femur was cut open at both ends. A 26G syringe was used to draw up RPMI-1640 medium from the dish and inserted into the bone. The bone marrow was flushed onto the cell sieve, and this was repeated several times until the bone appeared pure white. The bone marrow from the tibia was also flushed onto the cell sieve using the same method. The cell sieve was then scraped several times with the back of a 1-mL micropipette tip to break up any cell clumps in the bone marrow. The cell sieve was then rinsed several times with the culture medium from the dish. The culture medium from the dish was then collected into a centrifuge tube and centrifuged at 1500 rpm for 3 minutes. After centrifugation, the supernatant was removed and 1 mL of red blood cell lysis buffer was added to resuspend the red pellet. After standing for 5 minutes, the pellet was centrifuged again at 1500 rpm for 3 minutes. The supernatant was removed to obtain a nearly milky white pellet. The pellet was resuspended in DC culture medium and the resulting suspension was counted.

[0083] The cell concentration of the suspension was adjusted to 5 × 10 cells per ml. 5cells, and take a 24-well plate, add 1 mL of suspension to each well. The 24-well plate was cultured at 37 ° C and 5% CO2. After 2 days of culture, the culture medium was carefully removed to remove the unattached multinuclear balls (granulocytes), and 0.5 mL of DC culture medium was added to each well. After 3 days of culture, 0.5 mL of DC culture medium was replaced again and cultured for another 3 days. The 24-well plate was removed from the incubator, the culture medium was carefully removed, and 2 mL of PBS was used to gently rinse the bottom of each well (pipetting) several times. The rinsed PBS was collected into a centrifuge tube, the cells were counted, and the CD11c expression of the obtained cells was analyzed by flow cytometry.

[0084] As shown in FIG1 , flow cytometric analysis confirmed that the CD11c expression level of the obtained cells was greater than 70%, and they could be used as BMDCs for subsequent experiments.

[0085] C. Preparation of Rheumatoid Arthritis Rats

[0086] Lewis rats were housed at a temperature of 21°C to 25°C, a humidity of 40% to 70%, and a 12-hour light schedule (7:00 AM to 7:00 PM). The rats were allowed free access to food. After at least six days of observation, and confirming that the rats were healthy, the collagen-induced arthritis (CIA) model was prepared.

[0087] Collagen and IFA were homogenized in an ice-water bath at a 1:1 volume ratio using a homogenizer to produce an induction emulsion. On day 1 of the experiment, 200 μL of this emulsion was subcutaneously injected at the base of the rats' tails. On day 8 of the experiment, 100 μL of this emulsion was subcutaneously injected at a different injection site at the base of the rats' tails. Successful CIA induction was confirmed in all rats on day 17 of the experiment.

[0088] Example 1: Effect of amino acid chelated zinc on dendritic cell maturation

[0089] IL12p70 is secreted at very low levels by immature DCs but is secreted in large quantities after cell maturation, making it a useful indicator for monitoring DC maturation. In this example, BMDC surface markers (CD11c, CD40, CD83, and CD86) were analyzed by flow cytometry, and IL12p70 in BMDC culture medium was detected by ELISA to observe the effect of amino acid chelated zinc on dendritic cell maturation.

[0090] (1-1) Cell treatment

[0091] The BMDCs obtained in Preparation Example B were placed at 1×10 5The cells were seeded in a 24-well plate at a concentration of 10 cells / mL and cultured at 37°C, 5% CO2 for 48 hours in 500 μL of the following culture medium:

[0092] (1) Immature DC group: DC culture medium;

[0093] (2) Mature DC group: DC culture medium containing 1000 U / mL IFN-γ, 2.5 μg / mL R848, 20 μg / mL poly I:C, and 10 ng / mL LPS;

[0094] (3) Zinc sulfate (low dose) group: DC culture medium containing zinc sulfate with a zinc content of 3.27 mg / mL;

[0095] (4) Zinc sulfate (medium dose) group: DC culture medium containing zinc sulfate with a zinc content of 6.54 mg / mL;

[0096] (5) Zinc sulfate (high dose) group: DC culture medium containing zinc sulfate with a zinc content of 13.08 mg / mL;

[0097] (6) CMC-Zn (low dose) group: CMC-Zn with a zinc content of 3.27 mg / mL as provided in Preparation Example A;

[0098] (7) CMC-Zn (medium dose) group: CMC-Zn with a zinc content of 6.54 mg / mL as provided in Preparation Example A;

[0099] (8) CMC-Zn (high dose) group: CMC-Zn with a zinc content of 13.08 mg / mL as provided in Preparation Example A;

[0100] (9) Modified CMC-Zn (low dose) group: prepared from the modified CMC-Zn provided in Example A with a zinc content of 3.27 mg / mL;

[0101] (10) Modified CMC-Zn (medium dose) group: Modified CMC-Zn with a zinc content of 6.54 mg / mL as provided in Preparation Example A; and

[0102] (11) Modified CMC-Zn (high dose) group: The modified CMC-Zn provided in Preparation Example A with a zinc content of 13.08 mg / mL.

[0103] After 48 hours of culture, cells and culture medium from each group were collected for subsequent analysis.

[0104] (1-2) Flow cytometric analysis

[0105] Count the cells obtained in (1-1) and adjust the cell concentration to 1×10 cells per 100 μL using PBS. 5 cells to 5×10 5 Take 100 μL of the cell suspension and mix it evenly with 20 μL of FcR blocking reagent, and place the resulting mixture at 4 ° C for 10 minutes. Then, add CD11c antibody, CD40 antibody, CD80 antibody, CD83 antibody, CD86 antibody, MHC II antibody and corresponding isotype control antibody respectively, shake and mix for 1 second, and place it at room temperature in the dark for 15 minutes. Then, add 300 μL of PBS, shake and mix for 1 second, and centrifuge at 200xg for 5 minutes. Remove the supernatant after centrifugation and resuspend the cells with 300 μL of PBS. Finally, the obtained cell suspension was detected by flow cytometry, and each group of cell suspensions was kept on ice in the dark until detection.

[0106] As shown in Table 1 and Figures 2A to 2K below, flow cytometric analysis of dendritic cell maturation markers (CD40, CD80, CD83, CD86, and MHC II) following maturation induction and / or treatment with zinc sulfate, CMC-Zn, or modified CMC-Zn revealed elevated expression of each marker following maturation induction, demonstrating the stability of the research platform. The mature DC group and all zinc sulfate, CMC-Zn, and modified CMC-Zn treatment groups exhibited inhibitory effects on DC maturation, with the effect proportional to zinc concentration. CMC-Zn and modified CMC-Zn effectively inhibited DC maturation at low doses, with marker expression even lower than that of immature DC. Compared to the zinc sulfate-treated group, CMC-Zn and modified CMC-Zn significantly decreased CD80 and CD86 expression at both low and medium doses. This result demonstrates that CMC-Zn and modified CMC-Zn are superior to zinc sulfate in inhibiting DC maturation. The above experimental results prove that amino acid chelated zinc can indeed effectively inhibit DC maturation, so it can be used for immune regulation such as treating allergic diseases, treating autoimmune diseases, preventing organ transplant rejection, and promoting the generation of tolerant DC that can act as an immunosuppressant.

[0107] Table 1

[0108] (1-3) ELISA analysis

[0109] The culture fluids from each group obtained in (1-1) were placed in a centrifuge tube, centrifuged at 400 x g for 10 minutes at 4°C, and the supernatant was collected. The IL12p70 standard was serially diluted for use in drawing a standard curve. Each concentration of standard and each group of supernatants were added to a 96-well plate pre-coated with IL12p70 antibody (100 μL per well, duplicate), the 96-well plate was tapped to evenly mix the liquid in each well, and the 96-well plate was placed in a 37°C incubator for 1.5 hours. Next, 350 μL of 1x wash buffer was added to each well, soaked for 30 seconds, and the liquid in each well was removed. This washing step was repeated 4 times. After washing, 100 μL of 1X antibody conjugate was added to each well, the 96-well plate was tapped to evenly mix the liquid in each well, and the 96-well plate was placed in a 37°C incubator for 1 hour. 1X wash buffer was used again for 5 washing steps. Then, 100 μL of 1XHPR-streptavidin solution was added to each well, and after tapping to mix, the 96-well plate was placed in a 37°C incubator in the dark. After standing for 30 minutes, 5 washing steps were performed again. Then 100 μL of TMB reagent was added to each well, and after tapping to mix, the 96-well plate was placed in a 37°C incubator in the dark for 15 minutes. The 96-well plate was removed from the incubator, 100 μL of stop solution was added to each well, and the absorbance value (OD450nm) of each well at a wavelength of 450 nanometers (nm) was detected within 3 minutes. The standard curve drawn based on the absorbance value of each concentration standard was then used to obtain the IL12P70 content corresponding to each group of absorbance values. The results are shown in Table 2.

[0110] As shown in Table 2 below, IL12P70 was not detected in the cell culture medium of the mature DC group, nor in the immature DC group, zinc sulfate, CMC-Zn, or modified CMC-Zn groups. This experimental result further demonstrates that amino acid chelated zinc can effectively inhibit DC maturation, and therefore has potential applications in immunomodulation, such as the treatment of allergic diseases, autoimmune diseases, and the prevention of organ transplant rejection. It can also be used to promote the generation of tolerant DCs, which can act as immunosuppressants.

[0111] Table 2

[0112] *ND: not detected

[0113] Example 2: Effects of Amino Acid Chelated Zinc on Autoimmune Diseases

[0114] Rheumatoid arthritis is an autoimmune disease in which the patient's immune system becomes abnormally activated, attacking the body's own cells and tissues, leading to a long-term chronic inflammation. This example uses the CIA animal model to observe the therapeutic effect of amino acid chelated zinc on autoimmune diseases such as rheumatoid arthritis.

[0115] As described in [Preparation Example] C, successful CIA induction was confirmed on day 17 of the experiment. Therefore, the CIA rats were randomly divided into four groups starting on day 17 and administered the following solutions. Each group of rats was sacrificed on day 24 of the experiment. Healthy rats without arthritis induction served as the control group:

[0116] (1) Control group (5 healthy rats): 2 mL of PBS;

[0117] (2) CIA group (5 CIA rats): 2 mL of PBS;

[0118] (3) MTX group (3 CIA rats): 1 mg of MTX per kg of rat, dissolved in 2 mL of PBS;

[0119] (4) CMC-Zn group (5 CIA rats): CMC-Zn with a zinc content of 0.075 mg, dissolved in 2 mL of PBS; and

[0120] (5) Modified CMC-Zn group (5 CIA rats): Modified CMC-Zn with a zinc content of 0.075 mg was dissolved in 2 mL of PBS.

[0121] At the beginning of the experiment and on days 3, 7, 10, 14, 17, 21, and 24, the paws of each group of rats were observed and their volume measured. The severity of arthritis in the rats was assessed using the qualitative scoring system outlined in Table 3. Paw volume was measured based on Archimedes' principle of buoyancy. An appropriate amount of pure water was added to a beaker, placed on a scale, and the measurement was reset to zero. The rat's paw was then immersed in water to the desired depth, and the reading was recorded after stabilization.

[0122] Table 3

[0123] As shown in Figure 3, the paw weight percentage of all CIA rats, except for the healthy rats in the control group, increased significantly on day 17 of the experiment, indicating that arthritis was successfully induced in all CIA rats. Compared to the CIA rats, the paw weight percentage of rats in the MTX, CMC-Zn, and modified CMC-Zn groups gradually decreased after administration of MTX, CMC-Zn, and modified CMC-Zn, respectively. This indicates that arthritis symptoms in CIA rats treated with MTX, CMC-Zn, and modified CMC-Zn gradually improved, with modified CMC-Zn even achieving an effect comparable to MTX. Figure 4 shows that on day 24 of the experiment, the paw weight percentage of all CIA rats increased significantly compared to the control rats, and the paw weight percentage of rats in the MTX, CMC-Zn, and modified CMC-Zn groups decreased significantly compared to untreated CIA rats. The above experimental results indicate that administering amino acid chelated zinc to individuals can effectively alleviate the symptoms of rheumatoid arthritis and therefore can be used to treat autoimmune diseases such as rheumatoid arthritis.

[0124] Table 4 shows the mean arthritis severity scores for each group of rats at the start of the experiment (day 0) and on days 3, 7, 10, 14, 17, 21, and 24, as assessed using the criteria listed in Table 3. Compared to the CIA group, the rate of increase in arthritis severity in the MTX, CMC-Zn, and modified CMC-Zn groups significantly slowed or even decreased after day 17 (i.e., after administration of MTX, CMC-Zn, or modified CMC-Zn). In particular, the arthritis severity scores of rats in the modified CMC-Zn group were significantly lower than those in the MTX group.

[0125] Table 4

[0126] From the above experimental results, it can be seen that amino acid chelated zinc can not only inhibit DC maturation, but also effectively alleviate the related symptoms of rheumatoid arthritis individuals. Therefore, it can be used for immune regulation such as the treatment of allergic diseases, the treatment of autoimmune diseases, and the prevention of organ transplant rejection, and can be used to promote the generation of tolerant DCs that can serve as immunosuppressants.

[0127] The above embodiments are intended merely to illustrate the principles and efficacy of the present invention and to illustrate its technical features, and are not intended to limit the scope of protection of the present invention. Any modifications or arrangements readily achievable by one skilled in the art without violating the technical principles and spirit of the present invention are within the scope of the present invention. Therefore, the scope of protection of the present invention is as set forth in the claims.

Claims

1. Use of amino acid chelated zinc in the preparation of a composition, characterized in that, The composition is used for immunomodulation.

2. The use according to claim 1, characterized in that, The composition is an aqueous composition and has a pH value less than 7.

3. The use according to claim 1, characterized in that, The molar ratio of zinc to amino acid in the zinc amino acid chelate is 1:1 to 1:

10.

4. The use according to claim 1, characterized in that, The zinc amino acid chelate is zinc glycinate, zinc alaninate, zinc valinate, zinc leucinate and / or zinc isoleucinate.

5. The use according to any one of claims 1 to 4, characterized in that, The composition is a pharmaceutical composition, and the pharmaceutical composition is used for treating allergic diseases, treating autoimmune diseases, and / or preventing organ transplant rejection.

6. Use of amino acid chelated zinc in the preparation of a composition, characterized in that, The composition is used to inhibit the maturation of dendritic cells.

7. The use according to claim 6, characterized in that, The composition is an aqueous composition and has a pH value less than 7.

8. The use according to claim 6, characterized in that, The molar ratio of zinc to amino acid in the zinc amino acid chelate is 1:1 to 1:

10.

9. The use according to claim 6, characterized in that, The zinc amino acid chelate is zinc glycinate, zinc alaninate, zinc valinate, zinc leucinate and / or zinc isoleucinate.

10. A combination, characterized in that, It comprises: (1) A zinc amino acid chelate; and (2) A dendritic cell culture solution.

11. The combination according to claim 10, wherein, The molar ratio of zinc to amino acid in the zinc amino acid chelate is 1:1 to 1:

10.

12. The combination according to claim 10, wherein The zinc amino acid chelate is zinc glycinate, zinc alaninate, zinc valinate, zinc leucinate and / or zinc isoleucinate.

13. The combination according to claim 10, wherein It further comprises dendritic cells.

14. The combination according to any one of claims 10 to 13, characterized in that, Based on the content of the dendritic cell culture solution, the content of the zinc amino acid chelate is 1 mg to 25 mg of zinc per milliliter of the dendritic cell culture solution.

15. A composition for immunomodulation, characterized in that, It contains an effective amount of zinc amino acid chelate.

16. The composition according to claim 15, characterized in that, It is an aqueous composition and has a pH value less than 7.

17. The composition according to claim 15, wherein The molar ratio of zinc to amino acid in the zinc amino acid chelate is 1:1 to 1:

10.

18. The composition according to claim 15, characterized in that, The zinc amino acid chelate is zinc glycinate, zinc alaninate, zinc valinate, zinc leucinate and / or zinc isoleucinate.

19. The composition according to any one of claims 15 to 18, characterized in that, It is a pharmaceutical composition, and the pharmaceutical composition is used for treating allergic diseases, treating autoimmune diseases, and / or preventing organ transplant rejection.

20. A composition for inhibiting the maturation of dendritic cells, characterized in that, It contains an effective amount of zinc amino acid chelate.

21. The composition according to claim 20, wherein It is an aqueous composition and has a pH value less than 7.

22. The composition according to claim 20, wherein The molar ratio of zinc to amino acid in the zinc amino acid chelate is 1:1 to 1:

10.

23. The composition according to claim 20, wherein, The zinc amino acid chelate is zinc glycinate, zinc alaninate, zinc valinate, zinc leucinate and / or zinc isoleucinate.

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