Chemically modified polysaccharide and immunomodulatory composition comprising same

Abstract

The present invention relates to a chemically modified polysaccharide and an immunomodulatory composition comprising same. Specifically, the present invention relates to: a chemically modified immunomodulatory polysaccharide; an immunomodulatory composition comprising the chemically modified immunomodulatory polysaccharide as an active ingredient; and a pharmaceutical composition for preventing or treating cancer having immune-enhancing activity and a pharmaceutical composition for preventing or treating autoimmune diseases having immunosuppressive activity, both comprising the chemically modified immunomodulatory polysaccharide as an active ingredient, wherein the polysaccharide is modified by substituting at least one hydroxyl group with an aldehyde group, a hydroxyl group, an acetyl group, a sulfuric acid group, or a phosphoric acid group.

Description

Chemically modified polysaccharide and immunomodulatory composition containing the same

[0001] The present invention relates to a chemically modified polysaccharide and an immunomodulatory composition comprising the same.

[0002] The immune system is the body's defense system that protects against various pathogens (antigens) such as external bacteria, viruses, fungi, and various harmful substances. It is involved in every part and function of the body. However, an imbalance in the immune system can lead to various diseases. A weakened immune system can expose you to infections, colds, AIDS, and cancer. Conversely, an overactive immune system can lead to allergies, arthritis, and other hyperimmune and autoimmune diseases. Therefore, maintaining a healthy immune system is crucial.

[0003] Meanwhile, polysaccharides function as pathogen-associated molecular patterns (PAMPs) in the body, and are recognized by pattern recognition receptors (PRRs) of innate immune cells, triggering specific immune responses. Attempts have been made to utilize this specific immune response as disease-specific immunostimulators, but their low immunogenicity limits their ability to elicit sufficient immunogenicity. Therefore, the development of technologies that can overcome these limitations is urgently needed.

[0004] Accordingly, the inventors of the present invention have completed the present invention by finding that when a polysaccharide is chemically modified, branching and heterogeneous structures can be formed by the synthesized functional groups, thereby inducing complexity in the chemical structure, and thereby increasing recognition of pattern recognition receptors of innate immune cells compared to existing polysaccharides, thereby overcoming low immunogenicity, and elucidating the specific immunomodulatory ability according to the type of polysaccharide modification.

[0005] Therefore, the purpose of the present invention is to provide a chemically modified immunomodulatory polysaccharide, characterized in that the polysaccharide is modified by substituting at least one hydroxyl group with an aldehyde group, a hydroxyl group, an acetyl group, a sulfate group, or a phosphoric acid group.

[0006] Another object of the present invention is to provide an immunomodulatory composition comprising the chemically modified immunomodulatory polysaccharide of the present invention as an active ingredient.

[0007] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer having an immunostimulating activity, comprising a chemically modified immunomodulatory polysaccharide as an active ingredient, wherein the chemically modified immunomodulatory polysaccharide is characterized in that at least one hydroxyl group is modified by substituting an aldehyde group, a hydroxyl group, or an acetyl group.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an autoimmune disease having immunosuppressive activity, comprising a chemically modified immunomodulatory polysaccharide as an active ingredient, wherein the chemically modified immunomodulatory polysaccharide is characterized in that at least one hydroxyl group is modified by substituting a sulfate group or a phosphate group.

[0009] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined solely by the scope of the claims.

[0010] The terminology used herein is for the purpose of describing embodiments only and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise. As used herein, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components in addition to the mentioned components. Like reference numerals refer to like components throughout the specification, and "and / or" includes each and any combination of one or more of the mentioned components. Although "first", "second", etc. are used to describe various components, these components are not limited by these terms. These terms are only used to distinguish one component from another. Therefore, it should be understood that a first component mentioned below may also be a second component within the technical spirit of the present invention.

[0011] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in their common sense to those skilled in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0012]

[0013] The present invention relates to a chemically modified polysaccharide and an immunomodulatory composition comprising the same.

[0014] Specifically, the present invention is characterized in that it provides a chemically modified immunomodulatory polysaccharide, wherein the polysaccharide is modified by substituting at least one hydroxyl group with an aldehyde group, a hydroxyl group, an acetyl group, a sulfate group, or a phosphoric acid group.

[0015] As previously mentioned in the prior art, polysaccharides act as pathogen-associated molecular patterns (PAMPs) in our body and are known to be recognized by pattern recognition receptors (PRRs) of innate immune cells and induce specific immune responses. Therefore, attempts have been made to use them as disease-specific immune activators, but there has been a problem that their effectiveness is minimal due to the problem of low immunogenicity.

[0016] Accordingly, the inventors of the present invention, while conducting research to develop an improved polysaccharide having an excellent immunoactivator by improving low immunogenicity, have discovered that a chemically modified polysaccharide in which at least one hydroxyl group of the polysaccharide is substituted with an aldehyde group, a hydroxyl group, an acetyl group, a sulfate group, or a phosphate group has the activity of effectively regulating an immune response.

[0017] Therefore, the present invention can provide a chemically modified immunomodulatory polysaccharide, and the immunomodulatory polysaccharide according to the present invention has a modified form in which at least one hydroxyl group is substituted with an aldehyde group, a hydroxyl group, an acetyl group, a sulfate group, or a phosphoric acid group.

[0018] The above modification may be oxidation, reduction, acetylation, sulfation or phosphorylation.

[0019] In addition, the polysaccharide may be selected from the group consisting of, but not limited to, mannan, glucomannan, galactomannan, chitosan, chitin, pectin, galactan, and chondroitin.

[0020] In one embodiment of the present invention, modified mannan was manufactured by modifying mannan, a type of polysaccharide, through oxidation, reduction, acetylation, sulfation, and phosphorylation, and the immunomodulatory ability of each modified mannan was analyzed.

[0021] As a result, it was shown that the modified mannan by oxidation, reduction or acetylation had an immunostimulatory effect that increased immune activity.

[0022] Specifically, according to one embodiment of the present invention, when oxidized, reduced or acetylated mannan was treated on dendritic cells, the expression of CD86, CD80 and CD40, which are activation markers of dendritic cells, was found to significantly increase.

[0023] These results suggest that oxidized, reduced, or acetylated polysaccharides can enhance the activity of dendritic cells and thereby enhance the immune response.

[0024] Dendritic cells (DCs) are the most potent antigen-presenting cells and the only cells capable of inducing a primary cellular immune response. Originating in the bone marrow, they migrate immaturely through the bloodstream to all organs of the body. Dendritic cells collect antigens from their surrounding tissues and travel to lymphoid organs, where they present these antigens to T lymphocytes.

[0025] Therefore, dendritic cells can strongly induce antigen-specific T cell responses in adaptive immunity, and the stimulation of dendritic cells in T cell responses is known to be particularly important in antitumor immune responses. Furthermore, dendritic cells capture tumor antigens characteristic of tumor cells and transport them to the nearest lymph node (draining lymph node). In the lymph node, dendritic cells present the tumor antigens they harbor to cytotoxic T lymphocytes through cross-presentation, thereby playing a crucial role in antitumor efficacy and being utilized in cancer immunotherapy.

[0026] In addition, according to one embodiment of the present invention, it was confirmed that when oxidized, reduced or acetylated mannan was treated to macrophages, a conversion from M2 macrophages to M1 macrophages occurred.

[0027] Macrophages can be polarized from M0 type to M1 type or M2 type, with M1 type being polarized by LPS or IFN-γ, and M2 type being polarized by IL-4, IL-6, IL-10, etc.

[0028] In the case of M1 type macrophages, they secrete proinflammatory cytokines and enzymes that cause an inflammatory response, specifically TNF-α, IL-1β, IL-6, MCP-1, and O 2- They promote the secretion of cytokines, ultimately causing inflammation and fighting infection. In other words, they activate the immune response. In the case of M2 macrophages, they promote the secretion of anti-inflammatory cytokines such as IL-10 or TGF-β, and suppress the secretion of pro-inflammatory cytokines such as TNFα, IL-1β, IL-6, and MCP-1. Therefore, M2 macrophages induce an anti-inflammatory response and act to promote healing.

[0029] Therefore, it was found that the oxidized, reduced or acetylated polysaccharide according to the present invention activates the immune response by inducing a conversion from M2 macrophages to M1 macrophages.

[0030] In this respect, the chemically modified polysaccharide provided in the present invention, i.e., the polysaccharide modified by oxidation, reduction or acetylation, can increase the immune response by enhancing the activity of dendritic cells and macrophages, and thus can be used for the treatment and prevention of diseases such as infectious diseases and cancer caused by a decrease in the immune response.

[0031] In addition, it was shown that the polysaccharide modified by sulfation or phosphorylation according to the present invention has an immunosuppressive action that suppresses or reduces immune activity.

[0032] According to one embodiment of the present invention, modified mannan was prepared by sulfating or phosphorylating mannan, a type of polysaccharide, and when this was treated on dendritic cells and macrophages, it was found that the sulfated or phosphorylated mannan inhibited the activity of dendritic cells, and also that the expression of CD86 and iNOS, markers of M1 macrophages, was reduced in macrophages.

[0033] According to one embodiment of the present invention, oxidized mannan modified with an oxidation rate of 30 to 40% increased CD86 gene expression by about 60 times and iNOS gene expression by about 35 times in M2 macrophages compared to the original mannan. Reduced mannan modified with a reduction rate of 15% increased iNOS gene expression by about 5 times in M2 macrophages. Acetylated mannan modified with an acetylation rate of 13% increased CD86 gene expression by about 200 times and iNOS gene expression by about 20 times in M2 macrophages compared to the original mannan. Sulfated mannan modified with a sulfation rate of 17% decreased iNOS gene expression by about 5 times in M1 macrophages compared to the original mannan. Phosphorylated mannan modified with a phosphorylation rate of 13% decreased iNOS expression rate by about 70 times in M1 macrophages.

[0034] Therefore, it was found that polysaccharides modified by sulfation or phosphorylation have the activity of suppressing immune responses, and it was found that they can be used to treat and prevent autoimmune diseases caused by excessive immune responses.

[0035] Therefore, through the experimental results of the present invention, the inventors were able to find out that there are differences in immunomodulatory ability depending on the type of modification of the polysaccharide, and among the chemically modified immunomodulatory polysaccharides according to the present invention, the polysaccharide modified by oxidation, reduction or acetylation has an immune activity enhancing effect and can be usefully used as an immune enhancer or immune adjuvant, whereas the polysaccharide modified by sulfation or phosphorylation has an immune response suppressing effect and can be usefully used as an immunosuppressant.

[0036] Therefore, the present invention can provide a pharmaceutical composition for preventing or treating cancer having an immune-enhancing activity, which comprises a chemically modified immunomodulatory polysaccharide as an active ingredient, wherein the chemically modified immunomodulatory polysaccharide is characterized in that at least one hydroxyl group is modified by substituting an aldehyde group, a hydroxyl group, or an acetyl group.

[0037] Furthermore, the present invention can provide a pharmaceutical composition for preventing or treating an autoimmune disease having immunosuppressive activity, which comprises a chemically modified immunomodulatory polysaccharide as an active ingredient, wherein the chemically modified immunomodulatory polysaccharide is characterized in that at least one hydroxyl group is modified by substituting a sulfate group or a phosphate group.

[0038] The body's immune system constructs a defense system comprised of various cells to protect the body from internal and external pathogens. This defense system is achieved through the specific functions of each cell and the communication between them. The human immune system can be broadly divided into immune tolerance, which suppresses and regulates immunity, and the immune response, which promotes immunity. These two immune functions maintain a balance, achieving immunological homeostasis. However, if this balance is disrupted for any reason, and the immune response becomes overactive compared to tolerance, autoimmune diseases and other conditions can occur.

[0039] In particular, autoimmune diseases are diseases caused by an immune response to self-antigens, which causes the body to attack its own tissues. The types of autoimmune diseases include, but are not limited to, rheumatoid arthritis, osteoarthritis, Behcet's disease, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Goodfeiter syndrome, autoimmune meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, sarcoidosis, scleroderma, These may include spondyloarthritis, pernicious anemia, scleroderma, mitochondrial-related syndromes, and inflammatory bowel disease.

[0040] Conversely, if the immune system is weakened, the body's natural healing function is gradually weakened, which can lead to various diseases such as infection by external pathogens, and a representative example is cancer.

[0041] In the case of diseases caused by a weakened immune system, it is necessary to enhance the immune response to increase the body's immunity.

[0042] Therefore, the chemically modified polysaccharide provided in the present invention can provide a modified polysaccharide having immunostimulating activity depending on the type of modification, and can also provide a modified polysaccharide having immunosuppressive activity.

[0043] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier. The composition including the pharmaceutically acceptable carrier may be administered orally or parenterally in various dosage forms. When formulated, it is prepared using commonly used diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0044] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

[0045] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0046] The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.

[0047] Additionally, these pharmaceutical compositions can be administered to treat the target disease as described above.

[0048] The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0049] The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Considering all of the above factors, it is important to administer an amount that achieves maximum efficacy with the minimum amount possible without causing side effects. A typical dosage for the pharmaceutical composition of the present invention is 0.001-100 mg / kg for adults.

[0050] The pharmaceutical composition may be administered via any conventional route as long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonary, or rectally, depending on the intended purpose, but is not limited thereto. Furthermore, the composition may be administered via any device capable of transporting the active ingredient to target cells.

[0051] The chemically modified polysaccharide provided in the present invention has a superior immunomodulatory property compared to unmodified polysaccharides. Modified polysaccharides obtained by oxidizing, reducing or acetylating polysaccharides have superior immunostimulating activity and can be usefully used as immunostimulants or immunoadjuvants, and modified polysaccharides obtained by sulfating or phosphorylating polysaccharides have superior activity in suppressing excessive immune responses and can be usefully used as immunosuppressants.

[0052] Figure 1 is a schematic diagram showing the change in structural formula due to chemical modification of a polysaccharide according to the present invention.

[0053] Figure 2 shows the FT-IR results after purification of a mixture of dextran and sodium borohydride using a PD-10 column.

[0054] Figure 3 shows the FT-IR results after centrifugal filter purification of a mixture of dextran and acetic anhydride.

[0055] Figure 4 shows the FT-IR results after centrifugal filter purification of a mixture of dextran and sulfur trioxide pyridine salt.

[0056] Figure 5 shows the FT-IR results after centrifugal filter purification of a mixture of dextran, sodium trimetaphosphate, and sodium triphosphate.

[0057] Figure 6 shows the results of measuring the size and surface charge of the oxidized mannan produced by the present invention at different concentrations.

[0058] Figure 7 shows the results of measuring the size and surface charge of the reduced mannan produced by the present invention at different concentrations.

[0059] Figure 8 shows the results of measuring the size and surface charge of the acetylated mannan produced in the present invention at different concentrations.

[0060] Figure 9 shows the results of measuring the size and surface charge of the sulfated mannan produced in the present invention at different concentrations.

[0061] Figure 10 shows the results of measuring the size and surface charge of the phosphorylated mannan produced in the present invention at different concentrations.

[0062] Figure 11 shows the effect of dendritic cell activation by the oxidized mannan produced in the present invention.

[0063] Figure 12 shows the effect of improving inflammatory response by oxidized mannan produced in the present invention.

[0064] Figure 13 shows the effect of dendritic cell activation by the reduced mannan produced in the present invention.

[0065] Figure 14 shows the effect of improving inflammatory response by the reduced mannan produced in the present invention.

[0066] Figure 15 shows the effect of improving inflammatory response by acetylated mannan produced in the present invention.

[0067] Figure 16 shows the effect of suppressing inflammatory response by the sulfated mannan produced in the present invention.

[0068] Figure 17 confirms the inhibitory effect of inflammatory response by phosphorylated mannan produced in the present invention.

[0069] Hereinafter, the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes modifications of technical concepts equivalent thereto.

[0070]

[0071] <Example 1>

[0072] Chemical modification of polysaccharides

[0073] The present inventors prepared a chemically modified polysaccharide by the following method.

[0074]

[0075] <1-1> Production and identification of oxidized or reduced polysaccharides

[0076] The structural changes of polysaccharides due to oxidation and reduction of polysaccharides are as shown in the structural formula below.

[0077]

[0078] <Overview of Oxidation and Reduction of Polysaccharides>

[0079]

[0080] Specifically, oxidation of polysaccharides was performed using the following method. 100 mg each of polysaccharides, mannan and dextran, were dissolved in 1 ml of distilled water (100 mg / ml), and 1 ml of 0.01 M / 0.02 M / 0.04 M sodium periodate (NaIO4) solution was added. After vortexing for 1 hour at 4°C to react, the oxidized mannan was purified by passing it through a PD-10 column to obtain sodium periodate, and the oxidized mannan was freeze-dried for 2-3 days and then stored.

[0081] In addition, for the reduction of polysaccharides, 100 mg of oxidized mannan was dissolved in 1 ml of distilled water (100 mg / ml), and 1 ml of 0.026 M sodium borohydride (NaBH4) solution was added. After vortexing for 3 hours at 4°C, the reduced mannan was purified by passing it through a PD-10 column to obtain sodium borohydride. The reduced mannan was freeze-dried for 2-3 days and then stored.

[0082] In order to confirm the oxidized and reduced polysaccharides as described above, a mixture of dextran and sodium borohydride was passed through a PD-10 column, and then approximately 200 μl was sequentially obtained and subjected to FT-IR analysis. As a result, among a total of 15 samples, sodium borohydride was confirmed to be produced in the 15th sample, confirming that sodium borohydride was purified (Fig. 2).

[0083]

[0084] In addition, the oxidation rate and reduction rate according to the modification of the above polysaccharide were analyzed. The oxidation rate was calculated by reacting the polysaccharide with different concentrations of sodium periodate and quantifying the aldehyde group using Schiff's reagent that specifically reacts with the aldehyde group. The reduction rate was calculated by reacting the oxidized mannan with different oxidation rates with 0.026 M sodium borohydride and quantifying the reduced aldehyde group using Schiff's reagent. The results of the oxidation rate and reduction rate of the oxidized polysaccharide are as shown in Tables 1 and 2 below.

[0085] Oxidation rate of oxidized polysaccharides Polysaccharide*Reaction rate Oxidation rate (%) Dextran 1:0.018 2.75 ±0.93 1:0.09 0 9.64 ±0.56 1:0.18 15.45 ±0.61:0.36 6.7 Mannan 1:0.018 5.24 ±1.32 1:0.07 216.83 1:0.09 0 3 6.06 ±3.26 1:0.18 61.05 1:0.36 73.67

[0086] *Reaction ratio; monosaccharide: molar ratio of sodium periodate reacted

[0087] Reduction rate of reduced polysaccharide Polysaccharide oxidation rate (%) Reduction rate (%) Mannan 6.98 ± 1.09 4.02 ± 0.53 17.67 ± 0.92 15.37 ± 0.02 23.82 23.15 49.88 43.77

[0088]

[0089] <1-2> Preparation and identification of acetylated polysaccharides

[0090] The structural change of the polysaccharide due to acetylation of the polysaccharide is as shown in the structural formula below.

[0091]

[0092] Overview of Acetylation of Polysaccharides

[0093]

[0094] Specifically, for acetylation of polysaccharides, 100 mg of mannan was dissolved in 3 ml of distilled water, the pH was adjusted to 9.0 with 10 M NaOH, and equilibrated for 10 minutes. 0.1 ml / 0.5 ml / 0.7 ml of acetic anhydride was added, the pH was adjusted to 8.0-8.5, and the reaction was continued for 30 minutes. The pH was then adjusted to 7.0 to terminate the reaction, and acetic anhydride was purified by centrifugation at 3900 rpm for 6 hours using a 3K Centrifugal filter. The acetylated mannan was freeze-dried for 2-3 days and then stored.

[0095] In addition, to confirm the acetylated polysaccharide, a mixture of dextran and acetic anhydride was centrifuged at 3900 rpm for 6 hours using a centrifugal filter, and it was confirmed that acetic anhydride was purified as no specific peak of acetic anhydride appeared in the centrifuged sample (Fig. 3).

[0096]

[0097] In addition, in order to confirm the acetylation rate according to the above polysaccharide modification, the polysaccharide was reacted with acetic anhydride at different concentrations, and the synthesized acetyl group was quantified using 1H NMR analysis to calculate the acetylation rate, and the results are as shown in Table 3 below.

[0098] Acetylation rate of acetylated polysaccharides Polysaccharide*Reaction ratio Acetylation rate (%) Dextran 1:7.6 2 3.84 ±0.12 Mannan 1:7.6 2 3.6 ±0.67 1:15.2 4 7.73 ±2.43 1:30.48 14.22 ±1.77 1:60.96 6 0.49

[0099] *Reaction ratio; molar ratio of monosaccharide: acetic anhydride reacted

[0100]

[0101] <1-3> Production and identification of sulfated polysaccharides

[0102] The structural change of polysaccharides due to sulfation of polysaccharides is as shown in the structural formula below.

[0103]

[0104] <Overview of Polysaccharide Sulfation>

[0105]

[0106] Specifically, for the sulfation of polysaccharides, 100 mg of mannan was dissolved in 6 ml of dimethyl sulfoxide (DMSO), and 1 ml of 3 M / 6 M / 12 M sulfur trioxide pyridine salt solution was added. After stirring and reacting for 3 hours at 80°C, the pH was adjusted to 7.0. The sulfur trioxide pyridine salt was purified by centrifugation at 3900 rpm for 6 hours using a 3K Centrifugal filter, and the sulfated mannan was freeze-dried for 2-3 days and then stored.

[0107] In addition, to confirm the sulfation of polysaccharides, a mixture of dextran and sulfur trioxide pyridine salt was centrifuged at 3900 rpm for 6 hours using a centrifugal filter, and it was confirmed that no specific peak of sulfur trioxide pyridine salt appeared in the centrifuged sample, confirming that sulfur trioxide pyridine salt was purified (Fig. 4).

[0108]

[0109] In addition, to confirm the sulfation rate according to polysaccharide modification, the polysaccharide was reacted with different concentrations of sulfur trioxide pyridine salt, and the synthesized sulfate groups were quantified using XPS analysis to calculate the sulfation rate, and the results are shown in Table 4 below.

[0110] Sulfation rate of sulfated polysaccharide Polysaccharide*Reaction rate Sulfation rate (%) Mannan 1:5.66 18.43 1:11.32 26.38 1:22.64 38.77

[0111] *Reaction ratio; molar ratio of reacted monosaccharide:sulfur trioxide pyridine salt

[0112]

[0113] <1-4> Production and identification of phosphorylated polysaccharides

[0114] The structural change of polysaccharide due to phosphorylation of polysaccharide is as shown in the structural formula below.

[0115]

[0116] <Overview of Phosphorylation of Polysaccharides>

[0117]

[0118] Specifically, for the phosphorylation of polysaccharides, 600 mg / 1500 mg / 4000 mg of sodium tripolyphosphate (STPP): sodium trimetaphosphate (STMP) were dissolved in 5 ml of distilled water at a ratio of 6:1 to prepare a phosphorylation reagent. 100 mg of mannan was dissolved in 1 ml of distilled water, and the phosphorylation reagent was added. 5% (w / v) sodium sulfate was added to the mixture, and the pH was adjusted to 9.0. The mixture was stirred for 4 hours at 90°C for reaction, and then centrifuged at 3900 rpm for 6 hours using a 3K Centrifugal filter to purify the phosphorylated salt. The phosphorylated mannan was lyophilized for 2-3 days and then stored.

[0119] In addition, to confirm the phosphorylation of polysaccharides, a mixture of dextran and sodium trimetaphosphate and sodium triphosphate was centrifuged at 3900 rpm for 6 hours using a centrifugal filter, and it was confirmed that no specific peaks of sodium trimetaphosphate and sodium triphosphate appeared in the centrifuged sample, confirming that sodium trimetaphosphate and sodium triphosphate were purified (Fig. 5).

[0120]

[0121] In addition, to confirm the phosphorylation rate according to polysaccharide modification, the polysaccharide was reacted with different concentrations of sodium trimetaphosphate and sodium triphosphate, and the phosphorylation rate was calculated by quantifying the synthesized phosphate groups using XPS analysis, and the results are shown in Table 5 below.

[0122] Phosphorylation rate of phosphorylated polysaccharide Polysaccharide*reaction rate Phosphorylation rate (%) Mannan 1:5.00 13.82 1:7.50 20.50 1:10.00 24.36

[0123] *Reaction ratio; monosaccharide: molar ratio of sodium trimetaphosphate / sodium triphosphate reacted

[0124]

[0125] <Example 2>

[0126] Physicochemical properties of chemically modified polysaccharides

[0127] In order to analyze the physicochemical properties of the chemically modified polysaccharide in Example 1, the inventors of the present invention examined the trends in size and surface charge at various concentrations (1, 2, and 5 mg / ml) of chemically modified mannan. Mannan and chemically modified mannan were dissolved in distilled water and dynamic light scattering (DLS) analysis was performed.

[0128]

[0129] As a result, in the case of oxidized mannan, it was found that as the oxidation rate increased, the size increased due to the hydrophobic aldehyde group, and the surface charge did not change significantly (Fig. 6). In the case of reduced mannan, it was found that regardless of the reduction rate, no change was observed in the size and surface charge of mannan and reduced mannan (Fig. 7).

[0130] In addition, in the case of acetylated mannan, as the acetylation rate increased, the size increased due to the acetyl group having a high hydrophobicity, and the surface charge decreased and then increased rapidly in the 42% acetylated mannan, which appears to be a phenomenon that occurred as the hydrophobic acetyl group was rearranged internally to minimize contact with water (Fig. 8).

[0131] In addition, in the case of sulfated mannan, the size increased at 38% sulfated mannan and then rapidly decreased. This phenomenon appears to be due to the formation of particle-like shapes by sulfated mannan to minimize electrostatic repulsion, and it appears that the surface charge decreases as the sulfation rate increases due to the negatively charged sulfate groups (Fig. 9).

[0132] In addition, in the case of phosphorylated mannan, it was found that it increased by a constant size regardless of the phosphorylation rate, and the surface charge was found to decrease as the phosphorylation rate increased due to the negatively charged phosphate group (Fig. 10).

[0133]

[0134] <Example 3>

[0135] Analysis of the immunomodulatory properties of chemically modified polysaccharides

[0136] Furthermore, the inventors of the present invention performed an immunomodulatory efficacy analysis on the chemically modified polysaccharide of the present invention in Example 1. The immunomodulatory efficacy analysis was performed by analyzing the activation markers of dendritic cells (CD86, CD80, CD40) to determine whether dendritic cells were activated by treatment with the modified polysaccharide, and also analyzed the gene expression changes of CD86 and iNOS in macrophages treated with the modified polysaccharide. Here, CD86 is a co-stimulatory molecule essential for T cell activation and indicates a transition to an inflammatory state, and iNOS acts as a catalyst to generate nitric oxide (NO) from L-arginine.

[0137]

[0138] <3-1> Confirmation of the immunomodulatory ability of oxidized polysaccharides

[0139] After treating the oxidized mannan prepared in the above example to differentiated bone marrow-derived dendritic cells for 24 hours, changes in the activation markers (CD86, CD80, CD40) of the dendritic cells were analyzed using a flow cytometer.

[0140] As a result, compared to the control group that was not treated with oxidized mannan, the number of CD86, CD80, and CD40 positive cells, which are activation markers of dendritic cells, was significantly increased in the group treated with oxidized mannan, and compared to the group treated with unmodified mannan, the number of CD80 and CD40 positive cells was found to be greater in the group treated with chemically modified oxidized mannan (Fig. 11).

[0141]

[0142] In addition, when the expression rate of CD86 and iNOS genes was analyzed through RT-qPCR after 24 hours of treatment with oxidized mannan in M2 type macrophages, the group treated with oxidized mannan showed increased expression of CD86 and iNOS compared to the group not treated with oxidized mannan and the group treated with unmodified mannan. In particular, the oxidized mannan modified with an oxidation rate of 39.7% showed a significant increase in the expression of CD80 and CD40 (Fig. 12). These results imply that treatment with modified oxidized mannan resulted in a transition from M2 macrophages, which have an anti-inflammatory action and are involved in tissue repair, to M1 macrophages, which have a pro-inflammatory action and are involved in pathogen destruction.

[0143] Through this, the inventors of the present invention were able to find that oxidized mannan can induce the activity of dendritic cells and enhance inflammatory responses, thereby acting to increase immune activity.

[0144]

[0145] <3-2> Confirmation of the immunomodulatory ability of reduced polysaccharides

[0146] Next, the effect of reduced mannan on immunomodulatory ability was analyzed through an experiment similar to <3-1> above using reduced mannan.

[0147]

[0148] As a result, the group treated with reduced mannan to bone marrow-derived dendritic cells showed an increase in the expression rate of CD86, CD80, and CD40, which are activation markers of dendritic cells (Fig. 13), and the expression of CD86 and iNOS in M2 macrophages was found to increase, and in particular, the mannan reduced at a reduction rate of 15.8% showed a significant increase in the expression of iNOS (Fig. 14). These results imply that treatment with modified reduced mannan resulted in a transition from M2 macrophages, which have an anti-inflammatory action and are involved in tissue repair, to M1 macrophages, which have a pro-inflammatory action and are involved in the destruction of pathogens.

[0149]

[0150] Through this, the inventors of the present invention were able to find out that reduced mannan, like oxidized mannan, can induce the activity of dendritic cells and induce an enhancement of inflammatory response, thereby acting to increase immune activity.

[0151]

[0152] <3-3> Confirmation of the immunomodulatory properties of acetylated polysaccharides

[0153] The effect of acetylated mannan on immunomodulatory ability was analyzed through experiments similar to <3-1> above using acetylated mannan.

[0154]

[0155] As a result, in the group treated with acetylated mannan in M2 macrophages, the expression of CD86 and iNOS increased compared to the group not treated with mannan and the group treated with unmodified mannan. In particular, the acetylated mannan modified with an acetylation rate of 13.0% showed a significant increase in the expression of CD86 and iNOS (Fig. 15).

[0156] Through this, the inventors of the present invention were able to find out that acetylated mannan can also act to increase immune activity.

[0157]

[0158] <3-4> Confirmation of the immunomodulatory properties of sulfated polysaccharides

[0159] Next, the inventors of the present invention analyzed the effect of sulfated mannan on immunomodulatory ability through experiments such as the above <3-1> using sulfated mannan.

[0160]

[0161] As a result, in the group treated with sulfated mannan in M2 macrophages, the expression of CD86 and iNOS was found to be reduced compared to the group that was not treated with mannan (Fig. 16). These results indicate that the conversion to M1 macrophages, which have a pro-inflammatory effect and are involved in the destruction of pathogens, was reduced or inhibited by chemically modified sulfated mannan treatment. Through this, the inventors of the present invention were able to determine that sulfated mannan, unlike oxidized, reduced, and acetylated mannan, can act to suppress immune activity.

[0162]

[0163] <3-5> Confirmation of the immunomodulatory properties of phosphorylated polysaccharides

[0164] Next, the inventors of the present invention analyzed the effect of sulfated mannan on immunomodulatory ability through experiments such as <3-1> using phosphorylated mannan.

[0165]

[0166] As a result, in the group treated with phosphorylated mannan in M2 macrophages, the expression of iNOS was found to be reduced compared to the group that was not treated with mannan (Fig. 17). These results indicate that the conversion to M1 macrophages, which have a pro-inflammatory effect and are involved in the destruction of pathogens, was reduced or inhibited by chemically modified phosphorylated mannan treatment, and through this, the inventors were able to find out that phosphorylated mannan, unlike oxidized, reduced, and acetylated mannan, can act to suppress immune activity.

[0167]

[0168] Through these results, the inventors of the present invention were able to determine that polysaccharides manufactured by chemical modification have immunomodulatory properties, and in particular, polysaccharides that were chemically oxidized, reduced, and acetylated showed activity in increasing immune activity, whereas polysaccharides that were sulfated and phosphorylated showed activity in suppressing immune activity.

[0169] Therefore, it was found that when utilizing the different immunomodulatory effects of polysaccharides according to the type of modification, they can be used as more effective immunomodulators than existing polysaccharides.

[0170]

[0171] While the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, but can be manufactured in various different forms. Those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood to be illustrative in all respects and not restrictive.

Claims

1. As a chemically modified immunomodulatory polysaccharide, The above polysaccharide is a chemically modified immunomodulatory polysaccharide characterized in that at least one hydroxyl group is modified by substitution with an aldehyde group, a hydroxyl group, an acetyl group, a sulfate group, or a phosphoric acid group.

2. In paragraph 1, A chemically modified immunomodulatory polysaccharide, characterized in that the above modification is oxidation, reduction, acetylation, sulfation or phosphorylation.

3. In paragraph 1, A chemically modified immunomodulatory polysaccharide, characterized in that the polysaccharide is selected from the group consisting of mannan, glucomannan, galactomannan, chitosan, chitin, pectin, galactan, and chondroitin.

4. In paragraph 1, A chemically modified immunomodulatory polysaccharide characterized in that the above polysaccharide is modified by oxidation, reduction or acetylation and increases immune activity.

5. In paragraph 4, A chemically modified immunomodulatory polysaccharide characterized in that it increases immune activity through activation of dendritic cells and macrophages, wherein the immunomodulatory polysaccharide is modified by oxidation, reduction or acetylation.

6. In paragraph 1, A chemically modified immunomodulatory polysaccharide characterized in that the above polysaccharide is modified by sulfation or phosphorylation and suppresses immune activity.

7. In paragraph 6, A chemically modified immunomodulatory polysaccharide characterized in that it suppresses immune activity by inhibiting the activation of overly activated dendritic cells and macrophages, wherein the immunomodulatory polysaccharide is modified by sulfation or phosphorylation.

8. An immunomodulatory composition comprising the chemically modified immunomodulatory polysaccharide of paragraph 1 as an active ingredient.

9. In paragraph 8, An immunomodulatory composition characterized in that the composition contains an immunomodulatory polysaccharide modified by oxidation, reduction or acetylation, and increases immune activity.

10. In paragraph 8, An immunomodulatory composition characterized in that the composition contains an immunomodulatory polysaccharide modified by sulfation or phosphorylation, wherein the composition suppresses immune activity.

11. A chemically modified immunomodulatory polysaccharide comprising an active ingredient, wherein the chemically modified immunomodulatory polysaccharide is characterized in that at least one hydroxyl group is modified by substituting an aldehyde group, a hydroxyl group, or an acetyl group. A pharmaceutical composition for the prevention or treatment of cancer having immunostimulating activity.

12. A chemically modified immunomodulatory polysaccharide comprising an active ingredient, wherein the chemically modified immunomodulatory polysaccharide is characterized in that at least one hydroxyl group is modified by substituting a sulfate group or a phosphate group. A pharmaceutical composition for the prevention or treatment of autoimmune diseases having immunosuppressive activity.

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