Immunoactivating agents, pharmaceutical compositions and food products

Abstract

The present application provides an immune activator, and a pharmaceutical composition and food or drink containing the immune activator as an effective ingredient, the immune activator containing chitosan and / or chitosan derivative with a weight average molecular weight of 10k-1000k and an anionic surfactant, the immune activator being in a microparticle shape.

Description

Technical Field

[0001] This invention relates to immune activators, pharmaceutical compositions, and food and beverage products. Background Technology

[0002] Various substances are known to be useful as adjuvants used in vaccination. For example, many polysaccharides are known to have adjuvant activity, among which chitin and chitosan have been shown to be useful. They have a structure similar to peptidoglycan, which constitutes the bacterial cell wall, and are processed through the body's defense mechanisms, thus being inherently non-toxic. Wound dressings that effectively utilize the biocompatibility of chitin have also been put into practical use. On the other hand, chitin and chitosan are known to activate the immune system even when alone in animals as foreign substances, which plays one aspect of their adjuvant role. Chitosan, due to its free amino groups, is cationic and soluble under acidic conditions, and can form complexes through electrostatic interactions with anionic matrices (such as proteins or nucleic acids). The surface of all cells is anionic. Therefore, chitosan binds strongly to cell surfaces and mucous membranes in vivo. In this way, chitosan is considered more suitable as an immune adjuvant due to its cationic properties.

[0003] One of the problems with using chitosan as an immune adjuvant is its high crystallinity. For example, even when chitosan is temporarily dissolved in a solvent under acidic conditions, upon introduction into the body, the increased pH within the organism causes chitosan molecules to lose their charge and crystallize, precipitating as microparticles or gelling. Furthermore, it is believed that even complexes obtained by combining chitosan with other substances such as proteins in solution undergo dissociation along with crystallization. To date, various chitosan derivatives obtained by modifying the properties of chitosan have been reported, such as those described in Non-Patent Document 1. Additionally, Patent Document 1 describes a branched chitosan derivative developed to balance functionality such as water solubility and low viscosity, having chitosan side chains on the chitosan backbone. Patent Document 2 describes an aqueous solution of an immune adjuvant containing cationic chitosan.

[0004] Furthermore, techniques for using chitosan microparticles are also known. Patent Document 3 describes an immune adjuvant dispersion containing chitosan microparticles. Non-Patent Document 2 describes the use of chitosan microparticles made from tripolyphosphate as a drug delivery carrier. It is also known to induce antigen-specific immune responses by ingesting antigen-containing microparticles by phagocytes.

[0005] Chitosan microparticles are mostly prepared by mixing with polyanionic compounds such as tripolyphosphate or hyaluronic acid. Many examples of chitosan microparticles developed through electrostatic interactions have been reported.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Publication No. 2011-132369 (published on July 7, 2011)

[0009] Patent Document 2: Japanese Patent Publication No. 2009-29715 (published on February 12, 2009)

[0010] Patent Document 3: Japanese Patent Publication No. 2009-29714 (published on February 12, 2009)

[0011] Non-patent literature

[0012] Non-patent literature 1: Yalpani and Hall, Macromolecules, vol. 17, pp. 272-281, 1984

[0013] Non-patent literature 2: Xu et al., Int. J. Pharm., vol., 250, pp. 215-226, 2003 Summary of the Invention

[0014] The problem that the invention aims to solve

[0015] However, chitosan as an adjuvant for inducing immune responses has not yet been put into practical use. Compared with Freund's adjuvant used in animal experiments, it has not demonstrated sufficient effectiveness. To further enhance and improve the usefulness and immune-activating effects of chitosan as an immune adjuvant, it is desirable to develop novel amorphous chitosan derivatives that effectively utilize the aforementioned properties of chitosan, and to develop novel micronized chitosan.

[0016] Methods for solving problems

[0017] Based on the above, the inventors of this application conducted in-depth research and discovered that chitosan derivatives with a certain structure possess strong immunoactivating activity. Furthermore, the inventors of this application have newly discovered that by micronizing chitosan or chitosan derivatives with a certain range of weight-average molecular weight using an anionic surfactant, microparticles with high immunoactivating activity can be obtained. Moreover, the inventors have conceived of the chitosan derivatives of this application, immunoactivators containing chitosan derivatives, and pharmaceutical compositions and food and beverages containing the immunoactivator as an active ingredient.

[0018] To address the aforementioned issues, this invention includes the following solution.

[0019] An immune activator, characterized in that it comprises chitosan and / or chitosan derivatives with a weight-average molecular weight of 10k to 1000k, and an anionic surfactant, wherein the immune activator is in particulate form.

[0020] The effects of the invention

[0021] The present invention provides the following effects: it can provide chitosan derivatives with immune-activating effects, immune activators containing the chitosan derivatives, pharmaceutical compositions containing the immune activators as active ingredients, and food and beverages containing chitosan derivatives. Attached Figure Description

[0022] Figure 1 This indicates the antibody titer after intraperitoneal administration of various chitosan derivatives to mice in Example 2 of the present invention.

[0023] Figure 2 The antibody titer after intraperitoneal administration of chitosan, chitosan derivatives, micronized chitosan, or micronized chitosan derivatives of Example 3 of the present invention to mice is indicated.

[0024] Figure 3 The antibody titer of mice after intraperitoneal administration of the micronized chitosan derivative prepared using various additives in Example 4 of the present invention is shown.

[0025] Figure 4 The antibody titer of mice after intraperitoneal administration of micronized chitosan derivatives prepared using various fatty acid salts, as described in Example 5 of the present invention, is shown.

[0026] Figure 5 The antibody titer of each microparticle synthesized using chitosan of different molecular weights in the manufacture of the microparticles in Example 6 of the present invention is indicated after intraperitoneal administration to mice.

[0027] Figure 6 The antibody titer of each microparticle having different mixing ratios of chitosan and additives in Example 7 of the present invention after intraperitoneal administration to mice is shown.

[0028] Figure 7 The results show the average particle size and zeta potential measurements of Example 7 of the present invention.

[0029] Figure 8 The antibody titer of each microparticle, manufactured in Example 8 of the present invention by mixing solutions of chitosan derivatives, additives and antigens in different sequences, after being administered intraperitoneally to mice is shown.

[0030] Figure 9 The antibody titer is indicated by the administration of the micronized chitosan derivative of Example 9 of the present invention to rats via intramuscular or subcutaneous administration.

[0031] Figure 10 The antibody titer is indicated by the administration of the micronized chitosan derivative of Example 10 of the present invention to the intramuscular or subcutaneous tissue of rabbits.

[0032] Figure 11 The antibody titer is indicated by intraperitoneal administration of the micronized chitosan derivative of Example 12 of the present invention to mice.

[0033] Figure 12 This indicates the results of necropsy examination of mice immunized seven times with Freund's adjuvant according to Example 12 of the present invention. Detailed Implementation

[0034] The embodiments of the present invention will now be described in detail.

[0035] [Definitions of terms, etc.]

[0036] In this specification, "chitosan derivatives" refers to compounds with chitosan sugar units as the main chain and side chains on the main chain.

[0037] In this specification, "branching degree" refers to the proportion of units with side chains among the sugar units that make up chitosan. This is represented by the parameter z. Details about z will be described later.

[0038] In this specification, "degree of deacetylation" refers to the proportion of acetylamino groups bonded to the carbon at position 2 of the sugar unit constituting chitosan that are converted into free amino groups through deacetylation.

[0039] In this specification, the "degree of substitution" of the side chain refers to the extent to which the amino group bonded to the 2-carbon of the chitosan sugar unit is substituted by the side chain. It is expressed as the ratio of the substituted amino group bonded to the 2-carbon of the chitosan sugar unit to the total number of free amino groups and substituted amino groups. Here, it can be expressed as: Degree of substitution = Number of substituted amino groups in the chitosan sugar unit / (Number of free amino groups + Number of substituted amino groups).

[0040] In this specification, "microparticles" refers to particles with a diameter of less than tens of μm.

[0041] In this specification, "immunoactivator" refers to a molecule, substance, or composition that has the effect of specifically or non-specifically altering, amplifying, inducing, re-inducing, enhancing, or initiating an antigen-specific immune response. The immunoactivator of this invention comprises preparations capable of enhancing either or both of humoral and cellular immunity. "Immune activator" is sometimes also referred to as "adjuvant" or "immunoadjuvant" in this specification.

[0042] In this specification, "subject" refers to the object to which the chitosan derivative, immune activator, pharmaceutical composition, and food and beverage of the present invention are applied.

[0043] In this instruction manual, "prevention" means preventing the contracting of a disease, ailment, or disorder. "Treatment" means alleviating or eliminating an existing disease, ailment, or disorder, or its accompanying symptoms.

[0044] [1. Chitosan derivatives]

[0045] The chitosan derivative of the present invention will be described below.

[0046] In one embodiment of the present invention, the following chitosan derivative is provided.

[0047] The following general formula (I) represents a chitosan derivative with immune-activating activity.

[0048] [Chemical Formula 1]

[0049]

[0050] In equation (I), x, y, and z are molar ratios, x + y + z = 1, and x, y, and z are independently 0 ≤ x < 1, 0 ≤ y < 1, and 0 ≤ z < 1, respectively. <z≤1,R 1 The pullulan side chain is represented by the general formula (II) below.

[0051] [Chemical Formula 2]

[0052]

[0053] or,

[0054] R 1 The dextran side chain is represented by the following general formula (III).

[0055] [Chemical Formula 3]

[0056]

[0057] or

[0058] R 1 The glucosamine side chain is represented by the following general formula (IV).

[0059] [Chemical Formula 4]

[0060]

[0061] R 2 It is an acetyl group, or

[0062] R 1 The lysine side chain is represented by the following general formula (V).

[0063] [Chemical Formula 5]

[0064]

[0065] or

[0066] R 1 [The lactose side chain is represented by the following general formula (VI)]

[0067] [Chemical Formula 6]

[0068]

[0069] In this way, the chitosan derivatives of this invention have a structure in which a sugar with a free carboxyl terminus is bonded to an amino group bonded to the amino group at the 2-carbon position of the glucosamine unit constituting the sugar unit of chitosan via a Schiff base through reductive alkylation, or a structure in which an amino group with a free carboxyl terminus forms an amide bond with the amino group at the 2-carbon position of the glucosamine unit constituting the sugar unit of chitosan through a condensation reaction.

[0070] (Parameters related to the chitosan backbone)

[0071] The ratios of x and y described above vary depending on the type of chitosan used in the manufacture of the chitosan derivative according to the present invention. Furthermore, they can be adjusted by acetylation or deacetylation of the chitosan that will become the raw material or the generated chitosan derivative. It should be noted that the chitosan used as the raw material is described in the following (chitosan) section. z represents the proportion of units with side chains among the sugar units constituting the chitosan. Here, it is expressed as z = (number of sugar units with side chains in the chitosan derivative) / (total number of sugar units constituting the chitosan derivative). The value of z can be controlled by adjusting the amount of chitosan that forms the main chain and the amount of compounds that form the side chains.

[0072] (Length of the chitosan backbone)

[0073] The chitosan derivative of the present invention comprises a glucosamine unit, an N-acetylglucosamine unit, and the above-mentioned R 1 Chitosan, whose main chain is composed of N-bonded glucosamine units bonded to the carbon at position 2 of the glucosamine unit, is used as the main chain. The number of sugar units constituting the chitosan main chain is preferably 30 to 12,000, more preferably 60 to 6,000, and even more preferably 120 to 3,000.

[0074] (Side chain length and degree of substitution)

[0075] The chitosan derivatives of the present invention exhibit improved solubility over a wider pH range compared to chitosan without side chains due to the presence of hydrophilic side chains. For example, they also show high water solubility at pH 6.5–8.0. The degree of substitution of the side chain containing the amino group at position 2 of the sugar unit in the chitosan backbone can be appropriately set according to the type of chitosan derivative and its intended use. For example, in the case of use as an immunoadjuvant, the degree of substitution is preferably 0.005–0.5, more preferably 0.005–0.3. It should be noted that the degree of substitution can be determined based on known techniques (e.g., sugar composition analysis), and can also be calculated by NMR spectroscopy analysis.

[0076] Furthermore, the chitosan derivatives of the present invention can be modified by adding or substituting functional groups or functional molecules to impart other activities or functions, thereby designing them to simultaneously exert further activities or functions. Further modifications can be made to maintain the position and properties of the original chitosan derivative before modification. As an example of modification, amidation can be performed on the amino group bonded to the 2-carbon of the sugar unit in the chitosan backbone of the chitosan derivative and 4-maleimidebutyric acid. This modification, for example, allows the chitosan derivatives of the present invention to be coupled with cysteine-containing peptides.

[0077] Here, in R 1 When the pullulan side chain is as shown in formula (II) above, the a of the chitosan derivative of the present invention is preferably 20 to 600, more preferably 50 to 300. Furthermore, the degree of substitution is preferably 0.001 to 0.1, more preferably 0.002 to 0.05.

[0078] Additionally, in R 1 When the side chain is the dextran shown in formula (III) above, the a of the chitosan derivative of the present invention is preferably 15 to 300, more preferably 30 to 200. Furthermore, the degree of substitution is preferably 0.001 to 0.1, more preferably 0.002 to 0.05.

[0079] In R 1 When the chitosan derivative of the present invention has a glucosamine side chain as shown in formula (IV) above, the degree of substitution is preferably 0.05 to 0.5, more preferably 0.1 to 0.3. 2 H, acetyl, or R 1 All of R 2 They can be the same or different, depending on the H, acetyl, and R groups. 1 Among them, R is particularly preferred. 2 All are acetyl groups.

[0080] In R 1When the chitosan derivative of the present invention has a lysine side chain as shown in formula (V) above, the degree of substitution is preferably 0.02 to 0.5, more preferably 0.05 to 0.3. Alternatively, it may have the following structure: lysine with a free carboxyl terminus may be further reacted with R via a condensation reaction. 1 The amino group bonded to the 2-carbon of the lysine side chain forms an amide bond, thereby connecting at least one lysine molecule.

[0081] (The pH range in which chitosan derivatives are soluble)

[0082] As described above, chitosan with a degree of deacetylation of 70% or more is soluble only at acidic pH levels below pH 6. In contrast, the chitosan derivatives of the present invention have a wider soluble pH range than chitosan, being soluble in neutral to alkaline pH levels from 6.5 to 8.0. In one embodiment, the chitosan derivatives of the present invention are generally preferred to be used at a maximum pH below 8.0, and more preferably at pH levels from 5.0 to 7.5.

[0083] One embodiment of the chitosan derivative of the present invention is a chitosan derivative having pullulan side chains. As described in the following items (sugars present as side chains), pullulan is soluble in water and, despite its low viscosity, exhibits excellent lubricity and adhesiveness. In the case of pullulan side chains, the solubility is excellent, making it particularly suitable as a soluble adjuvant.

[0084] Furthermore, in one embodiment of the present invention, the chitosan derivative is a chitosan derivative having glucosamine side chains or dextran side chains. Glucosamine and dextran are biodegradable. Therefore, chitosan derivatives having these side chains exhibit excellent safety and are particularly suitable as components of immune adjuvants introduced into the body.

[0085] Furthermore, in one embodiment of the present invention, the chitosan derivative is a chitosan derivative having a lysine side chain. In the case of a lysine side chain, it is particularly suitable as a particulate adjuvant because it also undergoes cationization in plasma at pH 7.4.

[0086] The chitosan derivatives of the present invention are manufactured using chitosan as the main chain raw material and compounds such as sugars and amino acids as side chain raw materials. Hereinafter, the compounds that serve as raw materials for the chitosan derivatives of the present invention will be described sequentially.

[0087] (Chitosan)

[0088] Chitosan is typically prepared by deacetylation of chitin (poly-β1,4-N-acetylglucosamine) from the shells of crabs and shrimp using alkali treatment or enzymatic treatment. Hereinafter, in this specification, the substance obtained by deacetylation of at least a portion of chitin is referred to as chitosan. It should be noted that the chitosan of the present invention is not limited to chitosan of natural origin, but may also be chemically synthesized chitosan. The chitosan used as a raw material for the chitosan derivative of the present invention is chitosan in which the proportion of N-deacetylated molecules in the glucosamine units constituting the chitin molecule, i.e., the degree of deacetylation, is preferably in the range of 60-100%, more preferably 70-100%. It should be noted that the degree of deacetylation can also be quantified based on known techniques (e.g., colloidal titration). As an example of a known technique, NMR can be cited.

[0089] Alternatively, chitosan molecules of any molecular weight can be processed by hydrolyzing naturally sourced chitosan molecules, which are composed of many sugar residues and have a large molecular weight.

[0090] As described above, the number of sugar units constituting the chitosan backbone of the chitosan derivative is 30 to 12,000. Therefore, when the molecular weight average of the chitosan used as a raw material is set to 161 for the glucosamine residues, 203 for the N-acetylglucosamine residues, and 60% to 100% for the degree of deacetylation, the average molecular weight of the chitosan used as a raw material can be approximately 5k (5,000) to approximately 2,000k (2,000,000).

[0091] The properties of chitosan molecules, which are the starting material for the chitosan derivatives in this embodiment, are not particularly limited. Examples of chitosan molecules include powder, fibrous, film, sheet, hydrogel, and solution forms.

[0092] (Sugars present as side chains)

[0093] In one embodiment of the present invention, a chitosan derivative has sugars as side chains on the chitosan backbone. Examples of sugar chains with reducing ends suitable for chitosan derivatives include sugar chains derived from aldoses and ketoses. Sugar chains with one or more constituent sugar units are used. More specific examples of sugars, such as monosaccharides (i.e., pentasaccharides and hexasaccharides), include glucose, fucose, mannose, arabinose, galactose, xylose, erythrose, heptanose, hexanose, and pentanose. Additionally, examples of amino sugars include glucosamine, N-acetylglucosamine (e.g., N-acetyl-D-glucosamine), and galactosamine. Furthermore, examples of sugar derivatives include uronic acids and deoxyglucosides. Additionally, examples of sugars comprising disaccharides or polysaccharides consisting of sugar chains composed of these monosaccharides include maltose, isomaltose, lactose, melibiose, and maltotriose, as well as various oligosaccharides. Furthermore, examples include pullulan, inulin, amylose, amylopectin, dextran, dextrin, starch, and other natural polysaccharides, as well as their decomposition products, isomers, or derivatives. One or more combinations of these sugars can be used in the manufacture. Additionally, these sugars used as raw materials can also be hydrates. Among them, N-acetylglucosamine (e.g., N-acetylglucosamine D-glucosamine), dextran, and pullulan are particularly suitable as the sugars used in the chitosan derivatives of this invention. Pullulan, being water-soluble, exhibits excellent lubricity and adhesion despite its low viscosity, and is therefore used as a thickener or stabilizer as a food additive. Glucosamine and dextran are biodegradable and particularly suitable as components for use in immune adjuvants applicable to organisms.

[0094] It should be noted that, when melbiose is used as a side chain, the chitosan derivative of the present invention has a melbiose side chain as shown in the following formula (VII).

[0095] [Chemical Formula 7]

[0096]

[0097] (Amino acids or amino acid derivatives present as side chains)

[0098] Another embodiment of the chitosan derivative of the present invention has an amino acid as a side chain on the chitosan backbone. Examples of suitable amino acids or amino acid derivative side chains with reducing ends in the chitosan derivative include lysine (e.g., L-lysine), carnitine, arginine, proline, and histidine. Among these, lysine and carnitine are particularly suitable as amino acids used in the chitosan derivative of the present invention. It should be noted that when carnitine is introduced as a side chain, the chitosan derivative of the present invention has a carnitine side chain as shown in formula (VIII).

[0099] [Chemical Formula 8]

[0100]

[0101] In another embodiment of the present invention, the chitosan derivative has multiple different sugars and / or amino acids as side chains on the chitosan backbone.

[0102] (Other compounds introduced as side chains)

[0103] Another embodiment of the chitosan derivative of the present invention has polyethylene glycol as a side chain on the chitosan. Methoxylated PEG-aldehydes are examples of suitable chitosan derivatives with reducing ends.

[0104] (Methods for manufacturing chitosan derivatives)

[0105] <Method for manufacturing chitosan derivatives with sugar side chains>

[0106] The chitosan derivative of this embodiment can be manufactured by known manufacturing methods. As an example, when the side chain of the chitosan derivative is a sugar, the manufacturing method of the chitosan derivative of this embodiment is characterized by comprising the following steps: a chitosan dissolution step of dissolving chitosan to prepare a chitosan solution; a sugar dissolution step of adding sugar to the chitosan solution and dissolving it; and a reducing agent addition step of further adding a reducing agent after the sugar dissolution step.

[0107] In general, the reaction in the manufacturing method of chitosan derivatives with sugars as side chains is that the amino group bonded to the 2-carbon of the glucosamine unit constituting chitosan is bonded to the sugar via a Schiff base through reductive N-alkylation.

[0108] <Chitosan dissolution process>

[0109] The chitosan dissolution process is a step of preparing a chitosan solution by dissolving chitosan. In one embodiment, in the chitosan dissolution process, after dispersing chitosan in water, acetic acid is added to dissolve the chitosan. It should be noted that a step of partially deacetyling chitin can also be performed before or simultaneously with the chitosan dissolution process. Furthermore, the partial deacetylation reaction can be carried out by a known method of de-N-acetylation.

[0110] <Carbohydrate Dissolution Process>

[0111] The sugar dissolution step follows the chitosan dissolution step by further adding sugars to the chitosan solution and dissolving them. It should be noted that the sugar dissolution step can also be performed simultaneously with the chitosan dissolution step; that is, sugars can be added to the solvent at the same time as chitosan.

[0112] <Reducing Agent Addition Process>

[0113] The reducing agent addition step is performed after the sugar dissolution step. Here, when the chitosan derivative has sugars as side chains, the reducing agent can be 2-methylpyridineborane, sodium cyanoborohydride (NaCNBH3), or an amineborane-based reducing agent, such as ammoniaborane (NH3BH3) and monomethylamineborane (CH3(NH2BH3)), etc., preferably 2-methylpyridineborane. After adding the reducing agent, the reaction is carried out by stirring at room temperature. The reaction time is, for example, 2 hours to several days, or 6 hours to 24 hours, and preferably within 20 hours from the viewpoint of work efficiency. Furthermore, the <reducing agent addition step> can also be performed simultaneously with the <chitosan dissolution step> and / or the <sugar dissolution step>.

[0114] <Manufacturing method of chitosan derivatives with amino acid side chains>

[0115] As another example, the method for manufacturing chitosan derivatives in this embodiment, when the side chains of the chitosan derivatives are amino acids, is characterized by comprising the following steps: a chitosan dissolution step of dissolving chitosan to prepare a chitosan solution; an amino acid dissolution step of adding amino acids to the chitosan solution and dissolving them; and a condensing agent addition step of further adding a condensing agent after the amino acid dissolution step.

[0116] In general, the manufacturing process when the side chain is an amino acid involves a condensation reaction between the amino group bonded to the 2-carbon of glucosamine, the constituent sugar of chitosan, and the free amino acids such as lysine and carnitine at the carboxyl terminus, through a condensation agent to form an amide bond, thereby obtaining the product.

[0117] <Chitosan dissolution process>

[0118] The chitosan dissolution process is as described in the same process of the above-described method for manufacturing chitosan derivatives with sugars as side chains.

[0119] <Amino Acid Dissolution Process>

[0120] The amino acid dissolution step follows the chitosan dissolution step, involving the further addition and dissolution of amino acids into the chitosan solution. It should be noted that the amino acid dissolution step can also be performed simultaneously with the chitosan dissolution step; that is, amino acids can be added to the solvent at the same time as chitosan.

[0121] <Condensing agent addition process>

[0122] The condensing agent addition step is performed after the amino acid dissolution step. Here, when the side chain is an amino acid, the condensing agent can be 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride. After adding the condensing agent, the reaction is carried out by stirring at room temperature. The reaction time is, for example, 4 hours to several days, or 6 hours to 24 hours, and preferably within 12 hours from the viewpoint of work efficiency. Furthermore, the <condensing agent addition step> can also be performed simultaneously with the <chitosan dissolution step> and / or the <amino acid dissolution step>.

[0123] The chitosan derivatives obtained as described above can also be purified using known methods such as ultrafiltration, solvent washing, purification by precipitation, gel filtration, ion exchange column chromatography, activated carbon exchange column chromatography, cellulose dialysis membrane, and electrodialysis. Furthermore, the chitosan derivatives of this invention can be processed into powder, solution, and suspension forms using known methods. Additionally, the obtained chitosan derivatives can be dried using appropriate drying methods such as freeze-drying and stored in a dry state, as needed.

[0124] As shown in the following examples, the chitosan derivatives of the present invention exhibit significant activation of the biological immune system, i.e., immune activation. Therefore, the chitosan derivatives of the present invention are suitable for use as safe and effective immune activators. Furthermore, the chitosan derivatives of the present invention are also soluble in the neutral to alkaline region of pH 6.5 to 8.0. Consequently, they are stable in vivo and can be used for a wider range of applications compared to conventional chitosan and chitosan-modified forms.

[0125] [2. Immune activators]

[0126] (Immune activators containing chitosan derivatives)

[0127] Next, the immune activator of the present invention will be described. In one embodiment, the immune activator of the present invention is characterized by comprising at least one chitosan derivative represented by the above-described general formula (I) as an active ingredient. In another embodiment, the immune activator of the present invention may also comprise a combination of two or more chitosan derivatives represented by the above-described general formula (I). Because the immune activator of the present invention has a significant immune activating effect, it is useful, for example, for treating patients with diseases, disorders, or impairments in which the normal metabolic immune response is reduced or suppressed. Furthermore, the immune activator of the present invention can be used therapeutically or prophylactically to treat subjects such as animals or humans at increased risk of suffering from diseases, disorders, or impairments caused by states that adversely affect the immune system.

[0128] Furthermore, the inventors of this application have newly discovered that by micronizing chitosan or chitosan derivatives having a certain molecular weight range using an anionic surfactant, microparticles with high immunoactivating activity can be obtained. In one embodiment, the immunoactivator is in microparticle form. Hereinafter, the case where the immunoactivator of this application is in microparticle form (i.e., microparticles as immunoactivators) will be described in detail.

[0129] (Immune activators in the form of microparticles (microparticles as immune activators))

[0130] The immune activator of this embodiment comprises chitosan and / or chitosan derivatives with a weight average molecular weight of 10k to 1000k, and an anionic surfactant, wherein the immune activator is in particulate form.

[0131] The following describes the various components contained in the microparticles.

[0132] (The various components contained in the particles)

[0133] Chitosan and / or chitosan derivatives

[0134] The chitosan and / or chitosan derivatives contained in the microparticles have a weight-average molecular weight of 10k to 1000k, which is appropriately set according to the intended use of the final microparticles. The microparticles in this invention tend to exhibit the following tendency: the lower the molecular weight of the chitosan, the higher the immune activation effect.

[0135] However, low molecular weight chitosan and / or chitosan derivatives below a certain molecular weight, such as chitosan and / or chitosan derivatives with a molecular weight below 10k, tend to have more complex manufacturing processes. Furthermore, they are prone to aggregation in solutions after the addition of anionic surfactants. On the other hand, high molecular weight chitosan above a certain molecular weight, such as chitosan and / or chitosan derivatives with a molecular weight exceeding 1000k, tend to have higher viscosity.

[0136] Therefore, from the viewpoint that chitosan or chitosan derivatives used as raw materials for the manufacture of microparticles have a weight-average molecular weight of 10k to 1000k, more preferably 20k to 500k, and most preferably 30k to 100k, based on their high immune activation effect, ease of handling, ease of industrial production, easy availability in the market, and high commercial applicability.

[0137] Furthermore, the chitosan or chitosan derivative used as a component of the microparticles of the present invention has a chitosan backbone, i.e., chitosan, with a degree of deacetylation preferably in the range of 60 to 100%, more preferably in the range of 70 to 100%.

[0138] In one embodiment, the chitosan derivative included in the microparticles may be a chitosan derivative described in [1. Chitosan Derivatives] above, or a chitosan derivative having a side chain consisting of a sugar (as a side chain), an amino acid or amino acid derivative (as a side chain), or another compound introduced as a side chain. In one example, the sugar as a side chain may be selected from N-acetylglucosamine (e.g., N-acetylgD-glucosamine), dextran, and pullulan. In one example, the chitosan derivative included in the microparticles is dextran. Regarding the chitosan derivative, the chitosan derivative itself before micronization possesses an immunoactivating effect. Therefore, by including this chitosan derivative, the immunoactivating activity can be improved compared to microparticles containing only chitosan. Furthermore, by including this chitosan derivative, the crystallinity is reduced compared to chitosan, or it is coated with a neutral polysaccharide. A reduction in crystallinity and secondary aggregation after micronization, and improved stability, are expected.

[0139] In another embodiment, the chitosan derivative contained in the microparticles may also be a substance in which molecules such as peptides, oligonucleotides, and oligosaccharides containing 3 to 150 amino acids are covalently bonded to the chitosan backbone. In one example, these bonded molecules are polypeptides or fragments thereof (e.g., epitopes) encoding antigens described in the [antigen] section below, polynucleotides or fragments thereof encoding genes that encode the antigen polypeptide, or peptides or fragments thereof having cell adhesion activity.

[0140] <Anionic surfactants>

[0141] The microparticles of the present invention comprise anionic surfactants. The anionic surfactants contained in the microparticles possess both anionic and hydrophobic groups. The hydrophobic groups may be substituted or unsubstituted, and may be saturated or unsaturated alkyl groups having 2 to 22 carbon atoms.

[0142] In one embodiment, the anionic surfactant is selected from at least one of phospholipids and fatty acids having 10 to 22 carbon atoms and their salts. The anionic surfactant can be a single type of anionic surfactant or a combination of multiple different anionic surfactants. Furthermore, the microparticles comprise any one or both of phospholipids or fatty acids having 10 to 22 carbon atoms and their salts.

[0143] Phospholipids

[0144] Examples of phospholipids include glycerol phosphates, sphingolipids, cephalins and mixtures thereof, fully synthetic phospholipids, lecithin, and lysophosphatidylcholine. In the chemical field, lecithin has the same meaning as phosphatidylcholine; in this specification, it refers to phosphatidylcholine. Phospholipids may preferably be lecithin or lysophosphatidylcholine. More specifically, examples of lecithin include soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin (HSPC), and hydrogenated egg yolk lecithin (HEPC). Examples of glycerol phosphates include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, and phosphatidic acid. Examples of sphingolipids include sphingolipids. It should be noted that phospholipids that are not sufficiently water-soluble can be dissolved in organic solvents such as ethanol and then used for the manufacture of microparticles.

[0145] [Fatty acids and their salts]

[0146] Fatty acids and their salts contain fatty acids with 10 to 22 carbon atoms, as well as their salts and glycerides. Fatty acids can be saturated or unsaturated. The preferred number of carbon atoms in fatty acids and their salts is 10 to 22, more preferably 12 to 18.

[0147] Examples of fatty acids include myristic acid, palmitic acid, stearic acid, arachidic acid, benzyl acid, tetracosanoic acid, myristic acid, palmitoleic acid, oleic acid, isoleic acid, eicosaenoic acid, docosaenoic acid, tetracosenoic acid, linoleic acid, α-linolenic acid, eicosadienoic acid, eicosatraenoic acid, eicosatrienoic acid, eicosapentaenoic acid, docosahexaenoic acid, decanoic acid, lauric acid, gamma-linolenic acid, and arachidonic acid.

[0148] Sodium and potassium salts are examples of the salts of the aforementioned fatty acids. It should be noted that fatty acids that are not sufficiently water-soluble can be dissolved in organic solvents such as ethanol for use in the manufacture of microparticles.

[0149] In one example, the microparticles contain sodium oleate or sodium lauryl oleate as an anionic surfactant.

[0150] [Other additives]

[0151] The microparticles in one embodiment may further contain additives. For example, additives may include trehalose, glucose, sucrose, lactose, mannitol, dextran, xylitol, maltose, fructose, glycine, citric acid, and sodium chloride or nonionic surfactants. By including such additives, instability caused by freeze-drying or long-term low-temperature storage, such as aggregation or decreased immune activation activity, can be prevented. Other additives include vitamin C and vitamin E. Including these can inhibit the oxidative decomposition of fatty acids.

[0152] [Other ingredients]

[0153] In another embodiment, the microparticles can also have drugs loaded on their surface. Alternatively, the surface of the microparticles can be coated with anionic polymers such as polycarboxylic acids. Examples of polycarboxylic acids include pectin, alginate, polyacrylic acid, hyaluronic acid, chondroitin sulfate, and poly-γ-glutamic acid. Coating with these substances imparts high stability relative to digestive enzymes.

[0154] Properties of Particles

[0155] [Zeta potential]

[0156] Generally, most particles dispersed in a liquid are positively or negatively charged. Ions with opposite charges are strongly attracted to the particle surface and form a fixed layer (fixed layer) and a layer on the outside of it (diffusion layer), forming the so-called diffusion double layer. It is speculated that a part of the inner side of the diffusion layer and the fixed layer move with the particles.

[0157] The zeta potential is the potential of the aforementioned moving surface (sliding surface) when the potential is taken as a reference to the potential of the electrically neutral region sufficiently far from the particle. The zeta potential can be used as an indicator of the dispersion state of particles. If the absolute value of the zeta potential increases, the repulsive force between particles becomes stronger and the stability of the particles increases; conversely, as the zeta potential approaches 0, the particles become more prone to aggregation. From the viewpoint of increasing the adhesion of particles to the negatively charged cell wall and improving the intracellular migration of bioactive molecules, it is also preferable to charge the particles so that their surface has a positive zeta potential.

[0158] In one embodiment, the Zeta potential of the particles is 10mV to 100mV, more preferably 20mV to 50mV.

[0159] (Methods for manufacturing microparticles)

[0160] In one embodiment, the method for manufacturing the microparticles of the present invention includes a step of mixing chitosan and / or chitosan derivatives with an anionic surfactant in a solvent.

[0161] It is believed that the formation of microparticles is mediated by (1) the protonation of the amino groups (-NH3) of chitosan. + ) and the carboxyl carboxyl ion (-COO) of the deprotonated anionic surfactant. - (1) The electrostatic interaction of chitosan and anionic surfactants, the complexation of chitosan and anionic surfactants, and (2) the association of anionic surfactants caused by hydrophobic interactions (References: Takashi Kuroiwa, Urakami Foundation Research Report Vol.22, pp.16-20 (2015)).

[0162] The preferred conditions for mixing the components of the microparticles are a neutral to weakly acidic pH, which indicates the cationic nature of chitosan.

[0163] Solvent

[0164] In the manufacture of microparticles, the solvent can be any solvent known to be able to dissolve chitosan. Examples of solvents include aqueous solutions of acetate buffer, acetic acid, formic acid, propionic acid, malic acid, succinic acid, and lactic acid. Buffer solutions are, for example, acetate buffer (pH 5.0, physiological saline) with a final concentration of 5–50 mM.

[0165] Microparticles can be manufactured by mixing chitosan and / or chitosan derivatives with anionic surfactants in the solvents described above.

[0166] The mixing order of the components of chitosan or chitosan derivatives and anionic surfactants into the solvent can be arbitrary. Furthermore, as described in the pharmaceutical composition section, the mixing order of the antigen, chitosan or chitosan derivatives and anionic surfactants into the solvent can be arbitrary, or the components can be mixed simultaneously.

[0167] That is, when the particulate immune activator of the present invention is provided as an article, if the article is made into a mixture of chitosan or chitosan derivatives and anionic surfactant, then when using the article, the user only needs to add the desired antigen to the mixture, without the need for the complicated operation of mixing multiple components. Therefore, an article with a simpler usage procedure can be provided.

[0168] <Molar ratio of each component>

[0169] The ratio of chitosan or chitosan derivative to anionic surfactant in each microparticle varies depending on factors such as the molecular weight of the chitosan or the type of surfactant used. In one embodiment, the molar ratio of the amino group of chitosan or chitosan derivative to the anionic group of the anionic surfactant in each microparticle is preferably in the range of 1:0.05 to 1:1, more preferably 1:0.1 to 1:0.5, and even more preferably 1:0.15 to 1:0.3.

[0170] <Content of each component>

[0171] The amount of chitosan and / or chitosan derivatives and anionic surfactant contained in the microparticles is determined, for example, by determining the final concentration of chitosan and / or chitosan derivatives in the solvent. In one example, it is prepared such that the final concentration of chitosan and / or chitosan derivatives in the solvent is 0.1%.

[0172] The microparticles of this invention can be manufactured by mixing all the components in an aqueous solvent. Therefore, emulsification and other similar processes are not required, making them easy to manufacture.

[0173] [Particle size]

[0174] When preparing a particulate shape for administering the immune activator of the present invention into a living organism, the particle size can be set, for example, to 20 nm to 10 μm.

[0175] In this specification, when referring to particle size, unless otherwise specified, it refers to the average particle size. "Average particle size" means the average size of multiple (e.g., 100) particles.

[0176] The microparticles of this invention can be used not only as adjuvants but also as drug delivery carriers. Furthermore, because they utilize biodegradable chitosan or chitosan derivatives that are non-toxic to living organisms, they exhibit suitable sustained-release properties in vivo and do not remain in the body for extended periods, completely decomposing and disappearing after a suitable time. Therefore, they do not leave residues in the body and cause side effects to the body's health, making them safe to use.

[0177] Furthermore, Freund's adjuvant, which was previously used, was considered problematic from an animal welfare perspective because it caused inflammation and tissue necrosis at the administration site in the target animal. Therefore, regulations regarding the use of Freund's adjuvant have been established through various guidelines (ARAC. Guidelines for the Use of Adjuvants in Research, Revised 04 / 10 / 13 (http: / / oacu.od.nih.gov / ARAC / documents / Adjuvants.pdf), Canadian Council on Animal Care guidelines on: antibody production (2002) (http: / / www.ccac.ca / Documents / Standards / Guidelines / Antibody_production.pdf)).

[0178] In contrast, the immune activator of the present invention can be administered to animals without causing excessive suffering, and can be manufactured safely, inexpensively, and easily, thus having very high commercial value.

[0179] For example, previous methods using sodium tripolyphosphate micronized chitosan as an adjuvant and as a delivery carrier for drugs containing proteins or nucleic acids have also been studied. These methods utilize the ionic bonds between positively charged micronized chitosan and negatively charged surface substances.

[0180] In contrast, the microparticles of the present invention use a substance with a hydrophobic group as an anionic surfactant. Therefore, the bond between the adjuvant particle and the antigen carried by the particle is formed, in addition to ionic bonds, a hydrophobic bond is also formed. Thus, the adjuvant and antigen are firmly bonded. Therefore, the microparticles of the present invention exhibit significantly improved immunoactivating activity compared to conventionally known microparticles. It should be noted that the immunoactivating effect of the immunoactivator of the present invention can be evaluated, for example, by measuring antibody titers using known methods such as ELISA.

[0181] [3. Pharmaceutical Composition]

[0182] Next, the pharmaceutical composition of the present invention will be described. The present invention provides a pharmaceutical composition comprising the immune activator of the present invention as an active ingredient. In one embodiment, the pharmaceutical composition of the present invention is a composition for activating immunity in an organism, and is a composition intended to prevent and / or treat diseases, ailments, or disorders by enhancing or increasing immune activation.

[0183] (Pharmaceutical compositions containing immune activators with chitosan derivatives)

[0184] In one embodiment, the pharmaceutical composition may comprise two or more chitosan derivatives of the present invention. In another embodiment, the pharmaceutical composition further comprises at least one antigen. As an example, the pharmaceutical composition of the present invention is configured as a vaccine composition. As yet another embodiment, the pharmaceutical composition of the present invention further comprises at least one substance selected from suitable solvents, carriers, excipients, and adjuvants. Examples of adjuvants that serve as immune activators include wetting agents, emulsifiers, pH adjusters, and other adjuvants. The components constituting the pharmaceutical composition are described below.

[0185] [Chitosan derivatives]

[0186] The chitosan derivatives of the present invention contained in the pharmaceutical compositions of the present invention are suitable for use with the substances described in [1. Chitosan Derivatives] above.

[0187] [antigen]

[0188] Examples of antigens in the pharmaceutical compositions of the present invention include antigens comprising polypeptides, recombinant proteins, natural proteins, nucleic acids, and their partial degradation products. Antigens may be derived from pathogenic organisms or allergens, such as viruses (e.g., HIV), bacteria, fungi, and parasites. The pharmaceutical compositions of the present invention, by comprising an immune activator and an antigen, can be used as vaccine compositions. Furthermore, by comprising specific antigens, vaccine compositions can be formulated for the prevention and / or treatment of specific diseases, ailments, or disorders.

[0189] [solvent]

[0190] Examples of solvents for the pharmaceutical compositions of the present invention include water and buffer solutions. Examples of buffer solutions include physiological saline, phosphate buffer, and phosphate gel solution. Furthermore, the solvent used in the composition may be a mixture of two or more of the above-mentioned solvents.

[0191] pH adjuster

[0192] The pharmaceutical composition of the present invention may also contain at least one pH adjuster. As a substance used to adjust the pH of the pharmaceutical composition containing the chitosan derivative of the present invention, either an alkaline substance or an acidic substance may be used as needed. Examples of alkaline substances for adjusting pH include sodium hydroxide. Examples of acidic substances for adjusting pH include acetic acid and hydrochloric acid.

[0193] [Other adjuvants]

[0194] In another embodiment, the pharmaceutical composition of the present invention may also contain one or more other adjuvants. Examples of other adjuvants include, for instance, alum, Freund's complete adjuvant, Freund's incomplete adjuvant, oil adjuvants, saponins, cell wall skeletal components, lipopolysaccharides, endotoxins, abricin, liposomes, bacterial DNA, synthetic oligonucleotides, vitamin E, glycolipids, and squalene. Combining multiple adjuvants sometimes produces a synergistic effect compared to administering each adjuvant individually in the promotion of immune responses, etc.

[0195] [Content of chitosan derivatives]

[0196] The content of the chitosan derivative in the pharmaceutical composition of the present invention is preferably 0.01% to 10% by weight of the total mass of the composition, more preferably 0.1% to 5% by weight, and particularly preferably 0.5% to 2% by weight. By setting a concentration of 0.5% by weight or more, the effect as an immune adjuvant for promoting an immune response can be sufficiently obtained. Furthermore, by setting a concentration of 2% by weight or less, the composition can be maintained at a easily manageable viscosity, for example, a viscosity suitable for use as an injectable composition.

[0197] Furthermore, the viscosity of the pharmaceutical composition of the present invention is preferably 100 mPa·s or less, more preferably 10 mPa·s or less. The viscosity can also be measured using methods known in the art.

[0198] [Dosage Form]

[0199] The dosage form of the pharmaceutical compositions of the present invention is not particularly limited and can be formulated as liquid, solid, semi-solid, or semi-liquid. These dosage forms can be readily manufactured based on methods known in the art. In the case of a liquid dosage form, examples include preparations obtained by dispersing, suspending, or dissolving chitosan derivatives in a substantially aqueous solvent. Here, in one embodiment, a substantially aqueous preparation or formulation refers to a preparation containing one or more non-aqueous components or one or more pharmaceutically permissible components in a certain proportion. In the case of a solid dosage form, examples include powders, granules, tablets, and capsules. Furthermore, in the case of a semi-solid or semi-liquid dosage form, examples include ointments, lotions, creams, and gels.

[0200] [Route of administration]

[0201] Routes of administration for pharmaceutical compositions may include, but are not limited to, oral, local, subcutaneous, intramuscular, intravenous, intradermal, transmucosal, and transdermal administration.

[0202] [Administration Method]

[0203] The administration of the pharmaceutical composition of the present invention to the subject is based on known methods such as direct injection into the subcutaneous, intradermal, intravenous, intramuscular, or intraperitoneal cavity, spraying into the mucous membranes of the nasal cavity, oral cavity, lungs, vagina, or rectum, oral administration, and intravascular administration.

[0204] [Purpose of Use]

[0205] More specific uses of the pharmaceutical compositions of the present invention include the treatment or prevention of diseases such as cancer, infections, autoimmune disorders, or allergies. Examples include vaccine therapy, immunotherapy for cancer, and desensitization therapy for allergens.

[0206] [Target population for drug administration]

[0207] As subjects for administration of the pharmaceutical compositions of the present invention, all animals can be listed, preferably vertebrates such as mammals and birds, and more preferably mammals. Furthermore, among mammals, humans are preferred, but livestock, laboratory animals, or pets may sometimes also be subjects. Specifically, examples include livestock such as chickens, pigs, horses, goats, sheep, and cattle; pets such as cats, dogs, hamsters, rabbits, and guinea pigs; mice, rats, monkeys, fish, and birds.

[0208] (Pharmaceutical compositions containing particulate-shaped immune activators)

[0209] [antigen]

[0210] In another embodiment, the pharmaceutical composition of the present invention comprises a particulate immune activator and an antigen. The particulate immune activator is as described in the above-described item [particulate immune activator]. The amount of antigen contained in the particulate pharmaceutical composition may be appropriate depending on the application or site of administration.

[0211] The pharmaceutical composition of the present invention, compared with conventional adjuvants, can achieve very high immune activation activity and high neutralizing antibody titers even with small amounts of immune activators and antigens. It is possible to formulate vaccine compositions that are safe, inexpensive, and do not cause excessive stimulation to the organism due to reduced adjuvant and antigen dosages.

[0212] (Method for manufacturing a pharmaceutical composition containing antigens and microparticles)

[0213] In one embodiment, a pharmaceutical composition containing antigen and microparticles can be manufactured by mixing the antigen, chitosan and / or chitosan derivatives and anionic surfactant in a solvent.

[0214] At this point, the mixing order of the antigen, chitosan and / or chitosan derivatives, and anionic surfactant into the solvent can be arbitrary, or the components can be mixed simultaneously. In one example, the anionic surfactant is added after the mixture is pre-mixed with the antigen in a solvent such as a buffer solution. In another example, chitosan and / or chitosan derivatives are added after the mixture is pre-mixed with the anionic surfactant in a solvent. In yet another example, the antigen is added after the mixture is pre-mixed with the anionic surfactant in a solvent.

[0215] [Dosage Form]

[0216] The dosage form is the same as that of pharmaceutical compositions containing immune activators with chitosan derivatives.

[0217] [Route of administration]

[0218] Examples of routes of administration for the pharmaceutical composition include oral, topical, subcutaneous, intramuscular, intravenous, intradermal, transmucosal, and transdermal administration, but are not limited to these. Examples of routes of administration for the pharmaceutical composition in which the immune activator of the present invention is a microparticle include oral, subcutaneous, intramuscular, intradermal, transmucosal, and transdermal administration.

[0219] [Administration Method]

[0220] The pharmaceutical composition of the present invention, in which the immune activator is in the form of microparticles, can be injected directly into an intramuscular, intradermal, or intraperitoneal cavity. Alternatively, it can be sprayed onto the mucous membranes of the nasal cavity, oral cavity, lungs, vagina, or rectum. In another embodiment, the pharmaceutical composition of the present invention, in which the immune activator is in the form of microparticles, is administered orally. In the case of oral administration, the microparticles are stable in gastrointestinal fluids and can reach the intestine without decomposition, releasing antigens and the like from the composition.

[0221] [Dosage]

[0222] The pharmaceutical compositions of the present invention are generally administered to subjects such as animals and humans in amounts sufficient to enhance the immune system. For example, in the case of oral administration to humans, the dosage of the pharmaceutical compositions of the present invention can be appropriately determined by a physician based on observation of the patient's condition. In one embodiment, when administering a pharmaceutical composition comprising the above-described particulate immune activator and antigen to a subject, the dosage can be appropriately determined based on the type of control, the purpose, and the frequency of administration.

[0223] If the drug is administered at this dosage, it is effective because it will not cause adverse effects due to excessive immune activation in the subject, and it can maintain the safety of the organism and achieve sufficient immune activation activity.

[0224] [Uses and Target Patients]

[0225] The intended use and target population are the same as those for [pharmaceutical compositions containing immune activators with chitosan derivatives].

[0226] [4. Food and drink]

[0227] The present invention also provides a food product comprising the immune activator of the present invention. In one embodiment, the food product of the present invention comprises the chitosan derivative described in item [1. Chitosan derivative] above. In another embodiment, the food product of the present invention uses the immune activator described in item [2. Immune activator] above as an active ingredient, and contains an effective amount for activating the immune function of an organism that has ingested the food product.

[0228] The purpose of the dietary products of this invention is to activate the immune system through diet. In this specification, "dietary products" includes not only beverages, food, and health foods ingested by humans, but also feed or bait for livestock and pets.

[0229] The form of the food and beverage is not particularly limited; for example, it can be formulated into pharmaceutical preparations, processed foods, and various beverages. Examples of pharmaceutical preparations include health foods, functional foods, and foods for specific health purposes. When the "food and beverage" of the present invention is formulated into a pharmaceutical preparation, it can be based on the composition of a pharmaceutical composition. That is, the content of the immune activator in the immune-activating food and beverage can be set according to the content of the pharmaceutical composition, and the dosage form can be formulated as tablets, capsules, beverages, and powders based on the pharmaceutical composition. Furthermore, when the food and beverage is a processed food, an effective amount of the immune activator can be added to a known processed food. Examples of processed foods include bread, noodles, pastries, and animal feed. Furthermore, when the food and beverage is a beverage, an effective amount of the immune activator can be added to a known beverage. Examples of beverages include fruit juice, milk beverages, and syrups. By orally ingesting the food and beverage of the present invention, the immune system can be activated, which can prevent or improve diseases, ailments, or disorders. Due to the above facts, the immune activator of the present invention can also be ingested into the body long-term as a health food or pharmaceutical product.

[0230] The immune-activating effects of the chitosan derivatives of the present invention, the immune activators comprising the chitosan derivatives, the pharmaceutical compositions comprising the immune activators, and the diets comprising the chitosan derivatives are believed to be induced by antigen capture due to ionization, micronization caused by the addition of cofactor anions or retention caused by gelation at pH in vivo, and degradation caused by lysozyme or phagocytes. The chitosan derivatives of the present invention can be optimized for various desired properties by modifying the structure, length, molecular weight, and degree of substitution of the main chain and side chains. For example, they can be adapted to the optimal particle size for delivery into the body, and can be micronized as needed by forming liposomes or the like. The chitosan derivatives of the present invention are first discovered in this application and are expected to be used in various applications.

[0231] Therefore, in addition to being expected to be applicable to the basic uses of chitosan and its derivatives as medical raw materials such as cosmetics, pharmaceuticals and medical coverings, the chitosan derivatives of this application are also expected to be applicable to new uses such as drug delivery system (DDS) carriers and microcapsules.

[0232] Furthermore, the particulate immune activator of this invention has high safety in organisms and is easy to manufacture. At the same time, compared with conventional Freund's adjuvant, it has a very high immune activation effect and is suitable for use as an immune adjuvant in vaccine compositions, etc.

[0233] [5. Examples of specific solutions of the present invention]

[0234] As an example, the present invention includes any of the following inventions.

[0235] <1> An immune activator, characterized in that it comprises chitosan and / or chitosan derivatives with a weight-average molecular weight of 10k to 1000k, and an anionic surfactant, wherein the immune activator is in particulate form.

[0236] <2> according to <1> The immune activator is characterized in that the anionic surfactant is selected from at least one of phospholipids and fatty acids with 10 to 22 carbon atoms and their salts.

[0237] <3> according to <2> The immune activator is characterized in that the phospholipids are selected from the group consisting of lecithin and lysophosphatidylcholine, and the fatty acids and their salts are selected from the group consisting of sodium oleate and sodium laurate.

[0238] <4> according to <1> ~ <3> The immune activator according to any one of the following methods is characterized in that the chitosan derivative is a chitosan derivative with immune activating activity represented by the following general formula (I).

[0239] [Chemical Formula 9]

[0240]

[0241] In formula (I), x, y, z are molar ratios, x+y+z=1, x, y and z are each independently 0≤x<1, 0≤y<1, 0<z≤1, and the number of constituent sugar units of formula (I) is 30 to 12,000,

[0242] R 1 is a pullulan side chain represented by the following general formula (II),

[0243] [Chemical Formula 10]

[0244]

[0245] a represents an integer of 20 to 600, and the degree of substitution is 0.001 to 0.1, or

[0246] R 1 is a dextran side chain represented by the following general formula (III),

[0247] [Chemical Formula 11]

[0248]

[0249] a represents an integer of 15 to 300, and the degree of substitution is 0.001 to 0.1, or

[0250] R 1 is a glucosamine side chain represented by the following general formula (IV),

[0251] [Chemical Formula 12]

[0252]

[0253] R 2 is an acetyl group, and the degree of substitution is 0.05 to 0.5, or

[0254] R 1 is a lysine side chain represented by the following formula (V),

[0255] [Chemical Formula 13]

[0256]

[0257] the degree of substitution is 0.02 to 0.5]

[0258] <5>The immunostimulant according to <4>, characterized in that the number of constituent sugar units of the aforementioned formula (I) is 60 to 6,000.

[0259] <6> according to <4> or <5> The immune activator is characterized in that z is 0.001 to 0.5.

[0260] <7> according to <1> ~ <6> The immune activator according to any one of the above-mentioned methods is characterized in that the molar ratio of the amino group of the chitosan and / or chitosan derivative in each of the above-mentioned particles to the anionic group of the above-mentioned anionic surfactant is 1:0.05 to 1:1.

[0261] <8> A pharmaceutical composition, characterized in that it comprises <1> ~ <7> The immune activator mentioned in any one of these statements is the active ingredient.

[0262] <9> A food product, characterized in that it contains <1> ~ <7> The immune activator mentioned in any one of the above.

[0263] This invention is not limited to the embodiments described above. Various modifications can be made within the scope of the claims. Embodiments obtained by appropriately combining the technical solutions disclosed in different embodiments are also included within the technical scope of this invention. Furthermore, by combining the technical solutions disclosed in each embodiment, new technical features can be formed.

[0264] Example

[0265] [Example 1: Synthesis of chitosan derivatives]

[0266] (Synthesis of Ch10-Dex)

[0267] Chitosan 10 (1.00 g, amino 5.08 mmol) (manufactured by Wako Pure Chemical Industries, Ltd., degree of deacetylation 85%, Lot TLE3201) was dispersed in water (45 mL), dissolved by adding acetic acid (300 μL), and then dextran 40 (4.00 g) (manufactured by Meito Sangyo Co., Ltd., weight average molecular weight Mw 40,000, Lot CL-A594A) was added and dissolved. Then 2-methylpyridinium borane (100 mg, 0.93 mmol) was added and stirred for 25 hours. After ultrafiltration, the mixture was freeze-dried to obtain a chitosan derivative with dextran side chains (dextran-branched chitosan) Ch10-Dex (2.33 g).

[0268] 1 H-NMR (400MHz, D2O+TFA-d): δ2.07 (CH3 of NHCOCH3), 3.19 (H at C2 position of GlcN), 3.50-4.00 (H at C2, C3, C4, C5, and C6 positions of other sugar residues).

[0269] The comparison of the spectrum with that of chitosan 10, the raw material, showed that the dextran content in Ch10-Dex was 69%. At this point, with the molecular weight of chitosan set to approximately 100,000, the number of glucosamine units constituting the chitosan was 600 residues, the chitosan / dextran molecule ratio was 1 / 5.6, and the degree of substitution was 0.01 based on 5.6 substitutions per 600 residues. Furthermore, the number of disaccharide units constituting the dextran was approximately 120 (average molecular weight of dextran 40,000 / molecular weight of disaccharide unit 324).

[0270] (Synthesis of Ch10-Pul)

[0271] Chitosan 10 (500 mg, amino group 2.54 mmol) (manufactured by Wako Pure Chemical Industries, Ltd., degree of deacetylation 85%, LotTLE3201) was dispersed in water (22.5 mL), dissolved in acetic acid (150 μL), and then low molecular weight pullulan (2.00 g, reduced terminal residue 0.071 mmol) (weight average molecular weight Mw 73,000, number average molecular weight Mn 28,000) was added and dissolved. Then 2-methylpyridinium borane (50 mg, 0.47 mmol) was added and the mixture was stirred for 24 hours. Pullulan-branched chitosan Ch10-Pul (1.39 g) was obtained by ultrafiltration followed by freeze-drying.

[0272] 1 ¹H-NMR (400MHz, D₂O+TFA-d): δ 2.10 (CH₃ of NHCOCH₃), 3.20 (H at C2 position of GlcN), 3.40-4.10 (H at C2, C3, C4, C5, and C6 positions of other sugar residues).

[0273] The comparison of the spectra with those of the raw material chitosan 10 showed that the pullulan content in Ch10-Pul was 72%. At this point, with the molecular weight of chitosan set to approximately 100,000, the glucosamine units constituting chitosan comprised 600 residues, the chitosan / pullulan molecule ratio was 1 / 3.5, and the degree of substitution was 0.006 based on 3.5 substitution per 600 residues. Furthermore, the number of residues in the trisaccharide units constituting pullulan was approximately 150 (average molecular weight 73,000 / trisaccharide unit molecular weight 486).

[0274] (Ch10-GlcNAc synthesis)

[0275] Chitosan 10 (500 mg, amino 2.54 mmol) (manufactured by Wako Pure Chemical Industries, Ltd., degree of deacetylation 85%, LotTLE3201) was dispersed in water (20 mL), dissolved by adding acetic acid (200 μL), and then N-acetyl-D-glucosamine (550 mg, 2.48 mmol) was added and dissolved. Then, 2-methylpyridinium borane (266 mg, 2.49 mmol) and water were added to bring the total solution volume to 25 g, and the mixture was stirred for 18 days. After ultrafiltration, N-acetyl-D-glucosamine-branched chitosan Ch10-GlcNAc (642 mg) was obtained by freeze-drying. 1 ¹H-NMR (400MHz, D₂O+TFA-d): δ 2.10 (CH₃ of NHCOCH₃), 3.21 (H at C2 position of GlcN), 3.32 (H at C2 position of substituted GlcN), 3.50-4.40 (H at C2, C3, C4, C5, and C6 positions of other sugar residues).

[0276] The degree of substitution is 0.26 based on the ratio of H at the C2 position of substituted and unsubstituted GlcN.

[0277] (Ch10-Lys synthesis)

[0278] Chitosan 10 (500 mg, amino 2.54 mmol) (manufactured by Wako Pure Chemical Industries, Ltd., degree of deacetylation 85%, LotTLE3201) was dispersed in water (15 mL) and dissolved by adding 1M hydrochloric acid (1.9 mL). The pH was then adjusted to 6 by adding 1M sodium bicarbonate aqueous solution. L-lysine monohydrochloride (696 mg, 3.81 mmol) was added and dissolved. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride (1.12 g, 3.81 mmol) and water were added to bring the total solution volume to 25 g. The mixture was stirred for 24 hours. After ultrafiltration and freeze-drying, L-lysine-branched chitosan Ch10-Lys (506 mg) was obtained. 1 1H-NMR (400MHz, D2O+TFA-d): δ 1.52 (lysine γ-CH2), 1.73 (lysine δ-CH2), 1.90 (lysine β-CH2), 2.08 (CH3 from NHCOCH3), 3.00 (lysine ε-CH2), 3.19 (H at C2 position of GlcN), 3.40-4.40 (H at C2, C3, C4, C5, and C6 positions of other sugar residues). The degree of substitution is 0.09 based on the ratio of lysine to the H at C2 position of unsubstituted GlcN.

[0279] (Synthesis of FL80-Dex)

[0280] FL-80 (500 mg, amino 2.72 mmol) (manufactured by KOYO CHEMICAL CO.,LTD., degree of deacetylation 90%, weight average molecular weight Mw30k (3,000), Lot 0507-27) was dispersed in water (25 mL), dissolved by adding acetic acid (200 μL), and then dextran 40 (2.00 g) (manufactured by Meito Sangyo Co., Ltd., weight average molecular weight Mw29k (29,000), Lot CL-A594A) was added and dissolved. Then 2-methylpyridinium borane (50 mg, 0.47 mmol) was added and stirred for 24 hours. After ultrafiltration, the mixture was freeze-dried to obtain the chitosan derivative FL80-Dex (876 mg) with dextran side chains.

[0281] 1 ¹H-NMR (400MHz, D₂O+TFA-d): δ 2.07 (CH₃ of NHCOCH₃), 3.18 (H at C2 position of GlcN), 3.50-4.10 (H at C2, C3, C4, C5, and C6 positions of other sugar residues), 4.58 (H at C1 position of N-acetylglucosamine), 4.87 (H at C1 position of glucosamine), 4.98 (H at C1 position of dextran).

[0282] The result of comparing the spectrum with that of FL-80, the raw material, is that the dextran content in FL80-Dex is 50 mol% (monosaccharide ratio). At this time, when the molecular weight of chitosan is set to about 30,000, the glucosamine unit constituting chitosan is 180 residues, the ratio of chitosan / dextran molecules is 1 / 1.03, and the degree of substitution is 0.0057 based on 1.03 substitution / 180 residues.

[0283] [Example 2: Evaluation of antibody adjuvant activity of chitosan derivatives]

[0284] The chitosan derivative manufactured in Example 1 was used in the following activity evaluation test as an antibody adjuvant.

[0285] (Experimental materials)

[0286] <Examined animal>

[0287] Five-week-old female Balb / c mice were randomly divided into groups of three after one week of acclimatization.

[0288] <Immune antigens>

[0289] Mice administered the test solutions were divided into six groups (1-6) for the following experiments. Group 1 served as a control, using physiological saline; Group 2 served as a comparative example, using non-derivative chitosan (the raw material used in Example 1, namely chitosan 10); Group 3 used dextran-branched chitosan prepared in Example 1; Group 4 used pullulan-branched chitosan prepared in Example 1; Group 5 used N-acetylglucosamine-branched chitosan prepared in Example 1; and Group 6 used lysine-branched chitosan prepared in Example 1. Each group (2-6) was dissolved in 10 mM acetate buffer (pH 5.0) at a concentration of 2%, and 500 μL was aliquoted. Group 1 served as a control, using 500 μL of physiological saline. An equal amount of antigen solution (peptide conjugate KLH: Sigma-Aldrich, solvent: physiological saline) was added to each group's solution, and the resulting solution was used as the immunogenicity. It should be noted that the immunization dose was set at 100 μL per mouse.

[0290] (Experimental Methods)

[0291] <Immunization Schedule>

[0292] Mice were initially immunized at 6 weeks of age (administered intraperitoneally), and then booster immunizations were performed every 2 weeks using the same procedure. Starting one week after the second immunization, blood was collected from the tail vein to obtain serum.

[0293] <Antibody Titer Determination Methods>

[0294] Serum dilution steps

[0295] The serum obtained above was diluted 100-fold with diluent (0.1% BSA, 0.025% Tween-20 PBS (phosphate-buffered saline)) and used as a stock sample. The stock sample was diluted 10-fold to prepare a 1000-fold dilution, and then repeatedly diluted 3-fold to prepare a gradient dilution up to a 2,187,000-fold dilution.

[0296] Evaluation board production

[0297] KLH (Keyhole limpet hemocyanin) was dissolved in carbonate buffer at a concentration of 2 μg / mL, and 50 μL was dispensed into each well of a 96-well microplate to prepare a solid-phase plate for each antigen, which was then used as an evaluation plate.

[0298] Antibody titer assay

[0299] Add 50 μL of serially diluted serum buffer (1000-2187000-fold dilution) obtained from each mouse to each well in the vertical column of the evaluation plate, and perform antigen-antibody reaction overnight at 4°C. Afterward, wash the plate three times with 100 μL of washing buffer (T-PBS), and further react with POD-labeled anti-mouse IgG (Simple Stain MAX-PO(M): NICHIREICORPORATION / 100-fold dilution) or POD-labeled anti-mouse IgM (Sigma-Aldrich / 10000-fold dilution) as secondary antibodies. After 1 hour of reaction, wash and develop color with TMB as a matrix at room temperature for 10 minutes, stopping the reaction with 2N sulfuric acid. After the reaction is stopped, quickly measure the absorbance using a microplate reader (reading wavelength: 450 nm, reference wavelength: 650 nm). The antibody titer of IgM relative to KLH obtained as described above is shown in the figure. Figure 1 middle. Figure 1 The antibody titers after administration of each chitosan derivative are shown. The antibody titers in groups 1–6 after two and three immunizations are shown for serum at 1000-fold dilution (a) and 3000-fold dilution (b), respectively.

[0300] [Example 3: Potency increase caused by micronization]

[0301] The titers of antigen-specific antibodies after secondary immunization of mice were evaluated in the following groups.

[0302] (Experimental materials)

[0303] <Examined animal>

[0304] Similar to Example 2, for mice, 5-week-old female Balb / c mice were randomly grouped into groups of 3 after 1 week of acclimatization.

[0305] <Immune antigens>

[0306] Mice administered the test solutions were divided into groups 1–5 for the following experiments. Group 1 was a comparative example, using Freund's adjuvant; Group 2 used non-derivative chitosan (the raw material used in Example 2, i.e., FL80); Group 3 used dextran-branched chitosan (FL80-Dex) prepared in Example 2; Group 4 used micronized non-derivative chitosan (FL80+OA) using sodium oleate; and Group 5 used micronized dextran-branched chitosan (FL80-Dex+OA) using sodium oleate (OA).

[0307] For Group 1, the antigen solution was prepared by emulsifying a 1:1 mixture of ovalbumin (OVA) (manufactured by Wako Pure Chemical Industries, Ltd., 20 μg / mL physiological saline) and Freund's complete adjuvant (manufactured by Thermo Fisher Scientific KK) using two Luer lock syringes connected by biaxial needles. The dosage was 100 μL per mouse, and the antigen dosage was 1 μg per mouse. For Groups 2-5, the final chitosan concentration was set at 0.1%, and the additive dosage was set at 0.28 equivalents of chitosan amino groups. Acetate buffer (pH 5.0, physiological saline) was used with a final concentration of 5 mM. It should be noted that the dosage was 100 μL per mouse. For each group, the solution prepared by mixing ovalbumin (OVA) (manufactured by Wako Pure Chemical Industries, Ltd.) as the antigen solution with the test solution at a dosage of 1 μg per mouse was used as the immunogenic antigen.

[0308] (Experimental Methods)

[0309] <Immunization Schedule>

[0310] Mice were initially administered the drug intraperitoneally at 6 weeks of age, followed by a booster immunization 2 weeks later using the same procedure. Blood was collected from the tail vein one week after the second immunization to obtain serum.

[0311] <Antibody Titer Determination Methods>

[0312] Serum dilution steps

[0313] The serum obtained above was diluted 100-fold with diluent (0.1% BSA, 0.025% Tween-20 PBS (phosphate-buffered saline)) to prepare a stock sample. The stock sample was then diluted 10-fold to prepare a 1000-fold dilution, and then repeatedly diluted 3-fold to prepare a gradient dilution up to a 2,187,000-fold dilution.

[0314] Evaluation board production

[0315] OVA was dissolved in carbonate buffer at a concentration of 2 μg / mL, and 50 μL was dispensed into each well of a 96-well microplate to prepare a solid-phase plate for each antigen, which was then used as an evaluation plate.

[0316] Antibody titer assay

[0317] Add 50 μL of serially diluted serum buffer (1000-2187000-fold dilution) obtained from each mouse to each well in the vertical column of the evaluation plate, and perform antigen-antibody reaction overnight at 4°C. Then, wash the plate three times with 100 μL of washing buffer (T-PBS), and further react with POD-labeled anti-mouse IgG (Simple Stain MAX-PO(M): NICHIREICORPORATION / 100-fold dilution) as a secondary antibody. After 1 hour of reaction, wash the plate, develop color with TMB as a matrix at room temperature for 5 minutes, and stop the reaction with 2N sulfuric acid. After the reaction is stopped, quickly measure the absorbance using a microplate reader (reading wavelength: 450 nm, reference wavelength: 650 nm).

[0318] The antibody titer of IgG relative to OVA obtained by the above procedure is shown in the figure. Figure 2 According to Figure 2 The results showed that in groups 4 and 5, which had sodium oleate added, the potency was more than 400 times that of the FCA group.

[0319] [Example 4: Evaluation of various additives]

[0320] To investigate the additives used in micronization, the titers of antigen-specific antibodies after secondary immunization of mice were evaluated for the following groups.

[0321] (Experimental materials)

[0322] <Examined animal>

[0323] Similar to Example 2, for mice, 5-week-old female Balb / c mice were randomly grouped into groups of 3 after 1 week of acclimatization.

[0324] <Immune antigens>

[0325] Mice administered the test solutions were divided into groups 1–8 for the following experiments. Group 1 served as a control group and used Freund's adjuvant. Groups 2–8 used micronized dextran-branched chitosan (FL80-Dex) containing sodium oleate, sodium laurate, lysophosphatidylcholine, sodium cholate, sodium tripolyphosphate, sodium hyaluronate, and sodium alginate, respectively.

[0326] The antigen solution used in Group 1 was set the same as that in Group 1 of Example 3. For Groups 2-8, the final chitosan concentration was set to 0.1%, and the amount of additive added was set to 0.28 equivalents of chitosan amino groups. Acetate buffer (pH 5.0, physiological saline) was used, and the final concentration was set to 5 mM. It should be noted that the dosage was set to 100 μL per mouse. As the solution for each group, the ovalbumin (OVA) (solvent: physiological saline) used as the antigen solution was mixed with the test solution at a dosage of 1 μg / mouse to obtain the immunoantigen. The immunization schedule and antibody titer determination method were set the same as in Example 3.

[0327] The antibody titer of IgG relative to OVA obtained by the above procedure is shown in the figure. Figure 3 middle. Figure 3 The horizontal axis represents the serum dilution factor, and the vertical axis represents the absorbance. The absorbance of FCA at a 3000-fold dilution in group 1 (as a comparative example) was approximately equivalent to that at a 729000-fold dilution in groups 2–4. Therefore, it is concluded that approximately 250 times the amount of antibody produced in mice from the administration of FCA in groups 2–4 was obtained.

[0328] according to Figure 3 The results showed that anionic surfactants with alkyl chains, such as sodium oleate in group 2, sodium laurylate in group 3, and lysophosphatidylcholine in group 4, were particularly effective in increasing potency.

[0329] [Example 5: Evaluation of various fatty acid salts]

[0330] To further investigate the fatty acid salts used in micronization, the titers of antigen-specific antibodies after secondary immunization of mice were evaluated in the following groups.

[0331] (Experimental materials)

[0332] <Examined animal>

[0333] Similar to Example 2, for mice, 5-week-old female Balb / c mice were randomly grouped into groups of 3 after 1 week of acclimatization.

[0334] <Immune antigens>

[0335] Mice administered the test solutions were designated as groups 1–3 for the following experiments. Group 1 served as a control group and used Freund's adjuvant, while groups 2 and 3 used micronized chitosan (FL80) containing sodium laurate and decanoic acid, respectively.

[0336] The antigen solution used in Group 1 was the same as that in Group 1 of Example 3. For Groups 2 and 3, the final chitosan concentration was set to 0.1%, and the amount of additive added was set to 0.28 equivalents of chitosan amino groups. Acetate buffer (pH 5.0, physiological saline) was used, and the final concentration was set to 5 mM. It should be noted that the dosage was set to 100 μL per mouse. As the solution for each group, the ovalbumin (OVA) (solvent: physiological saline) used as the antigen solution was mixed with the test solution at a dosage of 1 μg / mouse to obtain the immunogenic antigen. The immunization schedule and antibody titer determination method were set to be the same as in Example 3.

[0337] The antibody titer of IgG relative to OVA obtained by the above procedure is shown in the figure. Figure 4 middle. Figure 4 The horizontal axis represents the serum dilution factor, and the vertical axis represents the absorbance.

[0338] according to Figure 4 The results showed that the micronization of sodium lauryl ester was particularly effective in increasing potency in both groups.

[0339] [Example 6: Evaluation of chitosan molecular weight]

[0340] To evaluate the effect of chitosan molecular weight on antibody titer, the titers of antigen-specific antibodies after secondary immunization of mice were evaluated for the following groups.

[0341] (Experimental materials)

[0342] <Examined animal>

[0343] Similar to Example 2, 5-week-old female Balb / c mice were randomly divided into groups of 3 after 1 week of acclimatization.

[0344] <Immune antigens>

[0345] Mice administered the test solutions were divided into groups 1–7 for the following experiments. Group 1 received Ch100 (Mw 1000kJ), Group 2 received FM-40 (Mw 500kJ), Group 3 received FM-40 (Mw 410kJ), Group 4 received Ch10 (Mw 65kJ), Group 5 received FL-80 (Mw 30kJ), Group 6 received low molecular weight chitosan (Mw 4.1kJ), and Group 7 received oligosaccharide (product name: KOYO Oligo Glucosamine WG (2–8 sugars (Mw 340–1306)) KOYO CHEMICAL CO.,LTD.).

[0346] For each group, Ch100 (manufactured by Wako Pure Chemical Industries, Ltd., degree of deacetylation 83%, Lot DPH0279), FM-40 (manufactured by KOYO CHEMICAL CO.,LTD., degree of deacetylation 92%, Lot 0402-27 and 95% deacetylation, Lot 0930-26), Ch10 (manufactured by Wako Pure Chemical Industries, Ltd., degree of deacetylation 85%, Lot TLE3201), and FL-80 (manufactured by KOYO CHEMICAL CO.,LTD., degree of deacetylation 90%, Lot 0507-27) were dissolved in 2.5% acetic acid solution and then freeze-dried to prepare acetates for use. Low molecular weight chitosan was prepared by hydrolyzing FL-80 in 7% hydrochloric acid at 80°C for 3 hours, followed by ultrafiltration and freeze-drying of the 3k to 10k fraction. KOYO Oligo Glucosamine WG (manufactured by KOYOCHEMICAL CO.,LTD., Lot 140910WG) was used directly. The amount of additive (sodium laurylate) was set to 0.28 equivalents of chitosan amino acid. A buffer solution prepared according to the total amount of chitosan derivative and additive, with a final concentration of 0.1%, was prepared using acetate buffer (pH 5.0, physiological saline) and the final concentration was set to 5 mM. The dosage was set to 100 μL per mouse. As the solution for each group, an immunogen was prepared by mixing ovalbumin (OVA) as the antigen solution into the test solution at a dosage of 1 μg / mouse. The immunization schedule and antibody titer assay method were set as in Example 3. The antibody titer of IgG relative to OVA obtained as described above is shown in [Figure / Table / Insert Table ... Figure 5 In the middle. By Figure 5 The results showed that chitosan with Mw30k~500k exhibited particularly high antibody titers.

[0347] [Example 7: Evaluation of the chitosan / additive mixing ratio]

[0348] To evaluate the effect of the chitosan / additive mixing ratio on antibody titer, the titer of antigen-specific antibodies after secondary immunization of mice was evaluated for the following groups.

[0349] (Experimental materials)

[0350] <Examined animal>

[0351] Similar to Example 2, 5-week-old female Balb / c mice were randomly divided into groups of 3 after 1 week of acclimatization.

[0352] <Immune antigens>

[0353] Mice administered the test solutions were divided into groups 1–5 for the following experiments. Groups 1–3 used micronized dextran-branched chitosan (FL80-Dex+OA) with a chitosan derivative / additive mixing ratio (chitosan derivative / additive) of 1 / 0.28, 1 / 1, and 1 / 7, respectively.

[0354] For each group using the chitosan derivative FL80-Dex, the amount of additive (sodium oleate) mixed was set to 0.28–7 equivalents of chitosan amino groups. The mixture was prepared with a final concentration of 0.12% based on the total amount of chitosan derivative and additive. Acetate buffer (pH 5.0, physiological saline) was used with a final concentration of 5 mM. The dosage was 100 μL per mouse. For each group, an immunogen was prepared by mixing ovalbumin (OVA) (the antigen solution) with the test solution at a dosage of 1 μg / mouse. The immunization schedule and antibody titer assay were set as in Example 3. The antibody titers of IgG relative to OVA obtained as described above are shown in [the table / image / image]. Figure 6 middle.

[0355] In addition, for each group with the current mixing ratio of chitosan derivative and additive, except that no antigen was used, a mixture system without antigen was prepared using the same preparation method as described above, and microparticles were prepared. The average particle size and zeta potential of these microparticles were measured. The average particle size was measured using a thick-film particle size analyzer (Otsuka Electronics Co., Ltd., FPAR-1000). The zeta potential was measured using a laser zeta potential meter (Otsuka Electronics Co., Ltd., ELS-8000). The results of the average particle size and zeta potential measurements are shown below. Figure 7 middle.

[0356] according to Figure 6 and Figure 7 As a result, antibody titers were significantly reduced in the three groups with excessive additives. It is believed that the negative Zeta potential is also a reason for the reduced antibody titers.

[0357] [Example 8: Evaluation of the mixing order of the solutions of chitosan, additives and antigen]

[0358] To evaluate the effect of the mixing order of chitosan, additives, and antigen solutions on antibody titers, the titers of antigen-specific antibodies after secondary immunization of mice were evaluated for the following groups.

[0359] (Experimental materials)

[0360] <Examined animal>

[0361] Similar to Example 2, 5-week-old female Balb / c mice were randomly divided into groups of 3 after 1 week of acclimatization.

[0362] <Immune antigens>

[0363] Mice administered the test solutions were divided into groups 1–5 for the following experiments. Group 1 used micronized dextran-branched chitosan (FL80-Dex+OA), which was micronized by adding sodium oleate (OA) after mixing chitosan derivative (FL80-Dex) with antigen (OVA). Group 2 used micronized dextran-branched chitosan (FL80-Dex+OA), which was micronized by adding chitosan derivative (FL80-Dex) after mixing sodium oleate (OA) with antigen (OVA). Group 3 used micronized dextran-branched chitosan (FL80-Dex+OA), which was micronized by adding antigen (OVA) after mixing chitosan derivative (FL80-Dex) with sodium oleate (OA).

[0364] The final concentration of chitosan FL80-Dex was set at 0.1%, and the additive mixing amount was set at 0.28 equivalents of chitosan amino groups. Acetate buffer (pH 5.0, physiological saline) was used, and the final concentration was set at 5 mM. The dosage was 100 μL per mouse. For each group, ovalbumin (OVA) was mixed with the test solution at a dosage of 1 μg / mouse as the antigen solution to obtain the immunoantigen. The immunization schedule and antibody titer assay method were set as in Example 3. The antibody titers of IgG relative to OVA obtained as described above are shown in [Figure / Table / Insert Table ... Figure 8 The results in the figure show that regardless of the mixing order of the chitosan, additives, and antigen solutions, all mixing orders exhibit equally high antibody titers.

[0365] [Example 9: Antibody production in rats]

[0366] To investigate the effects of antibody production in various animals, the titers of antigen-specific antibodies after secondary immunization of rats were evaluated in the following groups.

[0367] (Experimental materials)

[0368] <Examined animal>

[0369] Female SD rats were used.

[0370] <Immune antigens>

[0371] Each test solution was administered to individual rats via various routes. Individuals 1 and 2 were designated as comparative examples for intramuscular administration using Freund's adjuvant, individuals 3 and 4 were designated as intramuscular administration using micronized dextran-branched chitosan (FL80-Dex+OA) with sodium oleate (OA), and individual 5 was designated as subcutaneous administration using micronized dextran-branched chitosan (FL80-Dex+OA) with sodium oleate (OA).

[0372] The antigen solution used in individuals 1 and 2 was the same as that used in group 1 of Example 3, and the antigen solution used in individuals 3-5 was the same as that used in group 5 of Example 3. The dosage was 100 μL per rat. Following the immunization schedule, primary immunization was performed, followed by booster immunizations every two weeks using the same procedure. One week after the second immunization, blood was collected from the jugular vein to obtain serum. The antibody titer assay was performed using the same method as in Example 3. The antibody titers of IgG obtained as described above relative to OVA after secondary immunization are shown in the figure. Figure 9 In the middle. For example Figure 9 As the results show, adequate antibody production occurred not only in mice but also in rats. Furthermore, adequate antibody production was observed with both intramuscular and subcutaneous administration.

[0373] [Example 10: Antibody production in rabbits]

[0374] To investigate the effects of antibody production in various animals, the titers of antigen-specific antibodies after immunization of rabbits were evaluated in the following groups.

[0375] (Experimental materials)

[0376] <Examined animal>

[0377] Female Japanese white rabbits were used.

[0378] <Immune antigens>

[0379] Each test solution was administered to individual rabbits via various routes. Individuals 1 and 2 were designated as comparative examples of intramuscular administration using Freund's adjuvant, individuals 3 and 4 were designated as intramuscular administration using micronized dextran-branched chitosan (FL80-Dex+OA) with sodium oleate (OA), and individual 5 was designated as subcutaneous administration using micronized dextran-branched chitosan (FL80-Dex+OA) with sodium oleate (OA).

[0380] The antigen solution used in individuals 1 and 2 was the same as that used in group 1 of Example 3, and the antigen solution used in individuals 3-5 was the same as that used in group 5 of Example 3. The dosage was 100 μL per rabbit. Following the immunization schedule, primary immunization was performed, followed by booster immunizations every two weeks using the same procedure. One week after the second immunization, blood was collected from the jugular vein to obtain serum. The antibody titer assay was performed using the same method as in Example 3. The antibody titer of IgG relative to OVA obtained as described above is shown in the figure. Figure 10 In the middle. By Figure 10 The results confirmed that sufficient antibody production also occurred in the rabbits.

[0381] [Example 11: Antiviral Immunity in Chickens]

[0382] To investigate the effects of antibody production in various animals, and to confirm the effectiveness of immunization against viral antigens, the titers of antigen-specific antibodies and neutralizing antibodies after secondary or tertiary immunization of chickens were evaluated in the following groups.

[0383] (Experimental materials)

[0384] <Examined animal>

[0385] Two-week-old SPF Line-M strain chickens (white Leghorn, mixed sex) were grouped using stratified random sampling with equal weight and sex. Each group consisted of 5–6 chickens.

[0386] <Immune antigens>

[0387] The test solutions were administered to groups 1–3. Group 1 was set as a PBS control, group 2 was set as a micronized non-derivative chitosan (FL80+OA) using sodium oleate, and group 3 was set as Freund's adjuvant. In group 2, the final chitosan concentration was set at 0.1%, the buffer was acetate buffer (pH 5.0, physiological saline) with a final concentration of 50 mM, and the added dose was set at 0.25 equivalents of chitosan amino groups. The antigen solution contained 10 μL of Lukert strain of infectious bursal disease (IBD) virus (based on the viral load before inactivation). 6.48 PFU / mL was inactivated by β-propiolactone. Groups 2 and 3 were prepared by mixing each adjuvant at a 1:1 ratio. The dosage was set at 200 μL for the first immunization of each chicken and 500 μL for subsequent immunizations (250 μL at each of the two sites), administered intramuscularly to the foot.

[0388] (Experimental Methods)

[0389] <Immunization Schedule>

[0390] Chickens were initially immunized at 2 weeks of age, and a second immunization was given at 7 weeks of age. All chickens in group 1 and half of chickens in groups 2 and 3 were given a third immunization at 10 weeks of age (all administered intramuscularly in the foot). Blood samples were collected at 6, 9, 10, and 12 weeks of age to obtain serum.

[0391] <Antibody Titer Determination Methods>

[0392] The ELISA assay was performed using the IBD ELISA kit IDEXX IBD (manufactured by IDEXX Laboratories, Inc.) according to the instructions.

[0393] Neutralizing antibody titer assay involved inactivating 12-week-old serum at 56°C for 30 minutes and serially diluting it twofold using serum diluent. Equal volumes of virus solution containing 100–200 PFU for the neutralization test were mixed in 0.1 mL and incubated at 4°C for 18–24 hours. A virus control was prepared by mixing equal volumes of the virus solution with the serum diluent. 0.1 mL of each mixture was inoculated into two primary chicken embryo cells (CEF), incubated at 37°C with 5% CO2 for 60 minutes, then incubated on a double-layer agar medium for 3–4 days. A second double-layer agar medium was then incubated for 6–18 hours, and observation was performed. The highest dilution of serum that reduced plaque count by 50% was defined as the neutralizing antibody titer compared to the virus control.

[0394] The IBD antibody ELISA results and neutralizing antibody titers obtained as described above are shown in Table 1. The results in Table 1 show that the chitosan microparticle immunization group of the present invention exhibits significantly higher antibody titers compared to the Freund's adjuvant immunization group.

[0395] [Table 1]

[0396] Table 1. Chicken IBD Virus Immunization Test

[0397]

[0398] [Example 12: Effects of frequent drug administration in mice]

[0399] To evaluate the effects of frequent administration of chitosan micronized adjuvant and the shift in antibody titers, antigen-specific antibody titers were measured in mice for the following groups.

[0400] (Experimental materials)

[0401] <Examined animal>

[0402] Similar to Example 2, five-week-old female Balb / c mice were randomly divided into groups of five after one week of acclimatization.

[0403] <Immune antigens>

[0404] Mice administered the test solutions were divided into groups 1 and 2 for the following experiments. Group 1 served as a comparative example, using Freund's adjuvant, while group 2 used micronized non-derivative chitosan (FL-80+OA) using sodium oleate. The antigen solution used in group 1 was the same as that in group 1 of Example 3. Group 2 was the same as group 4 of Example 3. The dosage was 100 μL per mouse. For each group, ovalbumin (OVA), serving as the antigen solution, was mixed into the test solution at a dosage of 1 μg per mouse. The immunization schedule was the same as in Example 3, with additional immunizations every two weeks thereafter, and serum was obtained by collecting blood from the tail vein one week after each immunization. The antibody titer assay method was the same as in Example 3.

[0405] The antibody titer of IgG relative to OVA obtained by the above procedure is shown in the figure. Figure 11 middle. Figure 11 The horizontal axis represents the number of booster immunizations, and the vertical axis represents absorbance. Figure 11 The results showed that the chitosan microparticle group exhibited significantly higher antibody titers compared to the Freund's adjuvant group.

[0406] In addition, necropsy was performed on the immunized mice to observe their intraperitoneal condition. The results showed no abnormalities in the chitosan microparticle group after 10 immunizations (not illustrated). On the other hand, after 7 immunizations with Freund's adjuvant, as... Figure 12 As shown in the image, the body swells due to a large amount of ascites, confirming the presence of adjuvant residue.

[0407] Industrial availability

[0408] The chitosan derivatives of the present invention have immunoactivating effects and can be used as immunoactivators. Furthermore, microparticles comprising the chitosan and / or chitosan derivatives of the present invention, as well as anionic surfactants, can be used as immunoactivators.

Claims

1. A composition for immune activation, characterized in that, Said composition comprises an immunostimulant and a solvent; Said immunostimulant comprises a chitosan derivative having a weight-average molecular weight of 10k to 1000k, and an anionic surfactant, Said immunostimulant is in the form of microparticles; The chitosan derivative is micronized using an anionic surfactant; Said chitosan derivative is the chitosan derivative with immunostimulatory activity represented by the following general formula (I), In formula (I), x, y and z are molar ratios, x+y+z=1, x, y and z are each independently 0≤x<1, 0≤y<1, 0<z≤1, and the number of constituent sugar units of formula (I) is 30 to 12,000, R 1 The pullulan side chain is represented by the general formula (II) below. a represents an integer of 20 to 600, and the degree of substitution is 0.001 to 0.1, or R 1 The dextran side chain is represented by the following general formula (III). a represents an integer of 15 to 300, and the degree of substitution is 0.001 to 0.1, or R 1 The glucosamine side chain is represented by the following general formula (IV). R 2 It is an acetyl group with a degree of substitution of 0.05 to 0.5, or R 1 The lysine side chain is as shown in the following formula (V). The degree of substitution is 0.02 to 0.5; Said anionic surfactant is at least one selected from the group consisting of lysolecithin, fatty acids having 12 to 18 carbon atoms, and salts thereof.

2. The composition for immune activation according to claim 1, characterized in that, Said fatty acids and salts thereof are selected from the group consisting of sodium oleate and sodium laurate.

3. The composition for immune activation according to claim 1, characterized in that, The number of constituent sugar units of said formula (I) is 60 to 6,000.

4. The composition for immune activation according to any one of claims 1 to 3, characterized in that, The molar ratio of the amino groups of the chitosan derivative to the anionic groups of the anionic surfactant in each of said microparticles is 1:0.05 to 1:1; said z is 0.001 to 0.

5.

5. A pharmaceutical composition, characterized in that, Comprising the immunostimulatory composition according to any one of claims 1 to 4 as an active ingredient.

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