Polymer and use thereof

Abstract

The present disclosure relates to a polymer and a use thereof. Specifically, the present disclosure relates to a polymer, a polymer particle, and a composition comprising the polymer particle, as well as a use thereof. The polymer particle and the composition comprising the polymer particle have a use in the preparation of a drug for treating allergen-related diseases and / or autoimmune diseases.

Description

A polymer and its uses Technical Field

[0001] This disclosure pertains to the pharmaceutical field and relates to a polymer, polymer particles, and compositions comprising polymer particles, as well as the use of polymer particles and compositions comprising polymer particles in the preparation of medicaments for treating allergen-related diseases and / or autoimmune diseases. Background Technology

[0002] Although autoimmune diseases and allergic diseases have different mechanisms, they both involve abnormal reactions of the immune system. Autoimmune diseases are caused by the immune system mistakenly attacking the body's own healthy tissues (such as rheumatoid arthritis, lupus erythematosus, etc.), which manifests as a disruption of immune tolerance; allergic diseases are caused by the immune system overreacting to normally harmless substances (such as pollen, food, or dust mites), which manifests as immune activation.

[0003] The key to treating autoimmune diseases and allergic diseases lies in controlling the immune response and relieving symptoms. Common treatments for autoimmune diseases often rely on immunosuppressive and immunomodulatory drugs, while common treatments for allergies focus more on suppressing allergic reactions and building tolerance through immunotherapy.

[0004] Patent CN105555301B discloses a method for treating diseases related to immune system abnormalities by administering a composition containing PLGA microspheres encapsulated with antigens to the patient. Summary of the Invention

[0005] This disclosure provides a polymer as shown in Formula I.

[0006] in,

[0007] X is selected from -O-, -S-, or -N(R) 4a )-;

[0008] R 1a R 1b R 2a R 2b Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, carboxyl, N(R) 4b R 4c ), cyano, C 1-6 Alkyl, C 1-6 alkoxy, 3 to 10-membered cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally surrounded by one or more R A replace;

[0009] L is selected from alkylene or heteroalkylene, wherein the alkylene or heteroalkylene is optionally converted by one or more halogens, hydroxyl groups, mercapto groups, carboxyl groups, or N(R) groups. 4bR 4c ), cyano, C 1-6 Alkyl, C 1-6 Alkoxy or 3- to 10-membered cycloalkyl substituted, wherein the alkyl, alkoxy, or cycloalkyl group is optionally substituted with one or more R groups. B replace;

[0010] R 4a R 4b R 4c Each is independently selected from hydrogen and C. 1-6 Alkyl, 3- to 10-membered cycloalkyl, wherein the alkyl or cycloalkyl is optionally surrounded by one or more R C replace;

[0011] R A R B R C Each is independently selected from halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy group, wherein the alkyl group or alkoxy group is optionally replaced by one or more halogen, hydroxyl, mercapto, carboxyl, amino, or cyano groups;

[0012] The molar ratio of a:b is selected from 0:100 to 100:0.

[0013] In some embodiments, the molar ratio of a:b is selected from 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10. In some embodiments, the molar ratio of a:b is selected from 50:50.

[0014] In some implementations, R 1a R 1b Both are hydrogen.

[0015] In some implementations, R 2a It is hydrogen.

[0016] In some implementations, R 2b C 1-6 Alkyl groups, preferably methyl, ethyl, propyl, butyl, or isobutyl, with methyl being the most preferred.

[0017] In some embodiments, L is an alkylene group, said alkylene group optionally being converted by one or more halogens, hydroxyl groups, mercapto groups, carboxyl groups, N(R) 4b R 4c ), cyano, C 1-6 Alkyl, C 1-6 Alkoxy or 3- to 10-membered cycloalkyl substituted, wherein the alkyl, alkoxy, or cycloalkyl group is optionally substituted with one or more R groups. B replace,

[0018] R B R 4b R 4c As defined above.

[0019] In some embodiments, the polymer represented by Formula I is the polymer represented by Formula II.

[0020] in,

[0021] R 3a R 3b Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, carboxyl, N(R) 4b R 4c ), cyano, C 1-6 Alkyl, C 1-6 alkoxy, 3 to 10-membered cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally surrounded by one or more R A replace;

[0022] c is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0023] R A X, R 3a R 3b R 4b R 4c a and b are as defined above.

[0024] In some implementations, X is -N(R) 4a )-, R 4a As defined above.

[0025] In some embodiments, the polymer represented by Formula I is the polymer represented by Formula III.

[0026] Among them, a, b, R 4a c, R 3a R 3b As defined above.

[0027] In some implementations, R 4a It is hydrogen.

[0028] In some implementations, c is selected from 1, 2, 3, 4, 5, 6, with 2, 3, 4 being preferred and 2 being the most preferred.

[0029] In some implementations, R 3a R 3b Each of them is hydrogen independently.

[0030] In some embodiments, the polymer represented by Formula I is the polymer represented by Formula IV.

[0031] Where a and b are as defined above.

[0032] This disclosure also provides a polymer characterized in that it is formed by reacting polymer A of formula Ia with compound B of formula Ib.

[0033] Among them, R 1a R 1b R 2a R 2b a, b, X, and L are as defined above.

[0034] In some embodiments, the compound B is grafted onto polymer A via a condensation reaction with polymer A.

[0035] In some embodiments, the polymer A is as shown in formula IIa.

[0036] Where a and b are as defined above.

[0037] In some embodiments, the compound B is as shown in formula IIb.

[0038] Among them, X and R 3a R 3b c is as defined above.

[0039] In some embodiments, compound B is as shown in formula IIIb.

[0040] Among them, R 4a R 3a R 3b c is as defined above.

[0041] In some embodiments, the compound B is as shown in formula IVb.

[0042] This disclosure also provides a polymer characterized in that it is formed by reacting PLGA with compound B represented by formula Ib, IIb, IIIb or IVb.

[0043] In some embodiments, compound B, represented by formula Ib, IIb, IIIb, or IVb, is grafted onto PLGA via a condensation reaction with PLGA.

[0044] In some embodiments, the molar ratio of PLG to PLA in the PLGA is selected from 0:100 to 100:0. In some embodiments, the molar ratio of PLG to PLA is selected from 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10. In some embodiments, the molar ratio of PLG to PLA in the PLGA is selected from 50:50.

[0045] This disclosure also provides a polymer characterized by being formed by reacting PLGA with 2-aminoethanesulfonamide. In some embodiments, the 2-aminoethanesulfonamide is grafted onto the PLGA via a condensation reaction.

[0046] On the other hand, this disclosure also provides a polymer particle, characterized in that the particle surface is optionally functionalized, the polymer being as defined above.

[0047] In some implementations, the particles are biodegradable.

[0048] In some embodiments, the particles are surface-functionalized. In some embodiments, the functionalization is carboxylation.

[0049] In some embodiments, the carboxylating agent is selected from poly(ethylene-maleic anhydride) (PEMA). In some embodiments, the particles are surface-functionalized with a carboxylic ester. In some embodiments, the particles are polymer particles with PEMA surface carboxylation.

[0050] In some embodiments, PEMA is first hydrolyzed to convert the anhydride into a carboxyl group, and then ionized under alkaline conditions through a neutralization reaction to convert it into poly(ethylene-maleate) (e.g., sodium poly(ethylene-maleate)), which can be used as a surfactant to form particles as described in this disclosure.

[0051] On the other hand, this disclosure also provides polymer particles characterized in that they further contain a surfactant, said polymer as defined above. In some embodiments, the surfactant is an anionic surfactant. In some embodiments, the surfactant is sodium oleate.

[0052] The diameter of the particles described in this disclosure is from 100 to 1000 nm. In some embodiments, the diameter of the particles is selected from 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, and 900 nm.

[0053] In some embodiments, the z-potential of the particles is about 0 mV to about -100 mV. In some embodiments, the z-potential of the particles is about -10 mV, about -20 mV, about -30 mV, about -40 mV, about -50 mV, about -60 mV, about -70 mV, about -80 mV, about -90 mV, or about -100 mV.

[0054] In some embodiments, the particles further comprise one or more antigens. In some embodiments, the antigen is encapsulated in particles having a negative surface zeta potential. In some embodiments, the antigen is selected from heteroantigens, allogeneic antigens, autoantigens, or heterophilic antigens.

[0055] In some embodiments, the antigen is selected from autoantigens. In some embodiments, the antigen is selected from pyruvate dehydrogenase complex (PDC). In some embodiments, the antigen is selected from, but not limited to, dihydrolipoamide transacetylase (PDC-E2). In some embodiments, the antigen is a PDC-E2 polypeptide.

[0056] In some embodiments, the antigen is selected from allergens capable of causing type I hypersensitivity reactions. In some embodiments, the antigen is selected from food allergens, environmental allergens, lung allergens, skin allergens, and asthma allergens. In some embodiments, the antigen is selected from food allergens. In some embodiments, the antigen is selected from, but not limited to, gliadin.

[0057] In some embodiments, the particle is coupled to an antigen containing one or more epitopes. In some embodiments, the epitopes are associated with allergic reactions, autoimmune diseases, enzymes used in enzyme replacement therapy, lysosomal storage diseases, or inflammatory diseases or disorders. In some embodiments, the particle is coupled to an antigen containing only one epitope associated with one disease and / or disorder. In some embodiments, the particle is coupled to an antigen containing more than one epitope associated with the same disease and / or disorder. In some embodiments, the particle is coupled to an antigen containing more than one epitope associated with different diseases and / or disorders.

[0058] On the other hand, this disclosure also provides a composition comprising the foregoing defined particles and optionally pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable excipients based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable excipients.

[0059] In some embodiments, the composition is prepared by freeze-drying.

[0060] In some embodiments, the composition is prepared by spray drying.

[0061] In some embodiments, the excipient is selected from, but not limited to, sucrose and mannitol.

[0062] On the other hand, this disclosure also provides the use of the aforementioned particles or compositions comprising the aforementioned particles in the preparation of a medicament for treating allergic diseases. In some embodiments, the allergic diseases are selected from, but are not limited to, food allergies, environmental allergies, lung allergies, skin allergies, and asthma. In some embodiments, the allergic diseases are selected from food allergies, environmental allergies, and asthma. In some embodiments, the allergic diseases are selected from food allergies. In some embodiments, the allergic diseases are selected from celiac disease.

[0063] On the other hand, this disclosure also provides the use of the aforementioned particles or compositions comprising the aforementioned particles in the preparation of a medicament for treating autoimmune diseases. In some embodiments, the autoimmune diseases are selected from, but not limited to, celiac disease and primary biliary cholangitis.

[0064] On the other hand, this disclosure also provides a method for inducing antigen-specific tolerance in a subject, the method comprising: administering to the subject an effective amount of the aforementioned particles or a composition comprising the aforementioned particles, wherein the particles have a negative zeta potential, and wherein the particles and the antigen induce the subject's tolerance to the antigen. In some embodiments, the aforementioned particles or a composition comprising the particles are administered to treat or prevent a disease or condition selected from: autoimmune diseases or allergic diseases. In some embodiments, the disease or condition is selected from, but not limited to: celiac disease. In some embodiments, the method further comprises repeatedly administering the composition to the subject. In some embodiments, the administration is performed before or after the occurrence of a disease or condition caused by the antigen.

[0065] On the other hand, this disclosure also provides a method for treating celiac disease in a subject, the method comprising: administering to the subject an effective amount of the aforementioned particles or a composition comprising the aforementioned particles, wherein the particles have a negative zeta potential. In some embodiments, the antigen is gliadin. In some embodiments, the antigen is gliadin.

[0066] In some embodiments, the aforementioned particles encapsulating gliadin have an average particle size of about 300 to 1500 nm and an average charge of about -30 to about -80 mV. In some embodiments, the aforementioned polymer particles encapsulating gliadin have an average particle size of about 500 to 1200 nm and an average charge of about -40 to about -70 mV. In some embodiments, the aforementioned particles encapsulating gliadin have an average particle size of about 500 to 700 nm and an average charge of about -40 to about -60 mV.

[0067] In some embodiments, the aforementioned particles or compositions comprising the aforementioned particles are administered intravenously. In some embodiments, administration of negatively charged particles induces antigen-specific tolerance in a subject. In some embodiments, the aforementioned particles are carboxylated. In some embodiments, the aforementioned particles have a zeta potential of less than about -100 mV. In some embodiments, the aforementioned particles have a zeta potential between about -75 mV and 0 mV, for example, between -50 mV and 0 mV, or between -100 mV and -50 mV, or between -75 mV and -50 mV, or between -50 mV and -40 mV. In some embodiments, the aforementioned particles have an average diameter of about 100 nm to about 10000 nm, for example, about 200 nm to about 2000 nm, or about 300 nm to about 5000 nm, or 500 nm to about 3000 nm, or about 500 nm to about 1000 nm.

[0068] On the other hand, this disclosure also provides a method for preparing the aforementioned particles. The method includes contacting an immunomodified particle precursor with a buffer solution under conditions conducive to the effective formation of immunomodified particles with a negative zeta potential. In some embodiments, the immunomodified particle precursor is formed by copolymerization, and the particle microstructure may depend on the copolymerization method. In some embodiments, the immunomodified particle precursor is the aforementioned polymer, which is formed by reacting 2-aminoethanesulfonamide with PLGA. In some embodiments, the ratio of block PLG to block PLA in the PLGA is selected from 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In some embodiments, the aforementioned polymers may be periodic, statistical, linear, branched (including star, brush, or comb copolymers) polymers. In some embodiments, the end-capping groups (e.g., carboxyl, thiol, hydroxyl) of the polymer blocks may be further modified. In some embodiments, the ratio of block PLG to block PLA in the PLGA is 50:50.

[0069] In some embodiments, the particles contain a carboxylating agent. In some embodiments, the carboxylating agent is present at about 0.1% to about 2.0%. In some embodiments, the particles contain PEMA. In some embodiments, PEMA is present at about 0.1% to about 2.0%.

[0070] In some embodiments, the buffer solution has an alkaline pH. In some embodiments, the buffer solution is selected from, but not limited to, sodium bicarbonate.

[0071] In some embodiments, the aforementioned particles are prepared by adding a composition comprising the aforementioned polymer to a solution of poly(ethylene-maleic anhydride) (PEMA). In some embodiments, the concentration of PEMA in the solution is between about 0.1% and about 10%. In some embodiments, the concentration of PEMA in the solution is between about 0.2% and about 5%. In some embodiments, the concentration of PEMA in the solution is between about 0.1% and 4%. In some embodiments, the concentration of PEMA in the solution is between about 0.1% and 2%. In some embodiments, the concentration of PEMA in the solution is between about 0.5% and 1%. In some embodiments, the percentage of PEMA in the solution is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%. In some embodiments, the percentage of PEMA in the solution is about 0.5%. In some embodiments, the percentage of PEMA in the solution is about 1.0%.

[0072] In some embodiments, the particles contain a surfactant. In some embodiments, the surfactant is present in amounts from about 0.1% to about 2.0%. In some embodiments, the particles contain sodium oleate. In some embodiments, sodium oleate is present in amounts from about 0.1% to about 2.0%.

[0073] In some embodiments, the aforementioned particles are prepared by adding a composition comprising the aforementioned polymer to a solution of sodium oleate. In some embodiments, the concentration of sodium oleate in the solution may be between about 0.1% and about 10%.

[0074] On the other hand, this disclosure also provides a method for preparing the aforementioned composition. The method includes: (a) generating a primary emulsion by mixing an aqueous solution of an antigen (the antigen comprising, but not limited to, gliadin, or a PDC-E2 polypeptide) with an oil phase containing a polymer; (b) mixing the primary emulsion with a solution containing one or more surfactants and / or stabilizers to form a secondary emulsion; (c) hardening the secondary emulsion by evaporation to remove the solvent, thereby producing hardened polymer particles, the hardened polymer nanoparticles encapsulating the antigen within their core; (d) filtering, washing, and concentrating the particles; and (e) freeze-drying the particles. In some embodiments, the primary emulsion of step (a) is a water-in-oil emulsion. In some embodiments, the secondary emulsion of step (b) is an oil-in-water emulsion.

[0075] In some embodiments, the aqueous solution in step (a) contains a solvent.

[0076] In some embodiments, the surfactant and / or stabilizer solution in step (b) contains a solvent.

[0077] In some embodiments, the solvent is an organic solvent. In some embodiments, the organic solvent is selected from, but is not limited to, dichloromethane, acetone, ethanol, dichloromethane, dimethyl sulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid.

[0078] In some embodiments, the solvent is an inorganic solvent. In some embodiments, the inorganic solvent is selected from, but is not limited to, water, ammonia, sulfuric acid, carbon disulfide, bromine trifluoride, phosphorus oxychloride, hydrogen fluoride, and sulfur dioxide.

[0079] In some embodiments, the excipient is added to the aforementioned composition prior to freeze-drying in step (e). In some embodiments, the excipient is a buffer and / or a freeze-drying protectant. In some embodiments, the excipient is selected from, but is not limited to, sucrose, mannitol, trehalose, sorbitol, dextran, sodium citrate, lactose, L-arginine, or glycine.

[0080] In some embodiments, the freeze-drying method described in step (e) involves resuspending the purified particles in water containing sucrose and D-mannitol, rapidly freezing them in liquid nitrogen, and freeze-drying them until dry.

[0081] The average particle size and charge can change slightly during lyophilization; therefore, both the post-synthetic average and the post-lyophilized average are described. As used herein, the terms "post-synthetic size" and "post-synthetic charge" refer to the size and charge of the particles before lyophilization. The terms "post-lyophilized size" and "post-lyophilized charge" refer to the size and charge of the particles after lyophilization. The particle size of this disclosure does not need to be uniform, but the particles must generally be large enough to achieve the following effects: chelation in the spleen or liver, and triggering phagocytosis or uptake by antigen-presenting cells (including endothelial cells or other MPS cells) via receptor- or non-receptor-mediated mechanisms. In some embodiments, the particle size is in the micrometer or nanometer range to enhance solubility, avoid potential complications due to in vivo aggregation, and facilitate endocytosis. Particle size can be a factor in uptake from interstitial spaces into regions where lymphocytes mature. Particles with a diameter of about 0.1 μm to about 10 μm are capable of triggering phagocytosis.

[0082] Particle size and charge are crucial for tolerance induction. While particles vary in size and charge based on the antigen encapsulated within them, generally speaking, the particles of this disclosure are effective at inducing tolerance when they are between about 100 nm and about 1500 nm in size and have a charge of 0 to about -70 mV, and are most effective at inducing tolerance when they are 400–800 μm in size and have a charge between about -25 mV and -70 mV. Furthermore, the average particle size and charge can be slightly altered during lyophilization due to particle concentration and the presence of sucrose and D-mannitol.

[0083] The particles disclosed herein can be manufactured by any means commonly known in the art. Exemplary methods for manufacturing the particles include, but are not limited to, microemulsion polymerization, interfacial polymerization, and precipitation polymerization.

[0084] The particles disclosed herein can effectively suppress the inflammatory immune response in subjects requiring treatment. In some embodiments, approximately 10 [units of something] are provided to the individual. 2 To about 10 20 One particle. In some implementations, approximately 10 are provided. 3 To about 10 15 Particles between 10. In some implementations, approximately 10 are provided. 6 To about 10 12 Particles between 10. In some implementations, approximately 10 are provided. 8 To about 10 10 The particles are between 100 and 1000 μm in size. In some embodiments, the dosage is 0.1% solids content / ml. For 0.5 μm microbeads, the dosage is approximately 4 × 10⁻⁶. 9 0.05 μm microbeads. For microbeads, the dosage is approximately 4 × 10⁻⁶. 12 1 microbead. For 3 μm microbeads, the dose is 2 × 10⁷ microbeads. However, this disclosure covers any dose that is effective in treating a specific condition to be treated.

[0085] This disclosure can be used to treat immune-related disorders, including but not limited to autoimmune diseases and allergic reactions. Using synthetic biocompatible particle systems as an alternative to induce immune tolerance can lead to easier manufacturing, increased availability of therapeutic agents, increased homogeneity between samples, an increased number of potential treatment sites, and a significantly reduced likelihood of allergic reactions to carrier cells.

[0086] In the chemical structure of the compounds described in this disclosure, the bonds... No configuration was specified, i.e., key It can be Or simultaneously include Two configurations. In the chemical structure of the compounds described in this disclosure, the bonds... No configuration is specified, meaning it can be Z configuration, E configuration, or both configurations.

[0087] This disclosure also includes various deuterated forms of the polymer or pharmaceutically acceptable salts thereof, wherein each available hydrogen atom in the polymer can be independently replaced by a deuterium atom. Those skilled in the art will recognize how to synthesize deuterated forms of the polymers disclosed herein or pharmaceutically acceptable salts thereof.

[0088] This disclosure also includes isotopically labeled polymers, wherein one or more atoms in the polymer are replaced by atoms having a mass or mass number different from the most common atomic masses or mass numbers found in nature. Examples of isotopes that can be used in the polymers of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine, and chlorine, for example... 3 H, 11 C 14 C 18 F, 123 I or 125 I.

[0089] Unless otherwise specified, when a position is specifically designated as deuterium (D), that position should be understood as having a deuterium abundance of at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). The natural abundance of deuterium in the example compounds can be at least 1000 times, at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, or even higher. This disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials can be used to prepare the deuterated form of the compound of formula (I), or they can be synthesized using conventional techniques with deuterated reagents, including but not limited to deuterated boranes, trideuterated borane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0090] The polymers disclosed herein may be in the form of pharmaceutically acceptable salts. It should be understood that non-pharmaceutically acceptable salts also fall within the scope of this disclosure.

[0091] Terminology Explanation

[0092] The term "particle" refers to any composition of material not derived from tissue, which may be nanospheres or nanospherical entities, microspheres or microspherical entities, microbeads, or liposomes. Particles may have any particle shape or conformation. However, in some embodiments, particles that are unlikely to clump together in vivo are preferred. In some embodiments, the particles are spherical in shape. The terms "particle," "immunomodulated particle," "carrier," and "carrier particle" are used interchangeably depending on the context. Additionally, the term "particle" may be used to encompass microbeads, microspheres, nanoparticles, nanospheres, and nanobeads.

[0093] The term "negatively charged particles" refers to particles that have been modified to have a net surface charge of less than zero. For example, poly(ethylene-maleic anhydride) (PEMA) modification is used to carboxylate polymer particles, making their particle surfaces negatively charged.

[0094] The term "carboxylated particle" includes any particle that has been modified to contain carboxyl groups on its surface. In some embodiments, the addition of carboxyl groups enhances the uptake of particles from the bloodstream by phagocytes / monocytes, for example, through interaction with scavenger receptors such as MARCO. The carboxylation of particles can be achieved using any compound capable of adding carboxyl groups, including but not limited to poly(ethylene-maleic anhydride) (PEMA).

[0095] The term "degradation" refers to the process by which a polymer becomes soluble through a reduction in molecular weight or by converting hydrophobic groups into hydrophilic groups. Polymers containing ester groups generally undergo spontaneous hydrolysis; for example, polylactide and polyglycolic acid.

[0096] The terms "antigen" or "antigen fraction" are used interchangeably in this disclosure and refer to any part recognized by the host's immune system, such as a peptide. Based on their phylogenetic relationship to the organism, antigens can be classified as: ① Heterogeneous antigens: antigenic substances from another genus or species; ② Allogeneic antigens: antigenic substances from individuals of the same species but with different genotypes; ③ Autoantigens: components of the body's own tissues that can elicit an autoimmune response; ④ Heterophilic antigens: common antigens present on the surface of cells from different species of animals, plants, and microorganisms, with extensive cross-reactivity among them. Examples of antigen fractions include, but are not limited to, autoantigens, enzymes and / or bacterial or viral proteins, peptides, drugs, or components.

[0097] The term "hypersensitivity reaction" refers to an abnormal or pathological immune response that occurs when the body is stimulated by a certain antigen. Based on the mechanism and clinical characteristics of hypersensitivity reactions, they are divided into four types. Among them, type I hypersensitivity reaction, also known as allergic reaction or immediate hypersensitivity reaction, is the most common type of hypersensitivity reaction in clinical practice. This type of hypersensitivity reaction has significant individual differences and a genetic predisposition.

[0098] The term "allergen" refers to antigens that trigger type I hypersensitivity reactions, such as food, house dust, pollen, fungi, human and animal dander, feathers, insects, parasites, drugs, and other chemicals. These substances sensitize the body through inhalation, ingestion, injection, or contact. Common allergens include, but are not limited to, food allergens, environmental allergens, lung allergens, skin allergens, and asthma allergens.

[0099] The term "allergic disease" refers to type I hypersensitivity diseases, which include, but are not limited to, food allergies, environmental allergies, lung allergies, skin allergies, and asthma.

[0100] The term "autoimmune disease" refers to a pathological reaction to self-antigens. These reactions can be classified as autoimmune or autoreactive and can cause a variety of clinical diseases, such as type 1 diabetes.

[0101] The term "conjugation" refers to the fixation of an antigen to the outside of a particle or its encapsulation within the particle. Therefore, antigens conjugated to particles include both surface conjugation and encapsulation within the particle.

[0102] The term "immune response" includes T-cell-mediated and / or B-cell-mediated immune responses. Exemplary immune responses include T-cell responses, such as cytokine production and cytotoxicity of cells. Additionally, the term "immune response" includes immune responses indirectly influenced by T-cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells, such as macrophages. Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4+, CD8+, Th1, and Th2 cells); antigen-presenting cells (e.g., professional antigen-presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen-presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes); natural killer cells; and bone marrow cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the modified particles of this disclosure effectively reduce the transport of inflammatory cells to sites of inflammation.

[0103] The terms "unresponsive," "tolerant," or "antigen-specific tolerance" refer to the insensitivity of T cells to T cell receptor-mediated stimulation. This insensitivity is typically antigen-specific and persists even after exposure to the antigenic peptide is stopped. For example, T cell unresponsiveness is characterized by a lack of cytokine production, such as IL-2. T cell unresponsiveness can occur when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if re-exposure occurs in the presence of co-stimulatory molecules) results in the inability to produce cytokines and subsequently, the inability to proliferate. Therefore, the inability to produce cytokines prevents proliferation. However, unresponsive T cells can proliferate if cultured with cytokines (such as IL-2). For example, T cell unresponsiveness can also be observed due to a lack of IL-2 production by T lymphocytes, as measured by ELISA or by using a proliferation assay with an indicator cell line. Alternatively, a reporter gene construct can be used.

[0104] The term "immune tolerance" refers to a method performed on a subset of treated subjects compared to untreated subjects, wherein: a) the level of a specific immune response (believed to be at least partially mediated by antigen-specific effector T lymphocytes, B lymphocytes, antibodies, or their equivalents) is reduced; b) the onset and development of a specific immune response is delayed; or c) the risk of the onset or development of a specific immune response is reduced. "Specific" immune tolerance occurs when immune tolerance is preferentially induced against certain antigens compared to other antigens. "Non-specific" immune tolerance occurs when immune tolerance is indiscriminately induced against antigens that cause an inflammatory immune response. "Quasi-specific" immune tolerance occurs when immune tolerance is semi-discriminately induced against antigens that cause a pathogenic immune response, but not against other antigens that cause a protective immune response.

[0105] The term "PDC-E2 polypeptide" refers to a specific peptide chain sequence on the PDC-E2 protein molecule from amino acid position 155 to position 185. "PDC-E2 protein molecule" and "PDC-E2" are used interchangeably in this disclosure.

[0106] The term "surfactant" refers to a compound that can reduce the surface tension between two liquids or between a liquid and a solid.

[0107] The term "anionic surfactant" refers to a surfactant that has at least one negative charge and, in addition to the counterion (M) it binds to, is an anionic surfactant. + Surfactants that do not have a positive charge on the outside.

[0108] The term "CFA adjuvant" refers to Freund's complete adjuvant.

[0109] The term "IFA adjuvant" refers to Freund's incomplete adjuvant.

[0110] The terms "optionally" or "optionally" mean that the event or situation subsequently described may, but does not have to, occur; the description includes the possibility or possibility of the event or situation occurring or not occurring. For example, "optionally halogenated or cyano-substituted C..." 1-6 "Alkyl" means that halogens or cyano groups may or may not be present. This description includes cases where alkyl groups are substituted by halogens or cyano groups and cases where alkyl groups are not substituted by halogens or cyano groups.

[0111] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0112] The term "hydroxyl group" refers to -OH.

[0113] The term "thiol" refers to -SH.

[0114] The term "amino" refers to -NH2.

[0115] The term "cyano" refers to -CN.

[0116] The term "carboxyl group" refers to -C(O)OH.

[0117] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched groups with 1 to 6 carbon atoms. The alkyl group includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and their various branched isomers. The alkyl group can be substituted or unsubstituted; when substituted, the substituent can be replaced at any usable connection point, preferably one or more of the following groups, including, but not limited to, halogens, hydroxyl, mercapto, carboxyl, amino, cyano, C6, and C7. 1-6 Alkyl, C 1-6 Alkoxy groups, wherein the alkyl or alkoxy group is optionally substituted with one or more halogen, hydroxyl, mercapto, carboxyl, amino, or cyano groups.

[0118] The term "alkoxy" refers to -O- (alkyl), where alkyl is defined as described above. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexyloxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, including but not limited to: halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6Alkoxy groups, wherein the alkyl or alkoxy group is optionally substituted with one or more halogen, hydroxyl, mercapto, carboxyl, amino, or cyano groups.

[0119] The term "cycloalkyl" or "carbocyclic" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 10 carbon atoms. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and cyclohexadienyl; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0120] The cycloalkyl or carbocyclic group may be substituted or unsubstituted. When substituted, the substituent can be replaced at any usable linker, preferably one or more of the following groups, including but not limited to halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy groups, wherein the alkyl or alkoxy group is optionally substituted with one or more halogen, hydroxyl, mercapto, carboxyl, amino, or cyano groups.

[0121] The term "alkylene" refers to the portion of an alkane molecule remaining after the removal of two hydrogen atoms, comprising straight-chain and branched subgroups with 1 to 20 carbon atoms. Alkylenes containing 1 to 6 carbon atoms, in non-limiting examples, include methylene (-CH2-), ethylene (e.g., -CH2CH2- or -CH(CH3)-). Unless otherwise specified, alkylenes can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, including but not limited to: halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy or 3- to 10-membered cycloalkyl substitutions.

[0122] The term "heteroalkylene" refers to an alkylene group in which one or more -CH2- atoms are replaced by heteroatoms selected from N, O, and S; wherein the alkylene group is as defined above; the heteroalkylene group may be substituted or unsubstituted. Unless otherwise specified, the heteroalkylene group may be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, including but not limited to: halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy or 3- to 10-membered cycloalkyl substitutions.

[0123] The term "monovalent group" refers to a compound that has "formally" eliminated a single-valent atom or group.

[0124] The term "subunit" refers to a compound that "formally" eliminates two monovalent or one divalent atom or group of atoms.

[0125] The term "substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort.

[0126] The term "composition" or "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or physiologically pharmaceutically acceptable salts or prodrugs thereof, along with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0127] The term "medicinal excipient" includes, but is not limited to, any adjuvant, carrier, flow aid, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0128] The term "effective dose" or "therapeutic effective dose" refers to a quantity sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a quantity sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0129] The term "pharmaceutically acceptable" means that these compounds, materials, compositions, and / or dosage forms are suitable for contact with patient tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within reasonable medical judgment, have a reasonable benefit / risk ratio, and are effective for their intended use. Attached Figure Description

[0130] Figure 1 shows the results of ear swelling detection in each group of animals (*** represents the test reagent group vs. the model group, P < 0.001; ## represents the test reagent group vs. the control group, P < 0.01).

[0131] Figure 2 shows the results of ear swelling detection in each group of animals (*** represents the test reagent group vs. the model group, P < 0.001). Detailed Implementation

[0132] The present disclosure is further described and explained below with reference to embodiments, but these embodiments are not intended to limit the scope of the present disclosure.

[0133] Experimental methods not specifying specific conditions in the embodiments of this disclosure are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, conventional reagents.

[0134] The structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). Chemical shifts were expressed in 10⁻¹⁰ increments. -6 (ppm) is given as the unit.

[0135] MS measurements were performed using a Finnigan LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

[0136] The determination by high performance liquid chromatography (HPLC) was performed using an Agilent 1200DAD high performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high performance liquid chromatograph (Gimini C18 150×4.6mm column).

[0137] Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The silica gel plates used in TLC are 0.15mm to 0.2mm in diameter, while those used for TLC separation and purification are 0.4mm to 0.5mm in diameter.

[0138] Column chromatography typically uses 200-300 mesh silica gel from Yantai Huanghai as the carrier.

[0139] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0140] Unless otherwise specified in the examples, the reactions were carried out under an argon or nitrogen atmosphere.

[0141] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0142] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

[0143] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0144] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0145] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0146] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0147] Unless otherwise specified in the examples, the reaction temperature is room temperature.

[0148] Room temperature is the optimal reaction temperature, with a range of 20℃ to 30℃.

[0149] Preparation of PBS buffer solution with pH=6.5 in the example: Take 8.5g of KH2PO4, 8.56g of K2HPO4·3H2O, 5.85g of NaCl and 1.5g of EDTA and put them into a bottle, make up to 2L, sonicate to dissolve completely, and shake well to obtain the solution.

[0150] The eluent systems for column chromatography and the developing solvent systems for thin-layer chromatography used to purify the compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and small amounts of triethylamine and acidic or basic reagents can also be added for adjustment.

[0151] Some of the compounds disclosed herein were characterized by Q-TOF LC / MS. The Q-TOF LC / MS was performed using an Agilent 6530 Precision Mass Number Quadrupole-Time-of-Flight Mass Spectrometer and an Agilent 1290-Infinity Ultra-High Performance Liquid Chromatography System (Agilent Poroshell 300SB-C8 5 μm, 2.1 × 75 mm column).

[0152] Example 1: Preparation of Particle 1

[0153] Step 1)

[0154] 1.0 g of poly(lactic acid-co-glycolic acid) (purchased from Daigang Biotechnology Co., Ltd., where the molar ratio of a:b is 50:50, batch 2024100902) and 0.25 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (purchased from Adamas, batch L394683) were dissolved in 10.0 ml of anhydrous N,N-dimethylformamide. 0.16 ml of N,N-diisopropylethylamine (purchased from Adamas, batch P2803872) was added, and the mixture was stirred at room temperature for 20 min under nitrogen protection. Then, 0.29 g of 2-aminoethanesulfonamide hydrochloride (purchased from Adamas, batch P3000836) was added at room temperature, and the mixture was stirred until the reaction was complete. The resulting reaction solution was added dropwise to methyl tert-butyl ether, filtered to obtain a solid, and then added to acetonitrile solvent. This solid was then added dropwise to purified water, and the precipitate was lyophilized to obtain polymer 1.

[0155] 1 H-NMR (400MHz, d6-DMSO): δ8.21,-CONHCH2-; δ6.92,-CH2SOONH2; δ5.28-5.20,-OCH(CH3)CO-; δ4.96-4.81,-OCH2CO-; δ3.49,-NHCH2CN2-; δ3.14,-CH2CH2SOO-; δ1.53-1.47,-OCH(CH3)CO-.

[0156] Step 2)

[0157] 25 mg of gliadin (purchased from Aladdin, CAS: 9007-90-3, batch 12308255) was dissolved in 0.05 mol / L acetic acid solution to prepare a 25 mg / mL gliadin solution. Poly(ethylene-alt-maleic anhydride) (PEMA) (purchased from Polysciences, batch A842202) was prepared into 1% w / v and 0.5% w / v solutions using ultrapure water, and stirred at 50°C for 5 hours to ensure complete dissolution of PEMA. 150 μL of gliadin solution was added dropwise to a 2 mL, 20% w / v solution of polymer 1 obtained in step 1 in dichloromethane. The mixture was premixed using a vortex mixer (VORTEX-5), and then sonicated at 26 W for 20 seconds in an ice bath using a VC 130 ultrasonic processor. Add 11 mL of 1% w / v PEMA solution to the mixed solution, premix using a vortex mixer (VORTEX-5), and sonicate the mixture for 20 seconds at 26 W in an ice bath using a VC 130 ultrasonic processor. Then pour the resulting solution into 200 mL of PEMA solution (0.5% w / v) and stir overnight to obtain a hardened particle suspension.

[0158] Step 3)

[0159] The particle suspension obtained in step 2 was filtered using a 40 μm cell filter. 36 mL of the filtered particle suspension was added in batches to 50 mL centrifuge tubes containing 4 mL of cooled 10x sodium bicarbonate buffer and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was then aspirated. The particle precipitate was redispersed in 40 mL of cooled 0.1 M, pH 9.6 sodium bicarbonate buffer and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was then aspirated. The particle precipitate was redispersed in 40 mL of cooled ultrapure water and centrifuged at 4000 rpm for 15 minutes at 4°C. This washing process was repeated three times. The particles were dispersed in 20 mL of a mixed solution containing 3% w / v mannitol and 4% w / v sucrose and lyophilized overnight at -80°C to obtain particle 1.

[0160] Example 2: Preparation of Particle 2

[0161] The same method as in Example 1 was used for preparation, except that the ultrasonic intensity of the VC 130 ultrasonic processor in step 2) was 78W. The resulting particles are designated as Particle 2.

[0162] Example 3: Preparation of Particle 3

[0163] The same method as in Example 1 was used for preparation, except that the ultrasonic intensity of the VC 130 ultrasonic processor in step 2) was 104 W. The resulting particles were designated as particles 3.

[0164] Example 4: Preparation of blank particles

[0165] It was prepared using the same method as in Example 1, except that gliadin was not added in step 2).

[0166] Example 5: Preparation of control particles

[0167] Step 1)

[0168] 25 mg of gliadin (purchased from Aladdin, CAS: 9007-90-3, batch 12308255) was dissolved in 0.05 mol / L acetic acid solution to prepare a 25 mg / mL gliadin solution. Poly(ethylene-alt-maleic anhydride) (PEMA) (purchased from Polysciences, batch A842202) was prepared into 1% w / v and 0.5% w / v solutions using ultrapure water, and stirred at 50°C for 5 hours to ensure complete dissolution of PEMA. 150 μL of gliadin solution was added dropwise to a carboxyl-terminated PLGA (50:50) solution in dichloromethane (2 mL, 20% w / v), premixed using a vortex mixer (VORTEX-5), and then sonicated at 78 W for 20 seconds in an ice bath using a VC 130 sonic processor. Add 11 mL of 1% w / v PEMA solution to the mixed solution, premix using a vortex mixer (VORTEX-5), and sonicate the mixture for 20 seconds at 78 W in an ice bath using a VC 130 ultrasonic processor. Then pour the resulting solution into 200 mL of PEMA solution (0.5% w / v) and stir overnight to obtain a hardened particle suspension.

[0169] Step 2)

[0170] The particle suspension obtained in step 1) was filtered using a 40 μm cell filter. 36 mL of the filtered particle suspension was added in batches to 50 mL centrifuge tubes containing 4 mL of cooled 10x sodium bicarbonate buffer and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was then aspirated. The particle precipitate was redispersed in 40 mL of cooled 0.1 M, pH 9.6 sodium bicarbonate buffer and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was then aspirated. The particle precipitate was redispersed in 40 mL of cooled ultrapure water and centrifuged at 4000 rpm for 15 minutes at 4°C. This washing process was repeated three times. The particles were dispersed in 20 mL of a mixed solution containing 3% w / v mannitol and 4% w / v sucrose and lyophilized overnight at -80°C to obtain control particles.

[0171] Example 6 Preparation of Particle 4

[0172] Step 1)

[0173] 2 mg of PDC-E2 peptide (purchased from Guotai Biotechnology, batch GTB50127-0929) was dissolved in 150 μL of 0.5 mM sodium hydroxide solution. After sonication for 10 min, the solution was vortexed to obtain a clear solution, which was then placed in an ice bath for later use. 2.2 g of sodium oleate (purchased from Nanjing Weier, intravenous injection grade) and 1.1 g of sodium chloride were weighed and prepared into 220 mL of a suspension containing 1% w / v sodium oleate and 0.5% w / v sodium chloride using ultrapure water. The suspension was stirred at 40°C for 1.5 hours to ensure complete dissolution of the sodium oleate until clear. The solution temperature was then lowered to 30°C for later use. 150 μL of the PDC-E2 peptide solution was added dropwise to a dichloromethane solution containing polymer 1 (2 mL, 25% w / v). The mixture was premixed using a vortex mixer (VORTEX-5) and then sonicated at 78 W for 30 seconds in an ice bath using a VC 130 ultrasonic processor. Add 10 mL of 1% w / v sodium oleate and 0.5% w / v sodium chloride solution to the mixed solution, premix using a vortex mixer (VORTEX-5), and sonicate the mixture at 78 W in an ice bath for 30 seconds using a VC 130 ultrasonic processor. Then pour the resulting solution into 200 mL of 1% w / v sodium oleate and 0.5% w / v NaCl solution and stir overnight to obtain a hardened particle suspension.

[0174] Step 2)

[0175] In batches, 40 mL of the particle suspension was added to 50 mL centrifuge tubes and centrifuged at 4000 rpm for 15 minutes at 15°C. The supernatant was collected. The particle precipitate was redispersed in 40 mL of cooled purified water and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was collected, and the washing process was repeated three times. The particles were dispersed in 20 mL of a mixed solution containing 3% w / v mannitol and 4% w / v sucrose and lyophilized overnight at -80°C to obtain particle 4.

[0176] Biological evaluation

[0177] Test Example 1: Particle size and potential characterization

[0178] Particle size and potential were characterized by dynamic light scattering analysis. Analysis was performed using a Brookhaven Instruments laser particle size analyzer in ultrapure water or PBS buffer solution. The average diameter, polydispersity index, and potential of the surface-functionalized polymer particles are shown in the table below.

[0179] Test Example 2: Determination of Protein Content

[0180] The content of gliadin was determined using the BCA protein assay kit (purchased from Thermo-Fisher, batch YI374064). Three sets of samples were prepared in parallel for each condition, and the results were the average of the three sets. To avoid interference from DMSO in the protein content determination, the final protein assay solution was kept at 11% DMSO.

[0181] 1. Preparation of gliadin standard and sample solution

[0182] 1.1 Dissolve 5 mg of gliadin in DMSO to prepare protein solutions of 25, 125, 250, 500, 750, 1000, 1500, and 2000 μg / mL. Dissolve particles 1–3 (encapsulated with gliadin) and blank particles (blank control sample) in DMSO, add the corresponding volume of ultrapure water, and prepare solutions of 5, 30, and 60 mg / mL, respectively.

[0183] 1.2. Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) and mix thoroughly.

[0184] 2. Protein concentration determination

[0185] Add 25 μl of the above sample solution to each well of a 96-well plate. Add 200 μl of BCA working solution to each well, mix well, and incubate at 37°C for 30 minutes. Measure the absorbance at 562 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve and the sample volume used. The final protein content of particle 1 was determined to be 12.9 ± 2.1 μg / mg polymer.

[0186] Test Example 3: Study of a mouse delayed-type hypersensitivity (DTH) model induced by gliadin.

[0187] 1. Instruments and reagents

[0188] 2. Laboratory animals:

[0189] 3. Reagent preparation

[0190] 3.1 Preparation of the first immunization emulsion

[0191] Step 1) Dissolve gliadin in 50 mmol / L glacial acetic acid solution to prepare a gliadin suspension with a concentration of 5 mg / mL. After sonication for 10 minutes, further dilute the resulting suspension with PBS buffer to a concentration of 2 mg / mL for later use.

[0192] Step 2) Dilute the 5 mg / mL CFA adjuvant to 4 mg / mL using the IFA adjuvant stock solution, and set aside.

[0193] Step 3) Mix the 2 mg / mL gliadin suspension obtained in Step 1) and the 4 mg / mL CFA adjuvant obtained in Step 2) at a 1:1 ratio, and then use T10basic Emulsify the mixture in a disperser until it reaches a water-in-oil state to obtain the initial immune emulsion, which is then ready for use.

[0194] 3.2 Preparation of immune stimulation solution

[0195] Glycol protein was dissolved in 50 mmol / L acetic acid solution to prepare a 5 mg / mL glycol protein suspension. After sonication for 10 minutes, the resulting suspension was further diluted with PBS buffer to a concentration of 1 mg / mL to obtain the immune stimulation solution for later use.

[0196] 3.3 Preparation of Dosing Formulations

[0197] Test preparation: Disperse and dissolve 50 mg of Particle 2 in PBS buffer to prepare a solution with a concentration of 12.5 mg / mL, store at about 4°C, and sonicate for 5 minutes before use.

[0198] Control preparation: Disperse and dissolve 50 mg of control particles in PBS buffer to prepare a solution with a concentration of 12.5 mg / mL, and store at about 4°C. Sonicate for 5 minutes before use.

[0199] 4. Experimental Methods

[0200] The animals were randomly divided into four groups based on their body weight (as shown in the table below). On day 0, each group of mice was administered the corresponding dose of the test and control formulations (the normal and model groups received the same volume of PBS buffer) via tail vein injection. Simultaneously, 100 μL of the initial immunization emulsion was injected subcutaneously at three points on the back of each mouse. On day 7, each group of mice was administered the corresponding dose of the test and control formulations (the normal and model groups received the same volume of PBS buffer) via tail vein injection. On day 14, after measuring ear thickness using a skin thrombometer, 10 μL of the immunization solution was injected intradermally into each ear. Ear thickness was measured again 24 hours later using a skin thrombometer. The difference between the ear thickness measured 24 hours later and the ear thickness on day 14 was defined as ear swelling. The average ear swelling measured in both ears of the same animal was used as the average ear swelling for the animal's experimental results analysis. Note: The normal group and the model group were given the same volume of PBS buffer. The normal group did not undergo immunization, while the model group underwent immunization.

[0201] 5. Test Results

[0202] The experimental results are shown in Figure 1. Compared with the normal group, the ear swelling of the model group was significantly increased, indicating that the model was successfully established. Compared with the model group, the control group and the test group significantly alleviated the ear swelling of the animals, and the efficacy of the test group was about 28% higher than that of the control group.

[0203] Test Example 4: Particle size and potential characterization

[0204] Particle size and potential were characterized by dynamic light scattering analysis. Analysis was performed using a Brookhaven Instruments laser particle size analyzer in ultrapure water or PBS buffer solution. The average diameter, polydispersity index, and potential of the surface-functionalized polymer particles are shown in the table below.

[0205] Test Example 5: Determination of PDC-E2 peptide encapsulation amount

[0206] The content of PDC-E2 peptide encapsulated in PLGA nanoparticles was determined by high performance liquid chromatography.

[0207] 1. Solution preparation

[0208] Mobile phase A (0.1% TFA-H2O): Accurately transfer 1 ml of trifluoroacetic acid to 1000 ml of water and mix well. Mobile phase B (0.1% TFA-ACN): Accurately transfer 1 ml of trifluoroacetic acid to 1000 ml of acetonitrile and mix well. Diluents: Peptide (ACN:H2O = 4:6); Nanosphere (N,N-dimethylformamide-DMF) standard solution: Prepare a 200 μg / ml peptide stock solution, then dilute sequentially to 100 μg / ml, 50 μg / ml, 25 μg / ml; 5 μg / ml, and 1 μg / ml peptide standard solutions. Sample solution: Weigh approximately 2.5 mg of nanospheres into a small tube, add 400 μl-500 μl of DMF to dissolve, sonicate for 30 minutes, and then inject for analysis.

[0209] 2. Testing Process

[0210] Take blank solution (DMF), standard solution, and sample solution, and inject them into the liquid chromatograph. After the chromatographic system stabilizes, determine the encapsulation amount of PBC-E2 peptide according to the following procedure: First, take blank solution and inject it at least once; second, take standard solution and inject it once for each sample; third, take sample solution and inject it once for each sample.

[0211] 3. Calculation

[0212] A standard curve was plotted based on the standard solution. The actual concentration of the peptide in the nanospheres was calculated using a linear equation. The encapsulation amount was then calculated using the formula below: The final experimental result showed that the content of PBC-E2 polypeptide in particle 4 was 1.04 ± 0.12 μg / mg polymer.

[0213] Test Example 6: Study of PDC-E2 peptide-induced mouse DTH model

[0214] 1. Instruments and reagents

[0215] 2. Laboratory animals

[0216] 3. Reagent preparation

[0217] 3.1 Preparation of the first immunization emulsion

[0218] Step 1) Dissolve the PDC-E2 peptide in pure water to prepare a PDC-E2 peptide solution with a concentration of 4 mg / mL for later use.

[0219] Step 2) Dilute the 5 mg / mL CFA adjuvant to 4 mg / mL using the IFA adjuvant stock solution, and set aside.

[0220] Step 3) Mix the 4 mg / mL PDC-E2 peptide solution obtained in Step 1) and the 4 mg / mL CFA adjuvant obtained in Step 2) at a 1:1 ratio, and then use T 10 basic Emulsify the mixture in a disperser until it reaches a water-in-oil state to obtain the initial immune emulsion, which is then ready for use.

[0221] 3.2 Preparation of immune stimulation solution

[0222] The PDC-E2 peptide was dissolved in pure water to prepare a PDC-E2 solution with a concentration of 5 mg / mL, and then diluted to 1 mg / mL to obtain an immune stimulation solution for later use.

[0223] 3.3 Preparation of Dosing Formulations

[0224] Test formulation group: 20 mg of granules 4 were dispersed and dissolved in PBS buffer to prepare a solution with a concentration of 12.5 mg / mL, and stored at about 4°C.

[0225] 4. Experimental Methods

[0226] The animals were randomly divided into three groups based on their body weight (as shown in the table below). On day 0 of the experiment, each group of mice was administered the corresponding dose of the test preparation via tail vein injection (the normal group and the model group received the same volume of PBS buffer). Simultaneously, 100 μL of the initial immunization emulsion was injected subcutaneously at three points on the back of each mouse. On day 7 of the experiment, each group of mice was administered the corresponding dose of the test preparation via tail vein injection (the normal group and the model group received the same volume of PBS buffer). On day 14 of the experiment, after measuring ear thickness using a skin thrombometer, 10 μL of the challenge immunization solution was injected intradermally into the right ear. Ear thickness was measured again using a skin thrombometer 24 hours later. The difference between the right and left ear thicknesses measured 24 hours later was defined as ear swelling and used for experimental result analysis. Note: The normal group and the model group were given the same volume of PBS buffer. The normal group did not undergo immunization, while the model group underwent immunization.

[0227] 5. Test Results

[0228] The experimental results are shown in Figure 2. Compared with the normal group, the ear swelling of the animals in the model group was significantly increased, indicating that the model was successfully established. Compared with the model group, the test formulation group could significantly alleviate the ear swelling of the animals, and the efficacy of the test formulation group was about 73% higher than that of the model group.

Claims

1. A polymer as shown in Formula I, in, X is selected from -O-, -S-, or -N(R) 4a )-; R 1a R 1b R 2a R 2b Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, carboxyl, N(R) 4b R 4c ), cyano, C 1-6 Alkyl, C 1-6 alkoxy, 3 to 10-membered cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally surrounded by one or more R A replace; L is selected from alkylene or heteroalkylene, wherein the alkylene or heteroalkylene is optionally converted by one or more halogens, hydroxyl groups, mercapto groups, carboxyl groups, or N(R) groups. 4b R 4c ), cyano, C 1-6 Alkyl, C 1-6 Alkoxy or 3- to 10-membered cycloalkyl substituted, wherein the alkyl, alkoxy, or cycloalkyl group is optionally substituted with one or more R groups. B replace; R 4a R 4b R 4c Each is independently selected from hydrogen and C. 1-6 Alkyl, 3- to 10-membered cycloalkyl, wherein the alkyl or cycloalkyl is optionally surrounded by one or more R C replace; R A R B R C Each is independently selected from halogen, hydroxyl, mercapto, carboxyl, amino, cyano, C 1-6 Alkyl, C 1-6 Alkoxy group, wherein the alkyl group or alkoxy group is optionally replaced by one or more halogen, hydroxyl, mercapto, carboxyl, amino, or cyano groups; The molar ratio of a:b is selected from 0:100 to 100:0, preferably 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, and most preferably 50:

50.

2. The polymer according to claim 1, wherein R 1a R 1b Both are hydrogen.

3. The polymer according to claim 1 or 2, wherein R 2a It is hydrogen.

4. The polymer according to any one of claims 1 to 3, wherein R 2b C 1-6 Alkyl groups, preferably methyl, ethyl, propyl, butyl, or isobutyl, with methyl being the most preferred.

5. The polymer according to any one of claims 1 to 4, wherein L is an alkylene group, said alkylene group optionally being converted by one or more halogens, hydroxyl groups, mercapto groups, carboxyl groups, N(R) groups. 4b R 4c ), cyano, C 1-6 Alkyl, C 1-6 Alkoxy or 3- to 10-membered cycloalkyl substituted, wherein the alkyl, alkoxy, or cycloalkyl group is optionally substituted with one or more R groups. B replace, R B R 4b R 4c As defined in claim 1.

6. The polymer according to any one of claims 1 to 5, wherein the polymer of Formula I is a polymer of Formula II, in, R 3a R 3b Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, carboxyl, N(R) 4b R 4c ), cyano, C 1-6 Alkyl, C 1-6 alkoxy, 3 to 10-membered cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally surrounded by one or more R A replace; c is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; R A X, R 3a R 3b R 4b R 4c a, b are as defined in claim 1.

7. The polymer according to any one of claims 1 to 6, wherein X is -N(R 4a )-, R 4a As defined in claim 1.

8. The polymer according to any one of claims 1 to 7, wherein the polymer represented by Formula I is a polymer represented by Formula III, wherein a, b, R 4a As defined in claim 1, c, R 3a R 3b As defined in claim 6.

9. The polymer according to any one of claims 1 to 8, wherein R 4a It is hydrogen.

10. The polymer according to any one of claims 1 to 9, wherein c is selected from 1, 2, 3, 4, 5, 6, preferably 2, 3, 4, and most preferably 2.

11. The polymer according to any one of claims 1 to 10, wherein R 3a R 3b Each of them is hydrogen independently.

12. The polymer of any one of claims 1 to 11, wherein the polymer of Formula I is a polymer of Formula IV, wherein, a and b are as defined in claim 1.

13. A polymer, characterized in that, which is formed by reacting a polymer A represented by Formula la with a compound B represented by Formula lb, Among them, R 1a R 1b R 2a R 2b a, b, X, L are as defined in claim 1.

14. The polymer of claim 13, wherein the compound B is grafted onto polymer A via a condensation reaction with polymer A.

15. The polymer of claim 13 or 14, wherein said polymer A is represented by formula IIa, ###00007### IIa in, a and b are as defined in claim 1.

16. The polymer according to any one of claims 13 to 15, wherein compound B is as shown in formula IIIb. in, R 4a As defined in claim 1, R 3a R 3b c, as defined in claim 6.

17. A polymer, characterized in that, It is formed by reacting PLGA with 2-aminoethanesulfonamide.

18. A polymer particle, characterized in that, The surface of its particles may optionally be functionalized, and the polymer is as defined in claims 1 to 17.

19. The polymer particles according to claim 18, wherein the functionalization is carboxylation.

20. The polymer particles according to claim 18 or 19, wherein the carboxylating agent is selected from poly(ethylene-maleic anhydride).

21. The polymer particles according to claim 18 or 19, further comprising a surfactant, such as sodium oleate.

22. The polymer particles according to any one of claims 18 to 21, wherein the diameter of the particles is 100 to 1000 nm, preferably 200 to 800 nm, and most preferably 300 to 600 nm.

23. The polymer particles according to any one of claims 18 to 22, wherein the particle zeta potential is -10mV to -100mV, preferably -20mV to -80mV, and most preferably -30mV to -75mV.

24. The particles according to any one of claims 18 to 23, further comprising one or more encapsulated antigens.

25. The particle according to any one of claims 18 to 24, wherein the antigen is selected from heteroantigens, allogeneic antigens, autoantigens, or heterophilic antigens.

26. The particle according to claim 25, wherein the antigen is an autoantigen, preferably a pyruvate dehydrogenase complex (PDC), more preferably dihydrolipoamide transacetylase (PDC-E2), and most preferably a PDC-E2 polypeptide.

27. The particle of claim 25, wherein the antigen is selected from allergens capable of causing type I hypersensitivity reactions.

28. The particle according to any one of claims 27, wherein the antigen is selected from food allergens, environmental allergens, lung allergens, skin allergens and asthma allergens, preferably food allergens, and most preferably gliadin.

29. A composition comprising particles as defined in any one of claims 18 to 28, and optionally a pharmaceutically acceptable excipient.

30. The composition of claim 29, wherein the composition is a lyophilized composition.

31. Use of the particles according to any one of claims 18-25 and 27-28, or the composition according to claim 29 or 30, in the preparation of a medicament for treating allergic diseases.

32. The use according to claim 31, wherein the allergic disease is selected from food allergy, environmental allergy, lung allergy, skin allergy and asthma, preferably food allergy, environmental allergy, and asthma, and most preferably food allergy.

33. The use according to claim 31 or 32, wherein the allergic disease is celiac disease.

34. Use of the particles as described in claims 18 to 26, or the composition as described in claim 29 or 30, in the preparation of a medicament for treating autoimmune diseases.

35. The use according to claim 34, wherein the autoimmune disease is celiac disease or primary biliary cholangitis.

36. A method for preparing the polymer according to any one of claims 1 to 17, comprising the step of reacting formula Ia with formula Ib to form formula I. in, R 1a R 1b R 2a R 2b X, L, a, b are as defined in claim 1.

37. A method for preparing the polymer according to any one of claims 1 to 17, comprising the step of reacting formula IIa with IVb to form formula IV. in, a and b are as defined in claim 1.

38. A method for preparing the polymer according to any one of claims 1 to 17, comprising the step of reacting PLGA with 2-aminoethanesulfonamide.

Images