Anti-inflammatory drugs

Abstract

To provide an anti-inflammatory agent. [Solution] An anti-inflammatory agent containing a carboxyl group-introduced high molecular weight hyaluronic acid derivative.

Description

[Technical Field]

[0001] This disclosure relates to anti-inflammatory agents, etc. [Background technology]

[0002] Currently, various small-molecule steroid drugs and immunosuppressants exhibiting anti-inflammatory effects are being developed. When these immunosuppressants are distributed throughout the body, they not only suppress inflammation at the site of inflammation but also suppress the normal immune response, leading to other problems such as increased susceptibility to infection.

[0003] When hyaluronic acid has a low molecular weight, it interacts with a complex of the cell surface receptor CD44 and Toll-like receptor (TLR) 2, which is also a cell surface receptor. This interaction leads to signal transduction, ultimately promoting inflammation. Interestingly, the effects of HA on cells differ depending on its molecular weight, and high molecular weight hyaluronic acid has been reported to exhibit anti-inflammatory effects.

[0004] Non-patent document 1 reported that the uptake of liposomes surface-modified with low molecular weight hyaluronic acid derivatives to which carboxyl groups have been introduced decreased in fibroblasts and increased in inflammatory cells such as macrophages and dendritic cells. However, the effects of introducing carboxyl groups into high molecular weight hyaluronic acid, such as its anti-inflammatory effects and effects on specificity for inflammatory cells, were unknown. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Maiko Miyazaki et al., ACS Biomater. Sci. Eng., Development of pH-Responsive Hyaluronic Acid-Based Antigen Carriers for Induction of Antigen-Specific Cellular Immune Responses 5, 5790-5797, (2019). [Overview of the project] [Problems that the invention aims to solve]

[0006] The objective of this disclosure is to provide an anti-inflammatory agent. [Means for solving the problem]

[0007] In view of the above problems, the inventors have diligently conducted research and have found that an anti-inflammatory agent containing a carboxyl group-introduced high molecular weight hyaluronic acid derivative can solve the above problems. That is, this disclosure includes the following exemplary embodiments.

[0008] Item 1. An anti-inflammatory agent containing a carboxyl group-introduced high molecular weight hyaluronic acid derivative.

[0009] Item 2. The anti-inflammatory agent according to Item 1, wherein the weight-average molecular weight of the carboxyl group-introduced high molecular weight hyaluronic acid derivative is 200,000 or more.

[0010] Item 3. The anti-inflammatory agent according to item 1 or 2, wherein the molecular weight of the carboxyl group-introduced high molecular weight hyaluronic acid derivative is 700,000 or more and 10,000,000 or less.

[0011] Item 4. The carboxyl group-introduced high molecular weight hyaluronic acid derivative is of general formula (1):

[0012] [ka] [In the formula: R 1[The symbols represent monovalent groups containing a hydrogen atom or a carboxyl group, either identical or distinct. n represents a natural number.] An anti-inflammatory agent according to any one of claims 1 to 3, comprising at least one selected from the group consisting of compounds represented by, salts thereof, and solvates thereof.

[0013] Section 5. The monovalent group is given by the general formula (2): -C(=O)-R 11 -COOH(in the formula:R 11 The group represented by ) indicates a single bond or a linker, and is an anti-inflammatory agent as described in item 4.

[0014] Item 6. The anti-inflammatory agent according to item 5, wherein the linker is an alkylene group.

[0015] Item 7. The anti-inflammatory agent according to any one of items 4 to 6, wherein the carboxyl group introduction rate in the carboxyl group-introduced high molecular weight hyaluronic acid derivative is 50% or less.

[0016] Item 8. The anti-inflammatory agent according to any one of items 4 to 7, wherein the carboxyl group introduction rate in the carboxyl group-introduced high molecular weight hyaluronic acid derivative is 5% or more and 30% or less.

[0017] Item 9. A pharmaceutical anti-inflammatory agent as described in any of items 1 to 8.

[0018] Item 10. An IL-10 production promoter containing a carboxyl group-introduced high molecular weight hyaluronic acid derivative.

[0019] Item 11. A carboxyl group-introduced high molecular weight hyaluronic acid derivative having a weight-average molecular weight of 200,000 or more. [Effects of the Invention]

[0020] According to this disclosure, an anti-inflammatory agent can be provided. The anti-inflammatory agent of this disclosure may also exert an IL-10 production-promoting effect. [Brief explanation of the drawing]

[0021] [Figure 1] This document outlines the design of a hyaluronic acid derivative that possesses anti-inflammatory effects and specific affinity for inflammatory immune cells. [Figure 2] The synthesis scheme for hyaluronic acid derivatives is shown. [Figure 3] The 1H NMR charts are shown: HA (a), RITC-HA (b) (400 MHz, D2O). [Figure 4] The 1H NMR charts are shown: Suc17-HA (400 MHz, D2O) (a), Suc17-HA (400 MHz, D2O + NaOD) (b). The table shows the peak integral values ​​and inclusion rates. [Figure 5] The 1H NMR charts are shown: Glu19-HA (400 MHz, D2O) (a), Glu19-HA (400 MHz, D2O + NaOD) (b). The table shows the peak integral values ​​and inclusion rates. [Figure 6] The 1H NMR charts are shown: MGlu16-HA (400 MHz, D2O) (a), MGlu16-HA (400 MHz, D2O + NaOD) (b). The table shows the peak integral values ​​and inclusion rates. [Figure 7] 1H NMR chart is shown. RITC-Suc16-HA (a), RITC-Glu19-HA (b) and RITC-MGlu14-HA (c) (400 MHz, D2O). [Figure 8] (a) Fluorescence spectra of RITC-HA, RITC-Suc-HA, RITC-Glu-HA, and RITC-MGlu-HA in PBS at pH 7.4 and 37°C (concentration: 0.33 mg / mL, excitation wavelength: 558 nm). (b) Bar graph showing fluorescence intensity at the peaks of the fluorescence spectrum in (a). [Figure 9] This shows the viscosity of a 5 mg / mL HA derivative. The photo shows the flow of the sample immediately after tilting. [Figure 10]The levels of TNF-α production in RAW264.7 cells treated for 24 hours with and without LPS stimulation are shown after treatment with HA(a), dextran sulfate, chondroitin sulfate, and polyvinyl sulfate(b). [Figure 11] This shows the level of TNF-α production by RAW264.7 cells treated with HA in the presence of LPS stimulation. Cells were incubated with HA for 3 hours. Subsequently, the cells were washed and stimulated with LPS for 21 hours. [Figure 12] This study demonstrates LPS-mediated inflammatory response signaling and SEAP production induced by NF-κB activation in RAW-Blue cells. [Figure 13] This shows NF-κB activation in RAW-Blue cells treated with HA or MGlu-HA (1 mg / mL) at various introduction rates in the presence (a) or absence (b) of LPS stimulation. The expression level of NF-κB in cells exposed to LPS was set to 100%. [Figure 14] The images show the levels of TNF-α production in RAW264.7 cells treated with HA or MGlu-HA (1 mg / mL) at different introduction rates in the presence (a) or absence (b) of LPS stimulation. The images also show the levels of IL-6 production in RAW264.7 cells treated with HA or MGlu-HA (1 mg / mL) at different introduction rates in the presence (c) or absence (d) of LPS stimulation. [Figure 15] The average fluorescence intensity of RAW264.7 cells treated with RITC-HA, RITC-Suc15-HA, RITC-Glu11-HA, or RITC-MGlu14-HA and incubated at 37°C for 4 hours is shown. The fluorescence intensities of RITC-HA, RITC-Suc15-HA, RITC-Glu11-HA, and RITC-MGlu14-HA were measured using a flow cytometer. Autofluorescence of the cells was subtracted, and the fluorescence intensity of RAW264.7 cells was corrected for the RITC fluorescence intensity of each HA derivative. [Figure 16]The image shows confocal laser scanning microscope images of RAW264.7 cells treated with HA derivatives. HA derivatives (red), nuclei (blue), and lysosomes (green) were stained with rhodamine B isothiocyanate, Hoechst, and Lyso Tracker Green. [Figure 17] This shows the colocalization coefficient of HA derivatives localized together with lysosomes relative to the total HA derivative. [Figure 18] The percentage of RAW264.7 cells that incorporated the sample is shown. RAW264.7 cells were treated with CD44 and scavenger receptor (SR) and HA derivative inhibitors. The RAW264.7 cells were then incubated at 37°C for 3 hours. Fluorescence was measured using a flow cytometer. Autofluorescence of the cells was subtracted. [Figure 19] This shows the proportion of HA derivative-bound and unbound cells in splenocytes. Splenocytes were incubated with 5 mg / mL of RITC-HA and RITC-MGlu-HA for 3 hours. Subsequently, splenocytes were stained with fluorescent antibodies against specific cell surface markers and the cell populations were classified. [Figure 20] (a) With LPS stimulation, and (b) without LPS stimulation, NF-κB activation is shown in RAW-Blue cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL). The expression level of NF-κB in cells exposed to LPS was set to 100%. [Figure 21] Different carboxyl groups suppress the production of inflammatory substances, resulting in a preventative effect. [Figure 22](a) TNF-α production levels by RAW264.7 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) with and without LPS stimulation. (c) IL-6 production levels by RAW264.7 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) with and without LPS stimulation. (e) IL-10 production levels by RAW264.7 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) with and without LPS stimulation. [Figure 23] (a) TNF-α production levels by DC2.4 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) with and without LPS stimulation. (c) IL-6 production levels by DC2.4 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) with and without LPS stimulation. (e) IL-10 production levels by DC2.4 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) with and without LPS stimulation. [Figure 24] (a) Shows the level of TNF-α production by spleen cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) with and without LPS stimulation. (c) Shows the level of IL-6 production by spleen cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) with and without LPS stimulation. (e) Shows the level of IL-10 production by spleen cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) with and without LPS stimulation. [Figure 25](a) With LPS stimulation, and (b) without LPS stimulation, RAW264.7 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) exhibit reactive oxygen species removal ability. [Figure 26] This shows NF-κB activation in RAW-Blue cells 1 hour after LPS stimulation. NF-κB expression levels in cells exposed to LPS were set to 100%. [Figure 27] This shows the TNF-α, IL-6, and IL-10 production levels of RAW264.7 cells 1 hour after stimulation with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL). [Figure 28] This shows the TNF-α, IL-6, and IL-10 production levels of DC2.4 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) one hour after LPS stimulation. [Figure 29] This shows the TNF-α, IL-6, and IL-10 production levels of splenocytes treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (1 mg / mL) one hour after stimulation with LPS. [Figure 30] (a) TNF-α production levels by RAW264.7 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA (a) with and without LPS stimulation. (c) IL-6 production levels by RAW264.7 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA with and without LPS stimulation. (e) IL-10 production levels by RAW264.7 cells treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA with and without LPS stimulation. The concentrations of HA, Suc17-HA, Glu17-HA, and MGlu19-HA were 1 mg / mL, 8 mg / mL, 6 mg / mL, and 8 mg / mL, respectively. [Figure 31](a) TNF-α production levels by splenocytes treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA with and without LPS stimulation. (c) IL-6 production levels by splenocytes treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA with and without LPS stimulation. (e) IL-10 production levels by splenocytes treated with HA, Suc17-HA, Glu17-HA, and MGlu19-HA with and without LPS stimulation. The concentrations of HA and other HA derivatives were 1 mg / mL and 5 mg / mL, respectively. [Figure 32] The levels of TNF-α, IL-6, and IL-10 production in splenocytes treated with HA, Suc17-HA, Glu17-HA, or MGlu19-HA one hour after LPS stimulation are shown. The concentrations of HA and the other HA derivatives were 1 mg / mL and 5 mg / mL, respectively. [Figure 33] The images show in vivo imaging demonstrating the easy flow of hyaluronic acid derivatives from the tail. (a) Image taken 3 hours after administration. (b) Image taken 8 hours after administration. (c) Image taken 120 hours after administration. [Figure 34] This shows the product of the average fluorescence intensity and area in the image. [Figure 35] This shows in vivo imaging of various organs 3 hours after administration. [Figure 36] This study demonstrates the creation of CAIA model mice and the therapeutic effects of hyaluronic acid. [Figure 37] The clinical scores of the forelimbs and hindlimbs of CAIA mice at 13 days, representing the anti-inflammatory effects of hyaluronic acid, Suc17-HA, Glu19-HA, and MGlu16-HA, are shown. [Figure 38] This shows the proportion of each cell type within the spleen. (a) The ratio of M1 macrophages to M2 macrophages, (b) Neutrophils in monocytes, (c) Tregs in T cells, (d) B1a cells in B cells, and (e) MZ cells in B cells. [Figure 39]The levels of IL-10 and IL-6 produced in the popliteal lymph nodes on days 5 and 6 are shown. Cytokine levels are normalized to the protein levels in the lymph nodes. [Figure 40] The clinical scores of the forelimbs and hindlimbs of CAIA mice at 13 days, illustrating the anti-inflammatory effects of hyaluronic acid, Glu19-HA, and Glu27-HA, are shown. [Modes for carrying out the invention]

[0022] In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0023] In this specification, when multiple upper and lower limits are given for a given parameter, a range formed by any combination of any upper and lower limit is also uniquely disclosed.

[0024] In one aspect, this disclosure relates to a carboxyl group-introduced high molecular weight hyaluronic acid derivative (which may also be referred to herein as "HA derivative of this disclosure").

[0025] The HA derivatives of this disclosure are derivatives obtained by introducing a carboxyl group into hyaluronic acid, and are of high molecular weight.

[0026] The term "high molecular weight" is not particularly limited, as long as it is a molecular weight sufficient to exert an anti-inflammatory effect. The weight-average molecular weight of the HA derivative and / or high molecular weight hyaluronic acid before derivatization of this disclosure is, for example, 200,000 or more, preferably 500,000 or more, more preferably 700,000 or more, even more preferably 900,000 or more, and even more preferably 1,000,000 or more. The upper limit of the weight-average molecular weight is not particularly limited, and is, for example, 10,000,000 or less, 7,000,000 or less, 5,000,000 or less, or 3,000,000 or less. In one embodiment, the weight-average molecular weight is, for example, 200,000 to 1,000,000, preferably 500,000 to 1,000,000, more preferably 700,000 to 1,000,000, even more preferably 700,000 to 7,000,000, even more preferably 700,000 to 5,000,000, and particularly preferably 900,000 to 5,000,000. The weight-average molecular weight can be measured by GPC (Gel Permeation Chromatography).

[0027] The manner in which carboxyl groups are introduced into hyaluronic acid is not particularly limited, as long as some atoms or groups in the hyaluronic acid are replaced with monovalent groups containing carboxyl groups. From the viewpoint of anti-inflammatory effect, IL-10 production promoting effect, specificity to inflammatory cells, and viscosity reduction, the embodiment in which some oxygen atoms in the hydroxyl groups of hyaluronic acid are replaced with monovalent groups containing carboxyl groups is particularly preferred.

[0028] The HA derivatives of this disclosure are preferably of general formula (1):

[0029] [ka] [In the formula: R 1 [The symbols represent monovalent groups containing a hydrogen atom or a carboxyl group, either identical or distinct. n represents a natural number.] This includes compounds represented by , salts thereof, or solvates thereof.

[0030] The monovalent group containing a carboxyl group is not particularly limited as long as it is a monovalent group with a carboxyl group located at its terminus. From the viewpoint of anti-inflammatory activity, IL-10 production promoting activity, specificity to inflammatory cells, viscosity reduction, etc., the general formula (2):-C(=O)-R 11 -COOH(in the formula:R 11 This represents a single bond or linker.

[0031] Examples of linkers include alkylene groups, alkenylene groups, and heteroalkylene groups (for example, heteroalkylene groups containing -NH, -C(=O)-, -O-, etc., in the main chain). Linkers can be linear or branched, with branched being more preferable. The number of atoms constituting the main chain of the linker (in the case of alkylene and alkenylene groups, the number of carbon atoms) is not particularly limited, for example, 1 to 10, preferably 2 to 8, more preferably 3 to 6, and even more preferably 3 to 4. Preferably, the linker is an alkylene group, more preferably an alkylene group having 2 to 8 carbon atoms, even more preferably an alkylene group having 3 to 6 carbon atoms, and even more preferably an alkylene group having 3 to 4 carbon atoms. The number of atoms in the linker's main chain (the chain connecting the carbon atoms of -C(=O)- and -COOH) (or the number of carbon atoms in the case of an alkylene group) is preferably 2 to 8, more preferably 3 to 6, and even more preferably 3 to 4. By using the above-mentioned linker, effects such as anti-inflammatory activity, IL-10 production promotion, specificity to inflammatory cells, and viscosity reduction can be more effectively exhibited.

[0032] The carboxyl group introduction rate, that is, the ratio of the number of carboxyl groups introduced in the HA derivative of this disclosure to 100% of the number of hydroxyl groups in the hyaluronic acid before derivatization of the HA derivative of this disclosure, is not particularly limited as long as it can exert an anti-inflammatory effect, and is, for example, 50% or less, preferably 45% or less, more preferably 40% or less, even more preferably 35% or less, even more preferably 30% or less, particularly preferably 25% or less, and especially preferably 20% or less. The lower limit of this rate is, for example, 1% or more, preferably 2% or more, more preferably 5% or more, even more preferably 8% or more, and even more preferably 10% or more. By adjusting the carboxyl group introduction rate to the above range, effects such as anti-inflammatory effect, IL-10 production promotion effect, specificity to inflammatory cells, and viscosity reduction can be exerted more effectively. The carboxyl group introduction rate can be measured according to the method described in 2.1.3 of the Examples below.

[0033] n is, for example, 500 to 30000, preferably 1000 to 20000, and more preferably 2000 to 10000.

[0034] The HA derivatives of this disclosure may be in the form of salts. Salts are usually basic salts. Examples of basic salts include alkali metal salts such as sodium salts and potassium salts; and alkaline earth metal salts such as calcium salts and magnesium salts.

[0035] The HA derivatives of this disclosure may be in the form of solvates. The solvent is not particularly limited and includes, for example, water, ethanol, glycerol, acetic acid, etc.

[0036] The HA derivatives of this disclosure may be linked to other molecules, provided that the anti-inflammatory effect is not significantly impaired. Examples of other molecules include labeling substances and drugs. Examples of labeling substances include fluorescent substances and radioisotopes, and examples of drugs include peptides, nucleic acids, sugars, low molecular weight compounds, high molecular weight compounds, inorganic substances, and complexes thereof.

[0037] The HA derivatives of this disclosure can be synthesized, for example, according to the method described in Non-Patent Document 1, or according to the method described in Example 1.4 below.

[0038] The HA derivatives of this disclosure can exhibit anti-inflammatory effects, IL-10 production promoting effects, and the like. For this reason, the HA derivatives of this disclosure can be used as active ingredients in anti-inflammatory agents, IL-10 production promoting agents, and the like. This disclosure relates to anti-inflammatory agents, IL-10 production promoting agents, and the like (these may be collectively referred to as "the agents of the present invention") that contain the HA derivatives of this disclosure.

[0039] The agent of the present invention can be used as a pharmaceutical, reagent, or the like.

[0040] The agents disclosed herein can be used as prophylactic or therapeutic agents for inflammation. In this specification, "treatment" can include concepts such as cure, remission, reduction, alleviation, inhibition of progression, and improvement of symptoms. Furthermore, "prevention" can include not only preventing the onset of the disease, but also delaying its onset, suppressing symptoms if the disease does develop, preventing recurrence after cure or remission of symptoms, and suppressing exacerbation after reduction or alleviation of symptoms.

[0041] The agent of the present invention is not particularly limited as long as it contains an active ingredient, and may further contain additives as needed. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.

[0042] The manner in which the agent of the present invention is used is not particularly limited, and an appropriate manner of use can be adopted depending on the application. Depending on the application, the agent of the present invention can be used, for example, in vitro (e.g., added to the culture medium of cultured cells) or in vivo (e.g., administered to animals).

[0043] The agents of the present invention are not particularly limited in their application to mammals, but examples include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Examples of cells include animal cells. The types of cells are also not particularly limited, and examples include nerve cells, glial cells, blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, egg cells), fibroblasts, epithelial cells, vascular endothelial cells, hepatocytes, keratin-producing cells, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0044] The agent of the present invention can take any dosage form, such as parenteral formulations including injection preparations (e.g., intraventricular, lumbar, intravenous infusion preparations, intraintramuscular, subcutaneous, and intradermal injection preparations), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye drops, eye ointments, nasal sprays, and ear drops), or oral formulations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, and jelly drops), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), jellies, and films. Furthermore, the active ingredient can be administered in a compounded state with particles (e.g., lipid particles or exosomes) or encapsulated within such particles.

[0045] The content of the active ingredient in the agent of the present invention depends on the manner of use, the target of application, the condition of the target, etc., and is not limited, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0046] The dosage of the agent of the present invention when administered to animals is not particularly limited as long as it is an effective amount that produces a pharmacokinetic effect. Generally, the weight of the active ingredient is 0.1 to 1000 mg / kg body weight per day, preferably 0.5 to 500 mg / kg body weight per day, when administered orally, and 0.01 to 100 mg / kg body weight per day, preferably 0.05 to 50 mg / kg body weight, when administered parenterally. The above dosage and administration interval may be increased or decreased as appropriate depending on age, disease state, symptoms, etc. [Examples]

[0047] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0048] 1. Experimental Procedure 1.1 Reagents Sodium hyaluronate, succinic anhydride, pyridine, deuterium water, diethyl ether, ethanol, benzylpenicillin G potassium, streptomycin sulfate, minimal required medium (MEM) non-essential amino acid solution, L-glutamine solution, Dulbecco's modified Eagle medium (DMEM), Roswell Park Memorial Laboratory medium (RPMI-1640), Dulbecco's phosphate-buffered saline (PBS(-)), and the WST-8 kit were purchased from Nacalai Tesque. 4-dimethylaminopyridine, 3-methylglutaric anhydride, dimethyl sulfoxide (super-dehydrated DMSO), dibutyltin(IV) dilaurate, and potassium polyvinyl sulfate were purchased from Wako Pure Chemical Industries, Ltd. Glutaric anhydride and 2',7'-dichlorodihydrofluorescein diacetic acid (DCFH-DA) were purchased from Tokyo Chemical Industries, Ltd. Rhodamine B isothiocyanate was purchased from Cosmo Bio Inc. The dialysis membrane, Spectra / Pro 6 (molecular weight cutoff 12,000-16,000), was purchased from Viskase Companies Inc. Fetal bovine serum (FBS), sodium dextran sulfate, lipopolysaccharide (LPS), and sodium deuterium hydroxide were purchased from Sigma-Aldrich. The TNF-α ELISA kit, IL-10 ELISA kit, Hoechst solution, and Lyso Tracker Green were purchased from Invitrogen. The IL-6 ELISA kit was purchased from PEPRO TECH and Invitrogen. Normalin, zeosin, and QUANTI-Blue were also used. TM I purchased it from InvivoGen. BD Cytofix / Cytoperm TMI purchased the following from BD Biosciences: 2-mercaptoethanol from Gibco; T-PER Tissue Protein Extraction Reagent from Thermo Scientific; cOmplete tablets from Roche; and the Collagen-CIA Arthritogenic Monoclonal Antibody Cocktail kit from Chondrex.

[0049] 1.2. Cell culture 1.2.1. Mouse macrophage-derived cell line RAW264.7 RAW264.7 cells were cultured in a DMEM medium containing 10% FBS, 0.1 mg / mL benzylpenicillin G potassium, and 0.1 mg / mL streptomycin sulfate in a CO2 incubator (Thermo SCIENTIFIC, FORMA DIRECT HEAT CO2 Incubator) at a CO2 concentration of 5% and 37°C.

[0050] 1.2.2. RAW264.7 cells (RAW-Blue cells) equipped with an NF-κB reporter RAW-Blue cells were cultured in a CO2 incubator at 37°C with a CO2 concentration of 5%, using two DMEM mediums alternately for each passage: one containing 10% FBS, 0.1 mg / mL benzylpenicillin G potassium, 0.1 mg / mL streptomycin sulfate, and 0.1 mg / mL normalin, and the other containing 10% FBS, 0.1 mg / mL benzylpenicillin G potassium, 0.1 mg / mL streptomycin sulfate, 0.1 mg / mL normalin, and 0.2 mg / mL zeosin.

[0051] 1.2.3. Mouse dendritic cell-derived cell line DC2.4 DC2.4 cells were cultured in a CO2 incubator (Thermo SCIENTIFIC, FORMA DIRECT HEAT CO2 Incubator) at 5% CO2 concentration and 37°C using RPMI medium containing 10% FBS, 0.1 mg / mL benzylpenicillin G potassium, 0.1 mg / mL streptomycin sulfate, 2 mM L-glutamine, 0.1 mM MEM non-essential amino acid solution, and 0.55 mM 2-mercaptoethanol.

[0052] 1.3. Animals C57BL / 6N mice and BALB / cJ mice were purchased from Jackson Laboratory and reared and maintained in a conventional facility. Animal experiments were approved by the Osaka Metropolitan University Animal Experiment Committee and conducted in accordance with its regulations.

[0053] 1.4. Synthesis of hyaluronic acid derivatives 1.4.1. Desalting of sodium hyaluronate Sodium hyaluronate (Mw: ≥ 1,000 kDa) (pKa=3-4) was added to a round-bottom flask, and HCl and deionized water were added to adjust the pH to 1.0-2.0. Desalting was then performed by dialysis (MWCO=12,000-16,000) for 3 days with hydrochloric acid also adjusted to pH 1.0-2.0. After removing some of the water under reduced pressure using a rotary evaporator (EYELA, DPE-1150), the solution was freeze-dried overnight to recover a white solid.

[0054] 1.4.2 Synthesis of RITC-HA Hyaluronic acid (HA) and super-dehydrated DMSO (8 mL) were placed in a three-necked eggplant flask, which was equipped with a condenser tube and an Ar balloon. The mixture was stirred at 90°C to dissolve HA in DMSO. After confirming that HA was completely dissolved, pyridine, dibutyltin dilaurate (DBTDL), and rhodamine B isothiocyanate (RITC) dissolved in DMSO were added to the eggplant flask, and the mixture was stirred at 95°C. After 2 hours, the reaction solution was added dropwise to a mixed solvent of 360 mL of diethyl ether and 90 mL of ethanol (8:2). The resulting precipitate was centrifuged (Thermo SCIENTIFIC, Sorvall X4R Pro) (4000 rpm, 4°C, 10 min), and the supernatant was removed for reprecipitation. The precipitate was dissolved in DMSO, and reprecipitation was carried out once more following the same procedure. The obtained precipitate was re-dispersed in a mixed solvent of 360 mL of diethyl ether and 90 mL of ethanol (8:2), followed by centrifugation (4000 rpm, 4°C, 10 min), and purification was achieved by removing the supernatant. The same procedure was repeated three times. The precipitate was collected in a sample tube and vacuum-dried. Furthermore, in order to remove unreacted RITC and DMSO that could not be completely removed by reprecipitation, the obtained pale red solid was dialyzed against 30 mM aqueous sodium chloride solution for 2 days (7 external solution exchanges), and then dialyzed against distilled water for 2 days (4 external solution exchanges). After dialysis, water was removed under reduced pressure to an appropriate extent, followed by lyophilization to collect the pale red solid. Identification of the compound was 1 performed by 1H NMR (JEOL, ECX400).

[0055] 1.4.3. Synthesis of HA derivatives HA and super-dehydrated DMSO (10 mL) were placed in a three-necked round-bottom flask, and a condenser and Ar balloon were attached. The mixture was heated and stirred at 90°C to dissolve the HA in the DMSO. After confirming that the HA was completely dissolved, 4-dimethylaminopyridine (DMAP) and succinic anhydride (Suc An), glutaric anhydride (Glu An), or 3-methylglutaric anhydride (MGlu An) were added to the round-bottom flask, and the mixture was heated and stirred at 40°C. After 24 hours, the mixture was dialyzed against 30 mM aqueous sodium chloride for 2 days using a dialysis membrane with a molecular weight cutoff of 12,000-16,000 (7 phase changes), and then dialyzed against distilled water for 2 days (8 phase changes). After dialysis, the water was partially removed by vacuum distillation using a rotary evaporator (EYELA, DPE-1150), and the mixture was freeze-dried overnight to recover a white solid. Compound identification was performed. 1 The analysis was performed using 1H NMR (JEOL, ECX400 or Bruker, AV300N).

[0056] 1.5. Comparison of fluorescence intensity of fluorescently labeled HA derivatives RITC, RITC-Suc in a quartz cell 16 -HA, RITC-Glu 11 -HA, RITC-MGlu 14 -A solution of HA was placed in a fluorescence spectrophotometer (JASCO, FP-8600). The temperature inside the quartz cell was set to 37°C, the excitation wavelength to 558.0 nm, and the fluorescence intensity from 560 to 700 nm was measured at a scanning speed of 100 nm / min. The HA concentration was diluted with PBS to 0.33 mg / mL (final volume 3.0 mL).

[0057] 1.6. Observation of changes in fluidity due to the introduction of carboxyl groups The HA derivative was dissolved in PBS(-) to a concentration of 1 mg / mL, and 500 μL was placed in a screw-cap vial (NO. 01). The vial was placed on a stand tilted at an 18° angle, and the fluidity of the HA derivative aqueous solution was qualitatively observed.

[0058] 1.7. Inhibitory effect of hyaluronic acid on the production of inflammatory substances 1.7.1. Inhibitory effect of polyanions on inflammatory cytokine production 1.0 × 10 in a 96-well plate 4RAW264.7 cells were seeded in cells / well and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-), polyanions (dextran sulfate, chondroitin sulfate, potassium polyvinyl sulfate (concentrations unified in number of anions per 1 mg / mL of HA)), and 1 μg / mL of LPS) were added sequentially to each well, and incubated for 24 hours. The plates were centrifuged (himac centrifuge, CT6E) (1,000 rpm, 5 min), the supernatant was collected, and the production of TNF-α, a representative inflammatory cytokine, was examined by ELISA. Polyanions were dissolved in PBS(-), and LPS was dissolved in culture medium before use.

[0059] 1.7.2. Inhibitory effect of LPS on inflammatory cytokine production by endogenous HA derivatives 1.0 × 10 in a 96-well plate 4 RAW264.7 cells were seeded at a cell / well level and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-), and 1 mg / mL of HA derivative were added sequentially to each well, and incubated for 3 hours. After washing twice with PBS(-), 1 μg / mL of LPS was added, and incubated for 21 hours. The plates were centrifuged (himac centrifuge, CT6E) (1,000 rpm, 5 min), and the supernatant was collected. The production of TNF-α, a representative inflammatory cytokine, was examined by ELISA. The HA derivative was dissolved in PBS(-) and adjusted to pH 7.4, and the LPS was dissolved in culture medium before use.

[0060] 1.8. Inhibitory effect of differences in carboxyl group introduction rate on the production of inflammatory substances 1.8.1. Inhibitory effect of differences in carboxyl group introduction rate on NF-κB activation 1.0 × 10 in a 96-well plate 4RAW-Blue cells were seeded at cells / well and cultured for 48 hours. After washing with PBS, culture medium, PBS(-), 1 mg / mL of HA derivative, and 1 μg / mL of LPS were added sequentially to each well, and incubated for 24 hours. The plate was centrifuged (himac centrifuge, CT6E) (1,000 rpm, 5 min), and 20 μL of the supernatant was collected. 180 μL of QUANTI-Blue solution was added to this supernatant, and the plate was incubated in a CO2 incubator (astec, DIRECT HEAT CO2Incubator CPI-165R). After 1 hour, NF-κB activation was investigated by measuring the absorbance at a wavelength of 655 nm using a plate reader (CORONA, GRATING MICROPLATE READER SH-8000 Lab) to determine the amount of secreted alkaline phosphatase (SEAP) secreted from the cells. In all subsequent experiments, the HA derivative was dissolved in PBS(-) and adjusted to pH 7.4, and the LPS was dissolved in culture medium before use. The amount of SEAP at this time was set to 100%, and the relative NF-κB activity is expressed.

[0061] 1.8.2. Inhibitory effect of differences in carboxyl group introduction rate on inflammatory cytokine production 1.0 × 10 in a 96-well plate 4 RAW264.7 cells were seeded at a cell / well ratio and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-), 1 mg / mL of HA derivative, and 1 μg / mL of LPS were sequentially added to each well, and incubated for 24 hours. The plates were centrifuged (himac centrifuge, CT6E) (1000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines, TNF-α and IL-6, were examined by enzyme immunosorbent assay (ELISA) according to the Invitrogen Mouse TNF alpha Uncoated ELISA kit and the PEPRO TECH IL-6 Murine Recombinant kit.

[0062] 1.9. Evaluation of cell selectivity of HA derivatives 1.9.1. Evaluation of the interaction of HA derivatives with RAW264.7 cells using flow cytometry. 1.0 × 10 in a 12-well plate 5 RAW264.7 cells were seeded in cells / well and cultured for 24 hours. After washing with PBS, culture medium was added, and then PBS or 0.5 mg / mL RITC-HA, RITC MGlu-HA, RITC-Suc-HA, and RITC-Glu-HA were added. The cells were incubated for 4 hours to allow uptake of the HA derivatives. After washing twice with PBS, the cells were detached with a cell scraper and collected in tubes for flow cytometry. The fluorescence intensity of 5,000 cells was measured using a flow cytometer (Beckman Coulter CytoFlex).

[0063] 1.9.2. Evaluation of intracellular dynamics of HA derivatives in RAW264.7 using confocal microscopy. 1.0 x 10 in a glass-bottom dish 5 RAW264.7 cells were seeded in cells / well and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-) or a fluorescently labeled HA derivative was added. The HA derivative was incubated at 37°C for 4 hours to allow interaction with the RAW264.7 cells. After washing three times with PBS(-), phenol-red-free DMEM medium containing 10% FBS was added, and the nuclei or lysosomes were stained with 10 μg / mL Hoechst solution or 150 nM Lyso Tracker Green, respectively, and incubated for 15 minutes. After washing twice with PBS(-), phenol-red-free DMEM medium containing 10% FBS was added, and observation was performed using a confocal microscope, CLSM 5 EXCITER (Carl Zeiss).

[0064] 1.9.3. Evaluation of interactions with RAW264.7 cells during receptor blocking 4.0 × 10 in a 24-well plate 4RAW264.7 cells were seeded at cells / well and cultured for 48 hours. After washing with PBS, culture medium and PBS were added to each well in sequence. Sodium hyaluronate was added as a CD44 receptor inhibitor, and sodium dextran sulfate was added at 0.8 mg / well as a scavenger receptor inhibitor. After incubation for 1 hour, 1 mg / mL of a fluorescently labeled HA derivative was added and incubated for 3 hours. The amount of sample taken up by macrophages was measured using a flow cytometer (BD Accuri). TM The fluorescence intensity of 2,000 cells was measured using C6).

[0065] 1.9.4. Evaluation of cell selectivity in splenocytes C57BL / 6N mice (male) were euthanized by cervical dislocation, and their spleens were collected. The spleens were ground on a 70 μm cell strainer, 1 mL of R10 (RPMI medium containing 10% FBS, 0.1 mg / mL benzylpenicillin G potassium, and 0.1 mg / mL streptomycin sulfate) was added, and single cells were collected in a centrifuge tube.

[0066] The cell harvested solution was centrifuged at 350 g for 5 minutes (Thermo SCIENTIFIC, Sorvall X4R Pro), and the supernatant was discarded. 5 mL of ice-cold RBC Lysis Buffer was added to the suspension, and the mixture was incubated on ice for 5 minutes. 5 mL of PBS was added, and the mixture was centrifuged at 350 g for 5 minutes (Thermo SCIENTIFIC, Sorvall X4R Pro), and the supernatant was discarded. 5 mL of R10 was added to disperse the cells, and the mixture was centrifuged at 350 g for 5 minutes (Thermo SCIENTIFIC, Sorvall X4R Pro). After discarding the supernatant, 5 mL of R10 was added to the cell count. TM Measured using (Invitrogen by life technologies).

[0067] Place each cell suspension in a round-bottom 96-well plate in a 1.0 × 10⁶ 4Cells / well were added to the solution, and the sample was added. After 3 hours, 50 μL / well of an antibody cocktail diluted 200-fold with 2% FBS-containing PBS was added, mixed, and allowed to stand at 4°C in the dark. After 20 minutes, 150 μL / well of 2% FBS-containing PBS was added, and the mixture was centrifuged at 2,000 rpm for 2 minutes (Thermo SCIENTIFIC, Sorvall X4R Pro). The supernatant was discarded, and the mixture was redispersed with 200 μL / well of 2% FBS-containing PBS. The mixture was centrifuged at 2,000 rpm for 2 minutes, and 100-150 μL / well of 2% FBS-containing PBS was added. The mixture was measured using a flow cytometer (Beckman Coulter CytoFlex).

[0068] 1.10. Inhibitory effect of the production of preventive inflammatory substances due to differences in carboxyl groups 1.10.1. Evaluation of the inhibitory effect on prophylactic NF-κB activation in macrophages 1.0 × 10 in a 96-well plate 4 RAW-Blue cells were seeded at a cell / well ratio and cultured for 48 hours. After washing with PBS, culture medium, PBS(-), 1 mg / mL of HA derivative, and 1 μg / mL of LPS were added sequentially to each well, and incubated for 24 hours. The plate was centrifuged (himac centrifuge, CT6E) (1000 rpm, 5 min), and 20 μL of the supernatant was collected. 180 μL of QUANTI-Blue solution was added to this supernatant, and the plate was incubated in a CO2 incubator (astec, DIRECT HEAT CO2Incubator CPI-165R). After 1 hour, the absorbance at a wavelength of 655 nm was measured using a plate reader (Agilent BioTek Epoch 2 absorbance plate reader) to measure the amount of secreted alkaline phosphatase (SEAP) secreted from the cells, thereby investigating NF-κB activation. The amount of SEAP at this time is set to 100%, and the relative NF-κB activity is expressed as such.

[0069] 1.10.2. Evaluation of the effect of suppressing the production of prophylactic inflammatory cytokines in macrophages. 1.0 × 10 in a 96-well plate 4RAW264.7 cells were seeded at a cell / well ratio and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-), 1 mg / mL of HA derivative, and 1 μg / mL of LPS were sequentially added to each well, and incubated for 24 hours. The plates were centrifuged (himac centrifuge, CT6E) (1,000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines TNF-α and IL-6, and representative anti-inflammatory cytokine IL-10 were examined by ELISA according to Invitrogen's Mouse TNF alpha Uncoated ELISA kit, Mouse IL-6 Uncoated ELISA kit, and Mouse IL-10 Uncoated ELISA kit.

[0070] 1.10.3. Evaluation of the effect of suppressing the production of prophylactic inflammatory cytokines in dendritic cells 1.0 × 10 in a 96-well plate 4 DC2.4 cells were seeded at a cell / well ratio and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-), 1 mg / mL of HA derivative, and 1 μg / mL of LPS were sequentially added to each well, and incubated for 24 hours. The plates were centrifuged (himac centrifuge, CT6E) (1,000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines TNF-α and IL-6, and representative anti-inflammatory cytokine IL-10 were examined by ELISA according to Invitrogen's Mouse TNF alpha Uncoated ELISA kit, Mouse IL-6 Uncoated ELISA kit, and Mouse IL-10 Uncoated ELISA kit.

[0071] 1.10.4. Evaluation of the effect of suppressing the production of prophylactic inflammatory cytokines in splenic cells BALB / cJ mice (female) were euthanized by cervical dislocation, and their spleens were collected. The spleens were ground up using a cell strainer, and 1 mL of R10 was added to the splenic cells, which were then collected in a centrifuge tube.

[0072] The splenocyte harvested solution was centrifuged at 350 g for 5 minutes (Thermo SCIENTIFIC, Sorvall X4R Pro), and the supernatant was discarded. 5 mL of ice-cold RBC Lysis Buffer was added to the suspension, and the mixture was incubated on ice for 5 minutes. 5 mL of PBS was added, and the solution was centrifuged at 350 g for 5 minutes (Thermo SCIENTIFIC, Sorvall X4R Pro), and the supernatant was discarded. 5 mL of R10 was added to disperse the cells, and the solution was centrifuged at 350 g for 5 minutes (Thermo SCIENTIFIC, Sorvall X4R Pro), and the supernatant was discarded. Then, 5 mL of R10 was added to the solution to measure the cell count. TM Measured using (Invitrogen by life technologies).

[0073] Place the cell suspension in a flat-bottomed 96-well plate in a 1.0 × 10⁶ well. 5 Cells were seeded to a specific cell / well density, and 1 mg / mL of HA derivative and 1 μg / mL of LPS were added sequentially. After incubation for 24 hours, the plates were centrifuged (himac centrifuge, CT6E) (1000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines TNF-α and IL-6, and representative anti-inflammatory cytokine IL-10 were examined by ELISA according to Invitrogen's Mouse TNF alpha Uncoated ELISA kit, Mouse IL-6 Uncoated ELISA kit, and Mouse IL-10 Uncoated ELISA kit.

[0074] 1.10.5. ROS scavenging ability in macrophages 1.0 × 10 in a 96-well plate 4RAW264.7 cells were seeded at cells / well and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-), 1 mg / mL of HA derivative, and 1 μg / mL of LPS were sequentially added to each well, and incubated for 24 hours. After washing twice with PBS(-), 2',7'-dichlorodihydrofluorescein diacetic acid was added to a final concentration of 20 μM. After incubation for 1 hour, the fluorescence intensity of 2',7'-dichlorofluorescein in 2000 macrophages was measured using a flow cytometer (BD Accuri). TM Measurements were taken using C6).

[0075] 1.11. Inhibitory effect of therapeutic inflammatory substance production due to differences in carboxyl groups 1.11.1. Evaluation of the inhibitory effect of treatment on NF-κB activation in macrophages 1.0 × 10 in a 96-well plate 4 RAW-Blue cells were seeded at cells / well and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-), and 1 μg / mL of LPS were added to each well in that order. After incubation for 1 hour, 1 mg / mL of HA derivative was added and incubated for 24 hours. The plate was centrifuged (himac centrifuge, CT6E) (1000 rpm, 5 min), and 20 μL of the supernatant was collected. 180 μL of QUANTI-Blue solution was added to this supernatant, and the plate was incubated in a CO2 incubator (astec, DIRECT HEAT CO2Incubator CPI-165R). After 1 hour, NF-κB activation was investigated by measuring the amount of secreted alkaline phosphatase (SEAP) secreted from the cells using a plate reader (Agilent BioTek Epoch 2 absorbance plate reader) to measure the absorbance at a wavelength of 655 nm. The amount of SEAP at this time is set to 100%, and the result is expressed as relative NF-κB activity.

[0076] 1.11.2. Evaluation of the effect of therapeutic inflammatory cytokine production suppression in macrophages 1.0 × 10 in a 96-well plate 4RAW264.7 cells were seeded at a cell / well ratio and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-), and 1 μg / mL of LPS were added to each well in that order. After incubation for 1 hour, 1 mg / mL of HA derivative was added and incubated for 24 hours. The plates were centrifuged (himac centrifuge, CT6E) (1000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines TNF-α and IL-6, and representative anti-inflammatory cytokine IL-10 were examined by ELISA according to Invitrogen's Mouse TNF alpha Uncoated ELISA kit, Mouse IL-6 Uncoated ELISA kit, and Mouse IL-10 Uncoated ELISA kit.

[0077] 1.11.3. Evaluation of the inhibitory effect of therapeutic inflammatory cytokine production on dendritic cells 1.0 × 10 in a 96-well plate 4 DC2.4 cells were seeded at a cell / well ratio and cultured for 48 hours. After washing twice with PBS(-), culture medium, PBS(-), and 1 μg / mL of LPS were added to each well in that order. After incubation for 1 hour, 1 mg / mL of HA derivative was added and incubated for 24 hours. The plates were centrifuged (himac centrifuge, CT6E) (1000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines TNF-α and IL-6, and representative anti-inflammatory cytokine IL-10 were examined by ELISA according to Invitrogen's Mouse TNF alpha Uncoated ELISA kit, Mouse IL-6 Uncoated ELISA kit, and Mouse IL-10 Uncoated ELISA kit.

[0078] 1.11.4. Evaluation of the inhibitory effect of therapeutic inflammatory cytokine production in splenic cells The spleen was harvested and hemolyzed. The cell suspension was placed in a flat-bottomed 96-well plate in a 1.0 × 10⁶ format. 5Cells were seeded to a specific cell / well density, and 1 μg / mL of LPS was added. After incubation for 1 hour, 1 mg / mL of HA derivative was added. After incubation for 24 hours, the plate was centrifuged (himac centrifuge, CT6E) (1000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines TNF-α and IL-6, and representative anti-inflammatory cytokine IL-10 were examined by ELISA according to Invitrogen's Mouse TNF alpha Uncoated ELISA kit, Mouse IL-6 Uncoated ELISA kit, and Mouse IL-10 Uncoated ELISA kit.

[0079] 1.12. Inhibitory effect of high-concentration HA derivatives on the production of inflammatory substances. 1.12.1. Evaluation of the effect of high-concentration HA derivatives on suppressing the production of prophylactic inflammatory cytokines in macrophages. 1.0 × 10 in a 96-well plate 4 RAW264.7 cells were seeded at cells / well and cultured for 48 hours. After washing twice with PBS(-), the cells were added to culture medium, PBS(-), and 1 mg / mL of HA or 8 mg / mL of Sucrose. 17 - HA, 6 mg / mL of Glu 17 -HA, 8 mg / mL of MGlu 19 -HA was added, and then 1 μg / mL of LPS was added to each well. The mixture was incubated for 24 hours. The plates were centrifuged (himac centrifuge, CT6E) (1,000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines TNF-α and IL-6, and representative anti-inflammatory cytokine IL-10 were examined by ELISA according to Invitrogen's Mouse TNF alpha Uncoated ELISA kit, Mouse IL-6 Uncoated ELISA kit, and Mouse IL-10 Uncoated ELISA kit.

[0080] 1.12.2. Evaluation of the inhibitory effect of high-concentration HA derivatives on the production of prophylactic inflammatory cytokines in splenic cells. The spleen was harvested and hemolyzed. The cell suspension was placed in a flat-bottomed 96-well plate in a 1.0 × 10⁶ format.5 Cells were seeded to a density of cells / well, and 1 μg / mL of LPS was added. The cell suspension was then placed in a flat-bottomed 96-well plate in a 1.0 × 10⁶ arrangement. 5 Cells were seeded to a specific cell / well ratio, and 1 mg / mL of HA, 5 mg / mL of an HA derivative, and 1 μg / mL of LPS were added in sequence. After incubation for 24 hours, the plates were centrifuged (himac centrifuge, CT6E) (1000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines TNF-α and IL-6, and representative anti-inflammatory cytokine IL-10 were examined by ELISA according to Invitrogen's Mouse TNF alpha Uncoated ELISA kit, Mouse IL-6 Uncoated ELISA kit, and Mouse IL-10 Uncoated ELISA kit.

[0081] 1.12.3. Evaluation of the inhibitory effect of high-concentration HA derivatives on the production of therapeutic inflammatory cytokines in splenic cells. The spleen was harvested and hemolyzed. The cell suspension was placed in a flat-bottomed 96-well plate in a 1.0 × 10⁶ format. 5 Cells were seeded to a specific cell / well density, and 1 μg / mL of LPS was added. After incubation for 1 hour, 1 mg / mL of HA derivative was added. After incubation for 24 hours, the plate was centrifuged (himac centrifuge, CT6E) (1000 rpm, 5 min), and the supernatant was collected. The production levels of representative inflammatory cytokines TNF-α and IL-6, and representative anti-inflammatory cytokine IL-10 were examined by ELISA according to Invitrogen's Mouse TNF alpha Uncoated ELISA kit, Mouse IL-6 Uncoated ELISA kit, and Mouse IL-10 Uncoated ELISA kit.

[0082] 1.13. Evaluation of the in vivo pharmacokinetics of HA derivatives Nine BALB / cJ female mice were divided into three groups: one receiving RITC-HA (n=3), one receiving RITC-Glu-HA (n=3), and one receiving RITC-MGlu-HA (n=3). 100 mL of RITC-labeled HA derivatives with uniform fluorescence intensity was administered subcutaneously via the tail to each group. Three hours later, the fluorescence intensity of the HA derivatives remaining in the tail was observed using an in vivo imaging system (VIEWORKS VISQUE in vivoSmart-LF). Furthermore, one mouse from each group that received the same sample was selected, and organs retrieved by cervical vertebral dislocation were also examined for fluorescence intensity using an in vivo imaging system (VIEWORKS VISQUE in vivoSmart-LF).

[0083] 1.14. Evaluation using a collagen antibody-induced arthritis (CAIA) model mouse 1.14.1. Creation of CAIA model mice To investigate the therapeutic effects of HA derivatives in vivo, we created a CAIA model mouse.

[0084] On Day 0, 0.5 mg (0.5 mL) of a collagen antibody cocktail was administered to BALB / cJ mice (female), and they were then transferred to clean cages.

[0085] On Day 3, mice with swollen legs were removed from the experimental group (screening). The six mice used in the experiment were randomly divided into two groups: one receiving PBS (n=4) and the other receiving HA (n=2). 0.1 mL of PBS was administered subcutaneously via the tail. The group receiving the HA derivative received 0.5 mg (0.1 mL) of HA subcutaneously via the tail. Subsequently, all mice were administered 25 μg (50 μL) of LPS. I scored the data for Days 4-6. Scoring was performed on Day 7, and samples were administered in the same manner as on Day 3. I scored the points on days 8-13. Scoring: 0: No swelling, 1: One toe is swollen, 2: Multiple toes are swollen, 3: Multiple toes and both joints are swollen, 4: The entire foot is severely swollen. Maximum 16 for 4 legs x 4 levels. Maximum 8 for 2 hind legs x 4 levels.

[0086] 1.14.2. Evaluation of therapeutic effects in CAIA model mice based on differences in carboxyl groups The effects of introducing carboxyl group units into HA on anti-inflammatory activity were compared using CAIA model mice.

[0087] Similar to 1.14.1, CAIA model mice were prepared, and the mice used in the experiment (18 mice) were divided into three groups: one group administered PBS (n=3), one group administered HA (n=3), and one group administered Suc. 17 - Group receiving HA (n=4), Glu 19 - Group receiving HA (n=4), MGlu 16 The subjects were randomly divided into five groups (n=4) to receive HA. I scored the data for Days 4-6. Scoring was performed on Day 7, and samples were administered in the same manner as on Day 3. I scored the points from Day 8 to 12. I took more photos on Day 12. On Day 13, all mice were euthanized by cervical dislocation, and their spleens and popliteal lymph nodes (both left and right) were collected.

[0088] The spleen was crushed and passed through a 70 μm pore cell strainer to obtain a single-cell dispersion of splenocytes. The splenocyte dispersion was centrifuged at 350 g for 5 minutes, and the supernatant was discarded. 5 mL of ice-cold RBC Lysis Buffer was added to suspend the cells, and the mixture was incubated on ice for 5 minutes to lyse the red blood cells. 5 mL of PBS was added, and the mixture was centrifuged at 350 g for 5 minutes, and the supernatant was discarded. 5 mL of medium was added to adjust the cell concentration. TMMeasurements were taken using Invitrogen by Life Technologies. Cell suspension was seeded in a round-bottom 96-well plate, an antibody cocktail was added, and after mixing, the plate was left to stand at 4°C in the dark. After 30 minutes, the plate was centrifuged twice with PBS containing 2% FBS, and then 200 μL / well of PBS containing 2% FBS was added. Various immune cells in splenocytes were analyzed using a flow cytometer (Beckman Coulter, CytoFLEX).

[0089] Lymph nodes from the right leg were also crushed and passed through a 70 μm pore cell strainer to obtain a single-cell dispersion of splenocytes. The cell dispersion was centrifuged at 350 g for 5 minutes, and the supernatant was discarded. 5 mL of RPMI-1640 was added and dispersed, and the dispersion was centrifuged at 350 g for 5 minutes, discarding the supernatant. The cell suspension was then seeded into a round-bottom 96-well plate, an antibody cocktail was added, and after mixing, the plate was allowed to stand at 4°C in the dark. After 30 minutes, the plate was washed twice by centrifugation with PBS containing 2% FBS, and then 200 μL / well of PBS containing 2% FBS was added. Various immune cells in the lymph nodes were analyzed using a flow cytometer (Beckman Coulter, CytoFLEX).

[0090] To measure the production of inflammatory cytokines, lymph nodes in the left leg were crushed in PBS using a cell disruptor (Festprep-24 5G), the supernatant was collected, the total protein content was measured by BCA assay, and the amounts of IL-10 and IL-6 were measured by ELISA.

[0091] Similar experiments were conducted with PBS (n=4), HA (n=3), and Glu. 17 -HA (n=3), MGlu 19 -HA (n=3) was used, and scoring was performed up to Day 14.

[0092] Scoring: 0: No swelling, 1: One toe is swollen, 2: Multiple toes are swollen, 3: Multiple toes and both joints are swollen, 4: The entire foot is severely swollen. Maximum 16 for 4 legs x 4 levels. Maximum 8 for 2 hind legs x 4 levels.

[0093] 1.14.3. Evaluation of therapeutic effects in CAIA model mice based on differences in carboxyl groups The experiment was conducted using a similar procedure, with PBS (n=5), HA (n=4), and Glu 19 -HA (n=5), Glu 27 The mice were randomly divided into four groups (n=5) and the therapeutic effects of differences in the rate of carboxyl group unit introduction were investigated using CAIA model mice.

[0094] 1.15. Statistical analysis Statistical significance was assessed using GraphPad Prism 9.0 as mean ± standard deviation (SD). Confidence levels were set to ns (not significant), 95% (*p < 0.05), 99% (**p < 0.01), 99.9% (***p < 0.001), and 99.99% (****p < 0.0001).

[0095] 2. Results and Discussion 2.1. Synthesis of hyaluronic acid derivatives 2.1.1. Desalting of sodium hyaluronate HA was converted to HA by dissolving HA salt in water, adjusting the pH to 1.0-2.0, and then freeze-drying it (Figure 2). Table 1 shows the amount of HA salt used and the yield of HA.

[0096] [Table 1]

[0097] 2.1.2. Synthesis of RITC-HA RITC-HA was synthesized by introducing a fluorophore to the hydroxyl group of HA by reacting rhodamine B isothiocyanate with HA in the presence of dibutyltin dilaurate, a catalyst (Figure 2). Table 2 shows the type and amount of reagents used, reaction temperature, yield of the obtained compound, and the introduction rate of the RITC group. The obtained compounds were identified by 1H NMR. The NMR charts of each compound are shown in Figure 3. a (4.4 ppm), f (4.6 ppm), l (2.0 ppm), and be, gk (3.3-4.0 ppm) are proton peaks originating from HA. The introduction of RITC to HA was confirmed by the presence of proton peaks p (1.2 ppm) and q (3.6-3.7 ppm) originating from RITC. The introduction rate of RITC was calculated from the integral value of p of RITC, with the proton peak of l of HA as the reference.

[0098] The RITC introduction rates were 0.13% (5.2 molecules / 1000 sugar unit) and 0.15% (6.0 molecules / 1000 sugar unit), demonstrating the successful synthesis of RITC-HA with low introduction rates that minimized the impact on the inherent properties of HA (Table 2).

[0099] [Table 2]

[0100] 2.1.3. Synthesis of HA derivatives Based on previously reported synthesis methods using HA, Suc-HA, Glu-HA, MGlu-HA, RITC-Suc-HA, RITC-Glu-HA, and RITC-MGlu-HA were synthesized by reacting the hydroxyl groups of HA and the remaining hydroxyl groups of RITC-HA with succinic anhydride, glutaric anhydride, and 3-methylglutaric anhydride, respectively (Figure 2). Tables 3 and 4 show the types and amounts of reagents used, reaction temperatures, yields of the resulting compounds, and the rate of carboxyl group introduction. Compound identification was performed. 1The analysis was performed by 1H NMR. NMR charts for each compound are shown in Figures 4, 5, 6, and 7. In addition to the proton peaks a (4.4 ppm), f (4.6 ppm), l (2.0 ppm), and be, gk (3.3-4.0 ppm) derived from HA, and the peaks p (1.2 ppm) and q (3.6-3.7 ppm) of the RITC group, new proton peaks were present: m (2.6-2.8 ppm) derived from the Suc group, m (2.3-2.5 ppm) and n (1.8-1.9 ppm) derived from the Glu group, and m, n (1.9-2.3 ppm) and o (0.9 ppm) derived from the MGlu group. The broadening of the peaks confirmed the introduction of Suc, Glu, and MGlu into HA and RITC-HA. Furthermore, the introduction rate of the carboxyl group was calculated by taking the integral value of I when hydrated with NaOD as the baseline and using the integral values ​​of m, n, and o relative to that value.

[0101] In the following notation of compounds, the numbers following Suc, Glu, and MGlu indicate the percentage of Suc, Glu, and MGlu groups introduced. For example, HA with 16% MGlu groups introduced will be MGlu 16 It is written as -HA.

[0102] Based on the integral values, we successfully synthesized HA derivatives with a similar introduction rate (approximately 15%). Furthermore, because the introduction rate of RITC into HA was low, the subsequent introduction of carboxyl group units was also achieved with a reaction rate almost identical to that of HA.

[0103] [Table 3]

[0104] [Table 4]

[0105] 2.2. Comparison of fluorescence intensity of fluorescently labeled HA derivatives To correct for differences in fluorescence intensity between samples in cell uptake and in vivo imaging experiments, the fluorescence spectra of fluorescently labeled HA derivatives were measured. Figure 8 summarizes the fluorescence intensity at a wavelength of 558 nm for each sample. Although the RITC introduction rate was kept low, all HA derivatives showed a consistent fluorescence intensity. For the same weight, RITC-HA had the lowest fluorescence intensity, followed by RITC-Suc-HA, RITC-MGlu-HA, and RITC-Glu-HA, with increasing fluorescence intensity. In future experiments, this value will be used to correct for fluorescence intensity.

[0106] 2.3. Changes in fluidity due to the introduction of carboxyl groups To determine whether introducing a carboxyl group improves water solubility, the fluidity of HA derivatives dissolved in PBS was examined. Figure 9 shows the flow of a 5 mg / mL HA derivative. The HA derivative only flowed to about half the sample bottle immediately after being placed on the stand, indicating low fluidity. On the other hand, the HA derivative with the introduced carboxyl group flowed all the way to the bottom of the stand immediately after being placed on the stand at a concentration of 5 mg / mL, indicating high fluidity. This suggests that introducing a carboxyl group suppresses entanglement of the HA main chain due to electrostatic repulsion, improving the fluidity of the solution and enhancing water solubility.

[0107] 2.4. Inhibitory effect of hyaluronic acid on the production of inflammatory substances 2.4.1. Suppressive effect of polyanions on inflammatory cytokine production HA interacts with CD44 to exert anti-inflammatory effects. Cells in which inflammation has been induced with lipopolysaccharide (LPS) are used to evaluate this anti-inflammatory effect. The mechanism of LPS-induced inflammation is known to involve activation of NF-κB, a key transcription factor in the inflammatory response, through interaction with TLR4, thereby suppressing the production of inflammatory cytokines. Here, to investigate whether HA can suppress the production of inflammatory substances induced by LPS, and whether this effect is due to the structure of HA, we evaluated the LPS-induced inhibitory effect on inflammatory cytokine production using four types of polyanions containing HA.

[0108] Figure 10 shows the amount of TNF-α produced when polyanions and LPS were present for 24 hours. PBS was added as a control. Compared to LPS stimulation alone, the presence of HA suppressed TNF-α production. On the other hand, TNF-α production was not suppressed in the presence of any other polyanions. This suggests that HA derivatives do not suppress inflammatory cytokine production by inhibiting the binding of extracellular polymers to Toll-like receptor 4 (TLR4) expressed on the cell surface and LPS, but rather by interacting with CD44. Furthermore, chondroitin sulfate, like HA, is recognized and bound to CD44, but did not show an inhibitory effect on inflammatory cytokine production. This suggests that not only the interaction with CD44 but also the structure of HA is important for its anti-inflammatory effect.

[0109] 2.4.2. Inhibitory effect of LPS on the production of inflammatory cytokines by endogenous HA HA is known to exert anti-inflammatory effects by binding to the cell surface receptor CD44. Therefore, we investigated whether the inhibitory effect of HA described above is due to HA on the cell surface rather than HA taken into the cell. After contacting macrophages with HA, the HA on the cell surface was removed by washing, creating a state where only internalized HA could act. LPS was then added, and the amount of TNF-α produced was measured by ELISA.

[0110] Figure 11 shows the amount of TNF-α produced after incubation for 3 hours with HA, followed by washing and incubation for another 21 hours with LPS. Compared to LPS stimulation alone, internalization of HA into macrophages had almost no effect on TNF-α production. This indicates that internalized HA derivatives do not suppress LPS-induced inflammatory cytokine production, suggesting that the anti-inflammatory effect of HA is expressed through interaction with CD44 on the cell surface.

[0111] 2.5. Inhibitory effect of differences in carboxyl group introduction rate on the production of inflammatory substances 2.5.1. Inhibitory effect of differences in carboxyl group introduction rate on NF-κB activation To investigate the optimal introduction rate of carboxyl group units, the inhibitory effect of LPS stimulation on NF-κB activation was evaluated using MGlu-HA with various introduction rates.

[0112] When NF-κB is activated within cells, inflammatory cytokines are ultimately produced; therefore, suppressing NF-κB activation leads to suppression of inflammation. The RAW-Blue cells used here are RAW264.7 cells equipped with an NF-κB reporter, and as shown in Figure 12, when NF-κB is activated, they produce secreted alkaline phosphatase (SEAP), an indicator of the degree of inflammation. The higher the amount of SEAP produced, the more severe the inflammatory state.

[0113] Figure 13(a) shows the activity of NF-κB when HA derivatives and LPS were present for 24 hours. PBS was added as a control. Activation of NF-κB was confirmed in PBS (LPS(+)). This indicates that LPS triggered TLR4-mediated signaling, leading to NF-κB nuclear translocation. The amount of SEAP at this time is set to 100%, and the relative NF-κB activity is expressed. Compared to LPS stimulation alone, MGlu-HA with an MGlu group introduction rate of 43% or more had almost no effect on NF-κB activation. On the other hand, in the presence of HA, MGlu was present at 45% and 45%. 15 -50% in the presence of HA, MGlu 24 -In the presence of HA, NF-κB activation was suppressed by approximately 80%. This indicates that a lower rate of MGlu group introduction leads to a higher inhibitory effect on NF-κB activation by LPS.

[0114] Figure 13(b) shows the activation of NF-κB when only the HA derivative was added and incubated for 24 hours without the addition of LPS. HA and MGlu 52 - When HA was added, it hardly activated NF-κB, but the introduction rate of MGlu groups was moderate. 15 -HA, MGlu 24 -HA, MGlu 43-HA significantly activated NF-κB. This suggests that the LPS-mediated suppression effect of MGlu-HA on inflammatory cytokine production antagonized the inflammatory cytokine production effect of MGlu-HA itself, resulting in MGlu-HA being more effective despite its lower adoption rate. 52 -HA failed to suppress LPS-induced NF-κB activation, which is thought to be because its high introduction rate prevented it from maintaining its inherent anti-inflammatory effect.

[0115] 2.5.2. Inhibitory effect of differences in carboxyl group introduction rate on inflammatory cytokine production Figure 14 (a) shows the amount of TNF-α produced when HA derivatives and LPS were present together for 24 hours. PBS was added as a control. In the PBS group with LPS(-), there was almost no TNF-α production, while in the group with LPS(+), a large amount of TNF-α was produced. This indicates that LPS stimulated cells and triggered an inflammatory response. Furthermore, the results for LPS(+) showed that, compared to LPS stimulation alone, the presence of MGlu-HA with an MGlu group introduction rate of 24% or more had almost no effect on TNF-α production. On the other hand, HA and MGlu... 15 In the presence of -HA, TNF-α production was reduced. This suggests that, similar to the results regarding the inhibitory effect on NF-κB activation, a lower rate of MGlu group introduction leads to a greater inhibitory effect on inflammatory cytokine production by LPS.

[0116] Figure 14(b) shows the amount of TNF-α produced when only the HA derivative was added and incubated for 24 hours without the addition of LPS. When HA was added, almost no TNF-α was produced, but when MGlu-HA was added, TNF-α was produced, and the amount of TNF-α produced increased as the MGlu introduction rate increased. From this, similar to the results of the inhibitory effect on NF-κB activation, it is thought that MGlu-HA with a high introduction rate was unable to maintain the original anti-inflammatory effect of HA, and in addition, the inhibitory effect of MGlu-HA on inflammatory cytokine production by LPS antagonized the inflammatory cytokine production effect of MGlu-HA itself, resulting in MGlu-HA with a low introduction rate being more effective.

[0117] Furthermore, Figure 14(c) shows the amount of IL-6 produced when HA derivatives and LPS were present together for 24 hours. Similar to the case with TNF-α, the production of HA and MGlu is compared to LPS stimulation alone. 15 In the presence of -HA, IL-6 production was significantly reduced. On the other hand, IL-6 production was suppressed more effectively in the presence of MGlu-HA with a high MGlu group introduction rate of 52% than in the presence of MGlu-HA with a moderate introduction rate of 24%. From this, it was found that, similar to TNF-α, MGlu-HA with a low introduction rate of MGlu groups has a high inhibitory effect on inflammatory cytokine production by LPS. Furthermore, it was found that the inhibitory effect on inflammatory cytokine production was higher in MGlu-HA with a higher introduction rate than moderate MGlu groups, suggesting that MGlu-HA with an introduction rate of 52% or higher is effective. However, it was not possible to synthesize MGlu-HA with an even higher introduction rate. Considering that MGlu-HA with an introduction rate of about 90% was synthesized with low molecular weight HA, it is thought that high molecular weight HA has low solubility in the reaction solvent and reduced reactivity.

[0118] Figure 14 (d) shows the amount of IL-6 produced when only the HA derivative was added and incubated for 24 hours without the addition of LPS. 43 - Except for HA, no other substances produced IL-6, but MGlu 43 -When HA was added, IL-6 was produced. Therefore, MGlu 43 -HA's LPS-mediated suppression of inflammatory cytokine production, MGlu 43 -It is thought to be antagonizing the inflammatory cytokine production effect of HA itself.

[0119] 2.6. Evaluation of Cell Selectivity of HA Derivatives 2.6.1. Evaluation of the interaction of HA derivatives with RAW264.7 cells using flow cytometry. To correct the results of interactions with macrophages obtained by flow cytometry, RITC-HA and RITC-Suc 15 -HA, RITC-Glu 11 -HA, RITC-MGlu 14 -The fluorescence intensity of HA was measured.

[0120] Figure 15 shows RITC-HA and RITC-Suc 15 -HA, RITC-Glu 11 -HA, RITC-MGlu 15 -The interaction of HA with RAW264.7 cells was measured by flow cytometry, and the interaction between RITC-HA and RITC-Suc 15 -HA, RITC-Glu 11 -HA, RITC-MGlu 15 - The results were corrected for the ratio of fluorescence intensities of HA. Compared to RITC-HA, RITC-Glu-HA was slightly more readily taken up by macrophages. On the other hand, RITC-Suc-HA and RITC-MGlu-HA were less readily taken up.

[0121] 2.6.2. Evaluation of intracellular dynamics of HA derivatives in RAW264.7 using confocal microscopy Figure 16 shows RITC-HA and RITC-Suc 15 -HA, RITC-Glu 11 -HA, RITC-MGlu 14The results shown are from observing the nuclei and lysosomes after staining them with a confocal microscope following incubation of RAW264.7 cells with culture medium containing -HA for 4 hours and washing. All HA derivatives shown in red were observed near the nuclei and lysosomes, indicating that they were taken up via interaction with macrophages. Figure 17 shows the percentage of RITC-labeled HA derivatives that localized to lysosomes. While approximately 55% of HA localized to lysosomes, the localization rates for Glu-HA and MGlu-HA decreased to approximately 29% and 21%, respectively. This result suggests that HA derivatives are more easily escaped into endosomes than HA. In fact, it has been reported that when molecules with carboxyl groups in their polymer chains, namely aspartic acid (3 carbon atoms), glutaric acid (4 carbon atoms), and L-2-aminoadipic acid (5 carbon atoms), were introduced, the endosomal escape ability was highest in the order of L-2-aminoadipic acid, glutaric acid, and aspartic acid, in terms of carbon chain length. This is thought to be because an increase in the number of carbon atoms increases hydrophobicity, thereby enhancing membrane destabilization activity. In addition, our laboratory has previously prepared carboxylated polyglycidol derivatives with different hydrophobic properties by introducing succinyl groups, glutaryl groups, 3-methylglutaryl groups, and 1,2-cyclohexanedicarbonyl groups into polyglycidol, and modified liposomes with these derivatives. When the properties of these liposomes were evaluated, liposomes with highly hydrophobic polymers showed stronger responses, such as content release and membrane fusion, at weakly acidic pH, and achieved more efficient cytoplasmic delivery of membrane-impermeable dye molecules. This also indicates that improved hydrophobicity enhances membrane destabilization activity.

[0122] 2.6.3. Evaluation of interactions with RAW264.7 cells during receptor blocking To determine whether the introduction of a carboxyl group unit facilitates interaction with scavenger receptors and makes HA derivatives more easily taken up by macrophages, we inhibited CD44 and scavenger receptors and evaluated the interaction of HA derivatives with macrophages.

[0123] Figure 18 (a) shows the percentage of RAW264.7 cells that interacted with HA derivatives when CD44 and the scavenger receptor were inhibited, with the percentage of RAW264.7 cells that interacted with HA derivatives when the receptor was not inhibited set to 100%. For HA, the percentage of cells that took up HA did not change when either receptor was inhibited. On the other hand, when CD44 was inhibited, the percentage of cells that took up HA derivatives decreased by 12% and 7%, respectively, for Suc-HA and Glu-HA. However, MGlu-HA was actually more easily taken up. This has been observed in previous studies, suggesting that MGlu-HA interacts with HA and is more easily taken up by cells. Furthermore, when the scavenger receptor was inhibited, the percentages of Suc-HA, Glu-HA, and MGlu-HA decreased by 29%, 56%, and 30%, respectively. This indicates that introducing a carboxyl group unit makes it easier to interact with the scavenger receptor. Among these, Glu-HA was found to be the most effective. Similarly, Figure 18 (b) shows the amount of HA derivatives that interacted with RAW264.7 cells when the CD44 receptor and scavenger receptor were inhibited, with the average fluorescence intensity of RITC when the receptor was not inhibited set to 100%. When CD44 was inhibited, Suc-HA and Glu-HA interacted at 93% and 87%, respectively, which was almost equivalent to approximately 91% of HA. When the scavenger receptor was inhibited, HA uptake also decreased, but the overall trend was similar to (a). In other words, HA derivatives can increase the interaction with the scavenger receptor without impairing the interaction between HA and CD44. In particular, Glu-HA was found to have a strong interaction with the scavenger receptor.

[0124] 2.6.4. Evaluation of cell selectivity in splenic cells To investigate the cell selectivity of HA with MGlu introduced, fluorescently labeled HA and MGlu-HA were added to mouse splenocytes, and the uptake by each cell was measured. Figure 19 shows the ratio of cells that took up the sample to cells that did not, with the total number of cells set to 100%. In macrophages, there was almost no difference between MGlu-HA and HA. In dendritic cells and B cells, uptake became slightly less likely when MGlu was introduced. On the other hand, uptake was significantly reduced in T cells and NK cells. From these results, it was confirmed that HA derivatives with introduced carboxyl units are preferentially taken up by antigen-presenting cells, including macrophages expressing scavenger receptors.

[0125] 2.7. Inhibitory effect of the production of preventive inflammatory substances due to differences in carboxyl groups In this section, as shown in Figure 21, LPS was added immediately after the HA derivative was added to the cells, and the preventive effect of adding the sample before inflammation occurred was investigated.

[0126] 2.7.1. Evaluation of the inhibitory effect on prophylactic NF-κB activation in macrophages The amount of SEAP produced by RAW-Blue was used to evaluate the activation of NF-κB, the primary source of inflammatory cytokine production. The results are shown in Figure 20. PBS LPS(+) was used as the control, and its value is set to 100% to represent NF-κB activation. 17 -HA also Glu 17 -HA also MGlu 19 -HA also failed to suppress LPS-induced NF-κB activation. Furthermore, Suc was also not suppressed in LPS(-). 17 -HA, MGlu 19 -HA activated NF-κB. This indicates that the sample itself causes inflammation.

[0127] 2.7.2. Evaluation of the effect of suppressing inflammatory cytokine production in macrophages Figure 22 summarizes the amount of cytokines produced by macrophages when HA derivatives and LPS were added simultaneously.

[0128] Figure 22 (a) shows the results of the production amount of TNF-α, an inflammatory cytokine, when LPS and a sample are added simultaneously. HA significantly suppressed the production amount compared to PBS. In contrast, HA derivatives did not exhibit the suppressive effect. For IL-6 shown in Figure 22 (c), HA, Glu 17 -HA and MGlu 19 -HA significantly reduced the production amount compared with PBS, but the suppression effect was weaker than that of HA. Figures 22 (b) and (d) respectively show the results of TNF-α and IL-6 production when LPS was not added. For Suc 17 -HA, which failed to suppress IL-6 production in (c), TNF-α and IL-6 were produced even when LPS was not added, indicating that Suc 17 -HA itself promotes the production of inflammatory cytokines. Therefore, it is considered that the anti-inflammatory effect derived from HA was offset, and thus the production of inflammatory cytokines could not be suppressed. The results for IL-10, an anti-inflammatory cytokine, are summarized in Figures 22 (g) and (f). Regardless of the presence or absence of LPS, the production amount was low in all HA derivatives, and in particular, the anti-inflammatory effect of HA did not increase IL-10 production.

[0129] 2.7.3. Evaluation of the effect of suppressing inflammatory cytokine production in dendritic cells Figure 23 summarizes the amount of cytokines produced from dendritic cells when HA derivatives and LPS are added simultaneously.

[0130] As shown in Figure 23 (a), for TNF-α, similar to the results obtained in macrophages, HA significantly suppressed the production amount, while HA derivatives did not exhibit the suppressive effect. This is considered to be because when LPS was not added, Suc 17 -HA and MGlu 19 -HA induced TNF-α production. Different from the results obtained in macrophages for IL-6, Suc 17 -HA, as well as Glu 17 -HA and MGlu 19-HA production was also higher than with PBS. It is known that macrophages express more scavenger receptors than dendritic cells. Therefore, it is thought that less HA derivative was taken up, and the anti-inflammatory effect of HA was not as pronounced as in macrophages. In addition, almost no IL-10 was produced. This is thought to be because dendritic cells generally do not produce as much IL-10 as macrophages.

[0131] 2.7.4. Evaluation of the effect of suppressing inflammatory cytokine production in splenic cells The spleen is an organ rich in inflammatory cells such as macrophages and dendritic cells. Single-cell spleen cells, derived from the spleen, can be used to study the anti-inflammatory effects under conditions where diverse cells are present.

[0132] Figures 24 (a) and (c) show the production levels of TNF-α and IL-6 in splenocytes, respectively. HA suppressed production, while none of the HA derivatives suppressed it. Scavenger receptor expression is highest in macrophages, but macrophages make up only 3.5-5% of the spleen, while T cells and NK cells, which were poorly taken up in Figure 19, make up 21-35% and 1-5%, respectively. HA was equally taken up by all cells and suppressed inflammation in almost all cells, while HA derivatives mainly suppressed inflammation in macrophages, dendritic cells, and B cells. As a result, it is thought that HA was more effective overall in suppressing the production of inflammatory cyclocytokines. Furthermore, as shown in Figure 24 (b), Suc 17 -HA and MGlu 19 -HA promotes TNF-α production even in the absence of LPS, and it is thought that it could become an even more effective material if the production of inflammatory cytokines by HA itself can be suppressed. In the case of the anti-inflammatory cytokine IL-10, the results in Figure 24 (e) show that, unlike macrophages and dendritic cells, HA derivatives, especially MGlu, are effective. 19 -HA significantly induced production compared to PBS. Therefore, HA derivatives are expected to suppress inflammation by promoting the production of anti-inflammatory cytokines in vivo.

[0133] 2.7.5. ROS scavenging ability in macrophages ROS is one of the indicators of inflammation. When DCFH-DA is added to cells, it reacts with intracellular ROS to form 2',7'-dichlorodihydrofluorescein (DCF), a fluorescent molecule. The amount of intracellular ROS can be evaluated by measuring this fluorescence with a flow cytometer. The proportion of cells producing ROS is shown in Figure 25(a). While more than 50% of cells produced ROS in the PBS and HA groups, although there was no significant difference, HA derivatives reduced this proportion to about 40%. Furthermore, the amount of ROS produced in ROS-producing cells is shown in Figure 25(c). From this graph, compared with PBS, HA only reduced the production amount to about 80%, whereas Glu 17 -HA and MGlu 19 -HA reduced the production amount to about 65%, and for Suc 17 -HA, it was able to reduce the production amount to less than 50%. The result that these HA derivatives have higher intracellular ROS scavenging ability than HA is considered to be because introduction of carboxyl groups imparts endosomal escape ability as shown in Figure 17, thereby improving the intracellular ROS scavenging ability of HA. However, while Figure 17 showed that the longer the carbon chain of the carboxyl unit, the higher the endosomal escape ability, in the present experiment, Suc 17 -HA with shorter carbon chains and Glu 17 -HA scavenged ROS more effectively. This is considered to be because a shorter carbon chain gives a structure closer to that of native HA, thus maintaining the intrinsic ROS scavenging ability of HA. Glu 17 -HA is considered to have ROS scavenging ability equivalent to that of Suc 17 -HA due to the balance between endosomal escape ability and maintenance of the native HA structure.

[0134] 2.8. Inhibitory effect of inflammatory substance production due to differences in carboxyl groups Unlike Section 2.7, in this section, LPS was added to cells 1 hour before the addition of HA derivatives, to verify the therapeutic effect of adding the sample after inflammation has been induced.

[0135] 2.8.1. Evaluation of the inhibitory effect on NF-κB activation in macrophages Figure 26 shows a graph investigating the therapeutic effect of HA derivatives on NF-κB activity using RAW-Blue cells. Unlike Figure 20, which examined the preventive effect, HA significantly suppressed NF-κB activity. On the other hand, none of the HA derivatives were able to suppress it. This result suggests that HA is more likely to produce a therapeutic effect at the root cause of inflammation.

[0136] 2.8.2. Evaluation of the effect of suppressing inflammatory cytokine production in macrophages Unlike Figure 26, which showed the root cause of inflammation, Figure 27 summarizes the production levels of various cytokines. In Figure 27 (a), unlike the results for NF-κB, TNF-α production could not be significantly suppressed by HA, whereas MGlu 19 -HA production could be suppressed by up to 45%. On the other hand, in IL-6 shown in Figure 27 (c), HA was the most effective in suppressing Glu 17 -HA also showed significant suppression, though not as much as HA, but MGlu showed results with TNF-α. 19 -HA also Suc 17 -HA could not be suppressed either. In Figure 22, which shows the preventive effect, in contrast, HA derivatives could not suppress TNF-α, but Glu 17 -HA and MGlu 19 -HA production was suppressed by IL-6. 17 -HA also MGlu 19 -HA is also thought to be able to suppress inflammation in macrophages in both preventive and therapeutic ways. Regarding the anti-inflammatory cytokines in Figure 27 (e), HA was hardly produced, similar to the preventive effect, but HA derivatives, especially Suc, were produced. 17 -HA produced IL-10. Here, as shown in Figure 27 (f), PBS without the added sample also produced IL-10, just as in the case of the preventive effect. This is thought to be because inflammation is induced by LPS, and macrophages spontaneously produce IL-10 in an attempt to suppress the inflammation. Among them, Suc 17 -HA is produced significantly, and in addition to being produced spontaneously, Suc 17 -HA is thought to be having an effect.

[0137] 2.8.3. Evaluation of the effect of suppressing inflammatory cytokine production in dendritic cells Figure 28 is a graph showing the therapeutic effect on dendritic cells. Similar to Figure 23, which examined the preventive effect of dendritic cells, HA derivatives failed to suppress the production of inflammatory cytokines. Furthermore, unlike macrophages, HA did not show any therapeutic effect on dendritic cells. From this, it was found that the anti-inflammatory effect of HA is inherently less pronounced in dendritic cells. This is thought to be due to the difference in the functions of macrophages and dendritic cells. Macrophages are reversible cells that differentiate into M1-type macrophages, which have strong immune activity and inflammation, or M2-type macrophages, which have high immunosuppressive function, in response to environmental stimuli. When an inflammatory response occurs, they become M1 macrophages, but anti-inflammatory materials make them more likely to differentiate into M2 macrophages, reducing the production of inflammatory cytokines. On the other hand, dendritic cells are cells that give instructions to surrounding inflammatory cells by producing cytokines, and the effect of anti-inflammatory materials depends on the maturity of the dendritic cells. In fact, dendritic cells that were matured by LPS treatment showed less anti-inflammatory effect from corticosteroids than dendritic cells before treatment. Therefore, it is thought that there were differences in how the anti-inflammatory effect of HA manifested itself. From Figure 28 (c), it was found that, similar to the preventive effect, IL-10 was less likely to be produced from dendritic cells.

[0138] 2.8.4. Evaluation of the effect of suppressing inflammatory cytokine production in splenic cells Figure 29 shows the results of examining the cytokine production levels of spleen cells to investigate the therapeutic effects of HA derivatives.

[0139] Similar to the results for the preventive effect, neither HA derivative could suppress the production of either inflammatory cytokine. Furthermore, while HA did reduce production, the reduction was not significant. This suggests that the low anti-inflammatory effect of HA was the reason why the HA derivative also failed to suppress inflammation. Additionally, the trend in IL-10 production was exactly the same as in the preventive effect, and MGlu 19 -HA is the most produced, followed by Glu 17 -HA, Suc 17 -HA continued.

[0140] 2.9. Inhibitory effect of high-concentration HA derivatives on the production of inflammatory substances 2.9.1. Evaluation of the inhibitory effect of high-concentration HA derivatives on the production of inflammatory cytokines in macrophages. The introduction of a carboxyl group unit improved the water solubility of the HA derivative, increasing the amount that could be used. Therefore, we hypothesized that increasing the concentration of the HA derivative and allowing it to interact with more CD44 could lead to a greater anti-inflammatory effect.

[0141] Figure 30 summarizes the cytokine production levels of macrophages treated with high-concentration HA. Figure 30 (a) shows that, contrary to expectations, TNF-α was produced in greater quantities than with PBS. Figure 30 (b) shows the TNF-α production level when LPS was not added, and Suc 17 -HA and MGlu 19 -HA shows that, in addition to the influence of LPS, the pro-inflammatory effect of the sample itself plays a significant role. On the other hand, in the case of IL-6, as shown in Figure 30 (c), Glu 17 -HA and MGlu 19 -HA production could be suppressed to approximately 45% and 51% by increasing its concentration, and this was not different from that of HA. As can be seen in Figure 30 (d), the sample itself showed almost no inflammatory effect. Suc 17 - While the production of HA actually increased, the production of IL-10 was higher compared to other HA derivatives.

[0142] 2.9.2. Evaluation of the inhibitory effect of high-concentration HA derivatives on the production of inflammatory cytokines in spleen cells. The spleen was similarly treated with high concentrations of HA derivatives. The results are shown in Figure 31. From Figure 31 (a), neither HA nor HA derivatives could suppress TNF-α production. Nevertheless, as with Figure 24 (a), among the HA derivatives, Glu 17 -HA suppressed production the most. Also, as in Figure 30 (a), increasing the sample concentration allowed Suc 17 -HA and MGlu 19 -The inflammatory effect of HA itself became more pronounced. Figure 31 (c) shows that all HA derivatives significantly suppressed Il-6 production. In particular, MGlu 19 -HA reduced production more than HA.19 -HA is expected to suppress inflammation more effectively than HA in in vivo experiments. On the other hand, when the concentration of HA derivatives was increased, IL-10 levels became almost the same as those of PBS and HA. MGlu 19 -HA alone significantly increased the production of anti-inflammatory cytokines compared to HA alone.

[0143] 2.9.3. Evaluation of the inhibitory effect of high-concentration HA derivatives on the production of inflammatory cytokines in spleen cells. Similarly, high concentrations of HA derivatives were applied to assess therapeutic effects. The results are shown in Figure 32. As can be seen in Figure 32 (a), neither HA nor HA derivatives suppressed TNF-α production, similar to the preventive effect. Nevertheless, as with Figures 29 (a) and 31 (a), among the HA derivatives, Glu... 17 -HA was the most effective at suppressing production, and it suppressed production even more effectively than HA. As shown in Figure 32 (b), all HA derivatives significantly suppressed Il-6 production. Also, similar to Figure 31 (b), MGlu 19 -HA produced less IL-10 than HA. Furthermore, as shown in Figure 31 (c), when the concentration of the HA derivative was increased, the amount of IL-10 produced was the same as that of PBS and HA.

[0144] 2.10. Observation of HA derivative leakage from the administration site HA aqueous solution is more viscous than HA derivatives and is thought to be less likely to flow throughout the body from the injection site, while HA derivatives with improved water solubility are thought to be more likely to flow into other tissues from the injection site. Therefore, fluorescently labeled HA derivatives were administered and observed over time using an in vivo imaging device to compare the outflow of samples from the injection site. Suc-HA was excluded here because its inhibitory effect on inflammatory cytokines was not effective in cell experiments. Also, since there were limitations on the dosage when administering to the foot, which is closest to the arthritis site, subcutaneous injection into the tail was chosen to allow for a larger dose, considering actual treatment experiments.

[0145] Figure 33 shows imaging images taken 3 hours, 8 hours, and 120 hours after subcutaneous administration of HA derivatives via the tail. At 3 hours, strong fluorescence was observed from the administration site of HA. Glu-HA showed a darker and smaller spot than HA, indicating that it did not remain in the tail as much as HA. MGlu-HA had already disappeared from the tail at 3 hours, and weak fluorescence was observed in the rump. At 8 hours, HA still showed bright rhodamine fluorescence compared to the HA derivative. On the other hand, Glu-HA had a smaller glowing area and lower fluorescence intensity. Similar to 3 hours, no fluorescence was observed in the tail of MGlu-HA, and although the base of the tail glowed brightly, it was a slightly darker red, and the glowing area was smaller, suggesting that it was gradually spreading throughout the body. Furthermore, at 120 hours, no fluorescence from Glu-HA or MGlu-HA was observed in either the tail or the rump. The HA also changed to a darker red compared to 8 hours later, and the area of ​​light was smaller, indicating that it had moved away from the tail, but it did not completely disappear. This suggests that the HA derivative, whose water solubility was improved by the introduction of a carboxyl group, is easily washed away from the administration site.

[0146] Figure 34 summarizes the product of the average fluorescence intensity and area in the areas of the tail that glowed in the imaging images. Similar to Figure 34, HA showed the highest value at 3 hours after administration, decreased rapidly until 24 hours later, but the rate of decrease became more gradual after 48 hours. Glu-HA showed a similar trend, but its value was lower than HA at 3 hours after administration and never exceeded HA at any time. On the other hand, no areas of fluorescence were observed in the tail of MGlu-HA at 3 hours after administration. This result is thought to be related to the improved water solubility due to the introduction of the carboxyl group. As shown in Figure 9, all HA derivatives showed higher fluidity than HA. Also, Glu-HA became almost gel-like at a concentration of 25 mg / mL. On the other hand, MGlu-HA at 25 mg / mL had almost the same viscosity as MGlu-HA at 5 mg / mL. This suggests that MGlu-HA was able to improve viscosity more than Glu-HA, and as a result, the sample flowed more easily from the administration site.

[0147] Furthermore, to investigate where the HA derivative leaked from the tail and was distributed, organs were collected 3 hours after administration and observed using a bioimaging device. The results are shown in Figure 35. However, no rhodamine-derived fluorescence was detected in any organ, and it was below the detection limit (at the same level as autofluorescence from tissue). The liver and kidneys are organs involved in drug clearance, and the absence of fluorescence from these organs suggests that the drug is not rapidly excreted from the body.

[0148] 2.11. Evaluation using a collagen antibody-induced arthritis (CAIA) model mouse To investigate whether HA derivatives are more effective than HA in vivo, we examined their therapeutic effects in a mouse model of arthritis, a representative autoimmune disease. Collagen-induced arthritis (CIA) is widely used as a mouse model for rheumatoid arthritis. However, a typical study in the CIA model takes at least 6-8 weeks to complete. To shorten this timeline, a collagen antibody-induced arthritis (CAIA) model was developed using monoclonal antibodies against collagen antibodies, which can induce arthritis within a few days. This model has many advantages, including the ability to use various mice other than CIA-sensitive mice and the ability to rapidly screen and evaluate anti-inflammatory compounds. Therefore, in this experiment, we used CAIA model mice to evaluate therapeutic effects.

[0149] 2.11.1. Creation of CAIA model mice First, experiments were conducted using only PBS and HA to create CAIA model mice and to confirm whether HA could suppress inflammation. The results are shown in Figure 36. The horizontal axis represents the number of days, and the purple squares represent the sample administration days. The vertical axis represents the clinical score value, which indicates the degree of swelling of the finger joints and the top of the foot; a higher score indicates greater swelling of the foot and more severe symptoms. In both cases, the score began to rise from Day 4 and continued to rise similarly until Day 7, the day of the second administration. From there, the score for PBS continued to rise until Day 13, but the score for HA leveled off as the therapeutic effect became apparent. On Day 13, the final day of observation, the average score for PBS was 8.5, while for HA it remained at 5. From this, the dosage and method of sample administration that can create CAIA model mice and confirm the therapeutic effect of HA were determined.

[0150] 2.11.2. Evaluation of therapeutic effects in CAIA model mice based on differences in carboxyl groups The therapeutic effects of HA derivatives in arthritis model mice were investigated according to the scheme determined in 2.11.1. Figure 37 shows HA or Suc. 17 -HA, Glu 19 -HA, MGlu 16-This shows the changes in the clinical score of HA over time and photographs of the foot condition. Overall, the score began to rise around Day 4 or 5. For PBS, the score continued to rise until around Day 9, then leveled off, with an average score of 6 on the final day, Day 12. The photographs show swelling in the instep and toe joints, indicating a severe condition. The increase in the HA score was gradual, with a score of 5 on Day 12. The photographs show swelling in the toe joints, but compared to PBS, the instep was thinner and normal. Suc 17 -HA's score continued to rise, similar to PBS, reaching 7.5 on Day 12. The top of her foot was also significantly swollen, and her toe joints were red and swollen. Suc 17 -HA was hardly able to suppress the production of inflammatory cytokines, and was equivalent to PBS, so it is thought that it could not suppress them in in vivo experiments either. On the other hand, Glu 19 -HA showed almost no improvement in scores except for one mouse, and although the average score on Day 12 was 4.5, most mice had scores of 4 or less, demonstrating a very strong therapeutic effect. The photographs also showed them to be virtually indistinguishable from normal mice. MGlu 19 -HA showed a continuous increase in scores in some mice, similar to PBS, but other mice maintained low scores. Furthermore, unlike other samples, the score began to decline around Day 9 towards the end. 19 -HA was suggested to show therapeutic effects later in the treatment.

[0151] To elucidate the reasons for the changes in clinical scores over time as shown in Figure 37, the spleen, which contains many cells involved in inflammation, was collected, immunostained, and measured using a flow cytometer to investigate the proportion of inflammatory cells distributed within the spleen. The results are summarized in Figure 38. From Figure 38 (a), the proportion of M1 macrophages with high inflammatory function to M2 macrophages with high anti-inflammatory function was high with PBS, Suc-HA, and Glu-HA, while it was low with HA and MGlu-HA. This suggests that HA and MGlu-HA effectively differentiate macrophages into anti-inflammatory types. Figure 38 (b) shows the proportion of neutrophils, which are abundant at the site of inflammation, and Glu-HA and MGlu-HA showed lower values ​​than HA. This suggests that Glu-HA and MGlu-HA suppress neutrophil migration and thus suppress inflammation. Furthermore, Glu-HA, which showed the highest therapeutic effect, had the largest value in Figure 38 (c). This graph shows the proportion of regulatory T cells, which suppress inflammation, among the T cells. It is thought that the high therapeutic effect of Glu-HA was due to these regulatory T cells instructing many inflammatory cells to suppress their activity. On the other hand, Figures 38 (d) and (e) show the proportion of regulatory B cells among B cells. In both graphs, MGlu-HA showed a very high value compared to the other samples. In Figures 24 (e) and 29 (c), MGlu-HA produced significantly more IL-10 than the other samples. It is also known that regulatory B cells in the mouse spleen require IL-10 for their function. From these points, it can be concluded that MGlu-HA, which produces a large amount of IL-10, is effective. 19 -HA is thought to have increased the proportion of regulatory B cells.

[0152] Furthermore, to compare Glu-HA, which exhibited a high therapeutic effect, and MGlu-HA, for which therapeutic effects were observed starting from the latter half of the experiment, the popliteal lymph nodes, which are the regional lymph nodes for arthritis, were collected, and the amount of cytokine production in the lymph nodes, which is the cause of inflammation, was examined. The levels of the cytokines IL-6 and IL-10 in the popliteal lymph nodes at Day 5 and Day 13 are shown in Figure 39. Whereas neither IL-10 nor IL-6 was produced in any sample on Day 5, on Day 13 MGlu-HA resulted in a higher amount of IL-10 production than the other samples, and further suppressed IL-6 production. These results indicate that therapeutic effects of MGlu-HA can be expected in cases of chronic inflammation where inflammation persists over a long period of time.

[0153] 2.11.3. Evaluation of therapeutic effects in CAIA model mice based on differences in carboxyl group introduction rates From the results shown in Figures 13 and 14, it was found that in vitro, the lower the introduction rate of carboxy groups, the greater the inhibition of LPS-induced inflammation. However, it was considered that in vivo, cell selectivity contributes more greatly to the effect, and the higher the introduction rate of carboxy groups, the higher the therapeutic effect. Therefore, a similar experiment was conducted using Glu-HA with carboxy group unit Glu group introduction rates of 19% and 27%, which exhibited the highest therapeutic effect in Figure 37. The results of the clinical score obtained in this experiment are shown in Figure 40. Glu 19 -HA maintained an overall low score of 8 or lower, while Glu 27 -HA, although only one out of five mice was completely cured, exhibited high scores in most of the mice. These results suggest that a lower introduction rate of carboxy groups maintains the structure of HA and can exert an anti-inflammatory effect.

Claims

1. An anti-inflammatory agent containing a carboxyl group-introduced high molecular weight hyaluronic acid derivative.

2. The anti-inflammatory agent according to claim 1, wherein the weight-average molecular weight of the carboxyl group-introduced high molecular weight hyaluronic acid derivative is 200,000 or more.

3. The anti-inflammatory agent according to claim 1, wherein the molecular weight of the carboxyl group-introduced high molecular weight hyaluronic acid derivative is 700,000 or more and 10,000,000 or less.

4. The carboxyl group-introduced high molecular weight hyaluronic acid derivative has the general formula (1): 【Chemistry 1】 [In the formula: R 1 [The symbols represent monovalent groups containing a hydrogen atom or a carboxyl group, either identical or distinct. n represents a natural number.] The anti-inflammatory agent according to claim 1, comprising at least one selected from the group consisting of compounds represented by, salts thereof, and solvates thereof.

5. The aforementioned monovalent group is given by the general formula (2): -C(=O)-R 11 -COOH (in the formula: R 11 The anti-inflammatory agent according to claim 4, wherein the group is represented by (a single bond or a linker).

6. The anti-inflammatory agent according to claim 5, wherein the linker is an alkylene group.

7. The anti-inflammatory agent according to claim 4, wherein the carboxyl group introduction rate in the carboxyl group-introduced high molecular weight hyaluronic acid derivative is 50% or less.

8. The anti-inflammatory agent according to claim 4, wherein the carboxyl group introduction rate in the carboxyl group-introduced high molecular weight hyaluronic acid derivative is 5% or more and 30% or less.

9. An anti-inflammatory agent according to any one of claims 1 to 8, which is a pharmaceutical product.

10. An IL-10 production promoter containing a carboxyl group-introduced high molecular weight hyaluronic acid derivative.

11. A carboxyl group-introduced high molecular weight hyaluronic acid derivative with a weight-average molecular weight of 200,000 or more.

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