Composition containing poly (organophosphorus nitrile) polymer for preventing or treating inflammatory diseases

Abstract

The present invention relates to a drug-loaded temperature-sensitive poly (organophosphorus nitrile) polymer and a pharmaceutical composition for preventing or treating inflammatory diseases comprising the same, an injectable polymer nanoparticle hydrogel system comprising the hydrogel having a long-acting anti-inflammatory effect, capable of slowly releasing a therapeutically effective concentration of a drug, and capable of preventing or treating inflammatory diseases. The pharmaceutical composition can be used for preventing and treating various inflammatory diseases such as osteoarthritis and the like.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating inflammatory diseases containing a poly(organophosphazene) polymer. Background Art

[0002] Osteoarthritis (OA) is the most common degenerative joint disease. Degeneration and inflammation of the joints lead to chronic pain, stiffness, and progressive loss of articular cartilage, which is unable to repair itself. In the late stages of OA, prostheses can replace the degenerated joints, but prostheses have limitations and may require further joint replacement surgery. Other treatment strategies such as intra-articular (IA) administration of autologous chondrocytes, platelet-rich plasma, or supplementation of damaged cartilage with viscous substances such as hyaluronic acid (HA) are alternative solutions to regenerate damaged articular cartilage. However, these approaches have unsatisfactory results in alleviating OA due to poor target specificity and in vivo maintenance after intra-articular (IA) administration. Drug treatment includes oral analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), specific cyclooxygenase (COX)-2 inhibitors, and opioids, but these treatments are limited to symptomatic pain treatment and have little effect on local inflammation [Buchman AL Side effects of corticosteroid therapy. J. Clin. Gastroenterol. 2001; 33(4): 289–294.]. In addition, NSAIDs, cyclooxygenase (COX)-2 inhibitors, and opioids can also cause serious side effects on the gastrointestinal (GI) system, heart, and brain. Therefore, these oral medications should be used with caution in elderly people with underlying diseases of the GI system, heart, and brain. In order to increase the bioavailability of the drug at the target site and reduce possible systemic side effects, corticosteroids such as methotrexate, diclofenac, and triamcinolone acetonide (TCA) can be directly administered intra-articularly (IA). However, it is known that the half-lives of these drugs are 0.56 to 2.9, 5.2, and 1.47 hours, respectively, and they disappear rapidly from the body within their half-lives [Larsen C., Ostergaard J., Larsen SW, Jensen H., Jacobsen S., Lindegaard C., Andersen PH. Intra-articular depot formulation principles: role in the management of postoperative pain and arthritic disorders. J. Pharmacol. Sci. 2008; 97(11): 4622–4654. et al.].Because the drug does not stay in the body long enough, it cannot reduce the ongoing inflammatory response.

[0003] For these reasons, new approaches to the definitive treatment and early prevention of osteoarthritis are being investigated. Potential new osteoarthritis (OA) treatments using anti-osteoarthritis (OA) drug delivery systems with long-term activity in vivo include liposomes, nanoparticles, microparticles, and hydrogels. These new treatments have been found to release anti-osteoarthritis (OA) drugs for weeks to months, significantly extending the release time [Pradal J., Zuluaga MF, Maudens P., Waldburger JM, Seemayer CA, Doelker E., Gabay C., Jordan O., Allemann E. Intra-articular bioactivity of p38 MAPK inhibitor and development of an extended-release system. Eur. J. Pharm. Biopharm. 2015; 93: 110–117. et al.]. However, it has been further discovered that these systems are not suitable for precise drug delivery to the target site and have low release efficiency [Kopecek J. Hydrogel biomaterials: A smart future? (Hydrogel biomaterials: a smart future?), Biomaterials. 2007; 28(34):5185–5192.].

[0004] On the other hand, the advantages of hydrogel-based drug delivery systems are that they can prolong the drug release period and provide a local drug reservoir that can provide a three-dimensional (3D) physical support space similar to the synovial joint space. These properties can reduce physical friction in inflammatory arthritis, thereby further reducing synovial joint damage. In addition, the temperature-sensitive sol-gel transition property has the advantage of non-invasive drug delivery. However, the limitation of hydrogel-based anti-osteoarthritis drug delivery systems is that they only show drug release characteristics for a few days [Park CW, Ma KW, Jang SW, Son M., Kang M.J. Comparison of piroxicam pharmacokinetics and anti-inflammatory effect in rats after intra-articular and intramuscular administration. Biomol. Ther. (Seoul) 2014; 22(3): 260–266.].

[0005] Hybrid systems of nanoparticle drug carriers and 3D hydrogel drug carriers can be effective tools for the successful treatment of osteoarthritis (OA). Among them, poly(organophosphazenes) with substituted hydrophobic and hydrophilic side chains can be used as biodegradable drug carriers, which can form injectable in situ hydrogels, load drugs, and have the characteristics of long-term drug release [Hong KH, Kim YM, Song SC Fine-tunable and injectable 3D hydrogel for on-demand stem cell niche. Adv. Sci. 2019; 6(17): 1900597. doi: 10.1002 / advs.201900597.]. In aqueous solution, these polymers exist as extended chains or nanosized spherical particles according to the change of hydrophobicity based on temperature changes. Based on these properties, various drugs with hydrophobic moieties can be encapsulated in polymeric nanoparticles (PNs) by participating in the self-assembly process of polymeric nanoparticles (PNs). In addition, polymeric nanoparticles can form 3D hydrogel networks above a certain concentration.

[0006] The present inventors continued their research to achieve sustained local delivery of TCA using a poly(organophosphazene)-based PN hydrogel system, thereby completing the present invention. Summary of the Invention

[0007] Problems to be solved by the invention

[0008] The technical problem to be solved by the present invention is to provide a composition containing a poly(organophosphazene) polymer for preventing or treating inflammatory diseases.

[0009] However, the technical problems to be solved by the present invention are not limited to the above problems, and other problems not mentioned will be clearly understood by those skilled in the art through the following description.

[0010] Solutions for solving problems

[0011] In order to solve the above technical problems, a composition for preventing or treating inflammatory diseases is provided.

[0012] Include:

[0013] Polyphosphazene polymers; and

[0014] A therapeutically effective amount of a drug for controlling and treating inflammation,

[0015] The polyphosphazene polymer comprises the following components on the phosphorus atoms of the polyphosphazene main chain represented by the following chemical formula 1:

[0016] A first moiety, which is an amino acid ester represented by the following Chemical Formula 2;

[0017] a second moiety, which is polyethylene glycol represented by the following Chemical Formula 3; and

[0018] a third moiety comprising aminoethanol and an organic acid,

[0019] [Chemical Formula 1]

[0020]

[0021] [Chemical Formula 2]

[0022]

[0023] [Chemical Formula 3]

[0024]

[0025] In the chemical formula,

[0026] R1 is C 1-6 Alkyl, C 1-6 Alkenyl or C 6-10 Aryl-C 1-6 alkyl;

[0027] R2 is hydrogen, methyl, isopropyl, 1-methylpropyl, 2-methylpropyl, thiomethyl, methylthioethyl, benzyl, hydroxybenzyl or 2-indolylmethyl;

[0028] R3 is C 1-6 alkyl;

[0029] n is an integer from 3 to 100,000,

[0030] p is an integer from 1 to 20,

[0031] The molar ratio of a:b is 5.5:1 to 7.5:1,

[0032] a is 70 to 80 mol%.

[0033] According to one aspect, the organic acid in the third portion comprises glutaric acid.

[0034] According to one aspect, R1 is methyl, ethyl, propyl, butyl, benzyl or 2-propenyl.

[0035] According to one aspect, R3 is methyl.

[0036] According to one aspect, the drug for treating an inflammatory disease is hydrophobic.

[0037] According to one aspect, the hydrophobic drug for treating inflammatory diseases is at least any one selected from the group consisting of triamcinolone acetonide, methylprednisolone, dexamethasone, celecoxib, ibuprofen, naproxen, indomethacin, ketoprofen, etodolac, meloxicam, rofecoxib, etoricoxib, valdecoxib, lumiracoxib and diclofenac.

[0038] According to one aspect, the polymer slowly releases the drug for treating inflammatory diseases.

[0039] According to one aspect, the polyphosphazene polymer is dissolved in a solvent at a concentration of 1% to 50% by weight. The solvent is at least one selected from the group consisting of water, a buffer solution, an acidic solution, an alkaline solution, a saline solution, physiological saline, water for injection, a cell culture medium, and glucose saline, but is not limited thereto.

[0040] According to one aspect, the polymer exhibits a sol-gel transition behavior in the range of 5° C. to 70° C. and forms a hydrogel at a predetermined set temperature.

[0041] According to one aspect, the polymer gels when applied to an in vivo or in vitro environment and loses temperature sensitivity at a predetermined set temperature, thereby maintaining a gel state regardless of temperature.

[0042] According to one aspect, the inflammatory disease is any one selected from the group consisting of osteoarthritis, degenerative arthritis, rheumatoid arthritis, osteoporosis and Achilles tendinitis.

[0043] Effects of the Invention

[0044] The present invention relates to a drug-loaded temperature-sensitive poly(organophosphazene) polymer, a hydrogel containing the same, and a composition containing the same for preventing or treating inflammatory diseases. The injectable polymer nanoparticle hydrogel system containing the hydrogel can maintain an anti-inflammatory effect in the body for a long time, effectively regenerate the Achilles tendon including tendons, ligaments, muscles, cells, etc., and can be used to prevent and treat various inflammatory diseases such as osteoarthritis.

[0045] Furthermore, the present inventors have optimized the ratio of hydrophobic side chains to hydrophilic side chains constituting the poly(organophosphazene) polymer, thereby exhibiting ideal drug release characteristics in vivo and enabling administration to the body at a constant concentration for a long period of time.

[0046] The effects of the present invention are not limited thereto, and those skilled in the art can clearly understand other effects not mentioned from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 A schematic diagram showing the mode of action of TCA-loaded poly(organophosphazene) nanoparticles.

[0048] Figure 2 shows the poly(organophosphazene) 1 H NMR data.

[0049] Figure 3 Shown are the results of biocompatibility experiments of poly(organophosphazene).

[0050] Figure 4 Cytotoxicity data for carboxyl-terminated poly(organophosphazenes) are shown.

[0051] Figure 5 Characterization of poly(organophosphazene) nanoparticles is shown. (a) shows the structure and self-assembly behavior of poly(organophosphazene) nanoparticles; (b), (c), and (d) are TEM images of self-assembled PN, TCA aggregates, and TePN, respectively; (e) shows the hydrodynamic diameters of self-assembled PN, TCA aggregates, and TePN in PBS solution (n = 6).

[0052] Figure 6 This is a TEM image of micron-sized TCA aggregates.

[0053] Figure 7Shown are the hydrodynamic diameters of self-assembled PN, TCA, and TePN in PBS solution.

[0054] Figure 8 Figure 2 shows the characterization of the TePN hydrogel system. (a) shows the sol-gel transition and viscosity change with temperature; (b) shows the storage modulus and loss modulus of the PN solution with temperature; and (c) shows the storage modulus and loss modulus of the TePN solution with temperature and TCA content.

[0055] Figure 9 The biodegradability of PN hydrogel is shown, where (a) is a picture of a mouse experiment; (b) is a graph of hydrogel decomposition over time.

[0056] Figure 10 Pictures of the hydrogel at the injection site on different days are shown.

[0057] Figure 11 Predicted values of hydrogel volume from in vitro mass loss assays are shown.

[0058] Figure 12 The sustained release curve of TCA in TePN is shown, where (a) is the hydrogel morphology of TePN changing with time; (b) is the cumulative TCA release amount.

[0059] Figure 13 Shown are images of the cartilage 8 weeks after treatment.

[0060] Figure 14 Figure 3 shows the in vivo anti-inflammatory efficiency characteristics of TePN after IA injection, where (a) is X-ray and CT images; (b) is articular cartilage distance.

[0061] Figure 15 The in vivo anti-inflammatory efficiency of the TePN hydrogel system was demonstrated using tissue staining.

[0062] Figure 16 is real-time PCR analysis of anti-inflammatory cytokine expression.

[0063] Figure 17 The body weight changes of rats in each group are shown.

[0064] Figure 18 NMR data of the prepared PPZ are shown.

[0065] Figure 19 Shown are the changes in physical properties as the IleOEt / AMPEG molar ratio changes.

[0066] Figure 20 The expression levels of inflammation-related factors based on TCA concentrations are shown.

[0067] Figure 21 The confirmation process of the optimal IleOEt / AMPEG molar ratio is shown.

[0068] Figure 22 This is a schematic diagram of the process of administering the CXB-PPZ complex to AT.

[0069] Figure 23 Showing the PNP synthesis structure 1 H NMR and GPC results.

[0070] Figure 24 FT-IR results of PCNP, CXB, and PNP are shown.

[0071] Figure 25 CXB encapsulation and temperature-sensitive gelation process are shown.

[0072] Figure 26 Shown is the gelation process of PNP hydrogel.

[0073] Figure 27 The rheological properties of PNP and PCNP hydrogels are shown.

[0074] Figure 28 The characteristics of PNP and PCNP are shown.

[0075] Figure 29 Graph showing sustained release of CXB from PCNP hydrogels and maintenance of the hydrogel network for approximately one month.

[0076] Figure 30 In vitro disintegration of PNP hydrogels was shown.

[0077] Figure 31 Shows the stable modulus of PNP hydrogel at 37 degrees.

[0078] Figure 32 The in vivo biodegradation of PNP hydrogels was demonstrated.

[0079] Figure 33 Shown are the results of cytotoxicity experiments of PNP hydrogels by MTT assay.

[0080] Figure 34 Shown are the expression levels of inflammation-related factors as shown by real-time PCR analysis results of blood samples.

[0081] Figure 35 The anti-inflammatory effect of the long-term PCNP-releasing hydrogel system was demonstrated, which was confirmed by the regeneration of damaged Achilles tendons.

[0082] Figure 36The hydroxyproline content, stiffness, and tensile strength in the control, untreated, CXB solution-treated, and PCNP-treated groups are shown. DETAILED DESCRIPTION

[0083] To solve the above technical problems, the present inventors provide the following poly(organophosphazene) polymers and compositions containing the same for preventing or treating inflammatory diseases.

[0084] It is a composition for preventing or treating inflammatory diseases.

[0085] Include:

[0086] Polyphosphazene polymers; and

[0087] A therapeutically effective amount of a drug for controlling and treating inflammation,

[0088] The polyphosphazene polymer comprises the following components on the phosphorus atoms of the polyphosphazene main chain represented by the following chemical formula 1:

[0089] A first moiety, which is an amino acid ester represented by the following Chemical Formula 2;

[0090] a second moiety, which is polyethylene glycol represented by the following Chemical Formula 3; and

[0091] a third moiety comprising aminoethanol and an organic acid,

[0092] [Chemical Formula 1]

[0093]

[0094] [Chemical Formula 2]

[0095]

[0096] [Chemical Formula 3]

[0097]

[0098] In the chemical formula,

[0099] R1 is C 1-6 Alkyl, C 1-6 Alkenyl or C 6-10 Aryl-C 1-6 alkyl;

[0100] R2 is hydrogen, methyl, isopropyl, 1-methylpropyl, 2-methylpropyl, thiomethyl, methylthioethyl, benzyl, hydroxybenzyl or 2-indolylmethyl;

[0101] R3 is C1-6 alkyl;

[0102] n is an integer from 3 to 100,000,

[0103] p is an integer from 1 to 20,

[0104] The molar ratio of a:b is 5.5:1 to 7.5:1,

[0105] a is 70 to 80 mol%.

[0106] Generally, temperature-sensitive polymers are polymers whose solubility changes suddenly with temperature. As the temperature rises, the hydrogen bonds between the polymer and the solvent weaken and dehydration occurs, while the hydrophobic attraction between the polymers increases to produce a further hydrophobic structure. At the low critical solution temperature (LCST), polymer-polymer interactions and water-water interactions are more preferred than hydrogen bonding between the polymer and water, and therefore, the polymer is rapidly dehydrated to have a further hydrophobic structure. The low critical solution temperature (LCST) of temperature-sensitive polymers varies with the balance between the hydrophobic and hydrophilic groups bound to the polymer backbone. Generally, when the content of the hydrophilic group increases, the phase transition temperature increases, and when the content of the hydrophobic group increases, the phase transition temperature decreases. This feature is shown in the T of Example 9, which varies according to the ratio of the hydrophobic side chain to the hydrophilic side chain. max It can be confirmed in.

[0107] When the essential components of the poly(organophosphazene) polymer are in a predetermined ratio, the polymer solution undergoes a reversible sol-gel transition below a certain temperature. However, upon exposure to temperatures above a certain temperature, the rapid reversible transition is lost, and the gel remains in a gel-like state for a long time. This property change is specifically influenced by the ratio of substituents introduced during the initial polymer synthesis.

[0108] Especially, in the present invention, confirm that when described first portion and described second portion are within the scope of specific mol ratio, can realize slow release (referring to embodiment 9 below) when maintaining required optimal drug concentration in vivo.The basis of above-mentioned effect is this discovery that the hydrophobicity and phase transition temperature of the polymer that the present invention finds can change according to the ratio of constituent of hydrophobicity described first portion and hydrophilic described second portion.Along with the increase of the mol ratio of hydrophobic side chain, polymer has hydrophobicity gradually, thereby this strengthens the interaction with hydrophobic drug and allows less medicine to be released into body.Therefore, can control the drug release rate in body by regulating the mol ratio of hydrophobic side chain and hydrophilic side chain.

[0109] Both steroid inflammatory disease treatment drugs including triamcinolone acetonide (TCA) and non-steroidal drugs including celecoxib have a concentration range that is most suitable for their effects in the body, such as Figure 20 As shown in the results, when TCA exceeds a specific concentration range, it will increase the expression of inflammation-related factors.

[0110] As the present inventors have shown in the following Examples 9 and Figure 21 As shown in , the molar ratio of hydrophobic side chains to hydrophilic side chains is preferably in the range of 5.5:1 to 7.5:1, more preferably 6:1 to 7:1, and most preferably 6.13:1. Within this molar ratio range, the drug, preferably TCA, can be sustained-released within the above-mentioned appropriate concentration range. When the molar ratio exceeds this range, the pharmacological effect of TCA is reduced.

[0111] According to one aspect, the organic acid in the third part may include glutaric acid. The glutaric acid refers to the acid disclosed in Example 10-2 below. It is prepared according to the preparation method of Example 10-2, and its final product can be glutaric acid connected by a hydroxyl functional group and an ester bond.

[0112] Part or all of the functional groups of the third part may further include a fourth part, wherein the fourth part is formed by directly or through a linker combining the functional part, and the functional part is at least one selected from the group consisting of a substance capable of regulating the decomposition rate of the polymer, a substituent containing an ionic group capable of regulating the decomposition rate, a cross-linkable substituent, an additional compound that can induce tissue adhesion, a biologically active substance, and a composite substance formed by linearly connecting two or more functional substances therein.

[0113] At this time, the fourth part can include at least one selected from the group consisting of folic acid, hyaluronic acid, cyclodextrin, imidazole compounds, anticancer agents, histidine, lysine, arginine, cysteine, thioarylamine, spermine, spermidine, polyethyleneimine, polyhistidine, polylysine, polyarginine, protamine, heparin, chitosan and a peptide consisting of 1 to 20 amino acids.

[0114] According to one aspect, R1 is methyl, ethyl, propyl, butyl, benzyl or 2-propenyl.

[0115] According to one aspect, R3 is methyl.

[0116] According to one aspect, the drug for treating inflammatory diseases is hydrophobic and may further contain a functional substance.

[0117] The functional substance can be preosteoblast, chondrocyte, osteoblast, adult stem cell, Schwann cell, oligodendrocyte, insulin, oxytocin, vasopressin, adrenocorticotropic hormone, fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, transforming growth factor, brain nerve growth factor, antibiotics and angiogenesis inhibitors such as integrin α-5-β-1 antagonists or a combination thereof.

[0118] The hydrophobic drug for treating osteoarthritis may be at least one selected from the group consisting of triamcinolone, methylprednisolone, dexamethasone, ibuprofen, naproxen and diclofenac, with triamcinolone being most preferred.

[0119] According to one aspect, the polymer can slowly release the drug for treating osteoarthritis. As used herein, "slow release" refers to releasing the drug while maintaining it in the body for at least 21 days. Preferably, the released drug concentration is maintained at a therapeutically effective concentration. Alternatively, the sustained release can be achieved by releasing the drug at a concentration within a therapeutically effective range, preferably between 9.375 μg / ml and 75 μg / ml, and more preferably between 10 μg / ml and 50 μg / ml.

[0120] According to one aspect, the polyphosphazene polymer can be dissolved and contained in a solvent at a concentration of 1% to 50% by weight. The solvent can be at least one selected from the group consisting of water, a buffer solution, an acidic solution, an alkaline solution, a saline solution, physiological saline, water for injection, a cell culture medium, and glucose saline, but embodiments are not limited thereto. The polyphosphazene polymer of the present invention can hydrogel in the solvent and exhibit the following sol-gel transition behavior.

[0121] According to one aspect, the polymer exhibits a sol-gel transition behavior in the range of 5° C. to 70° C. and can form a hydrogel at a predetermined set temperature.

[0122] According to one aspect, when the polymer is applied to a living body or an in vitro environment, the polymer can gel, but loses temperature sensitivity at a predetermined temperature, thereby maintaining the gel state regardless of temperature.

[0123] The inflammatory disease is preferably any one selected from the group consisting of osteoarthritis, degenerative arthritis, rheumatoid arthritis, osteoporosis and Achilles tendinitis, and most preferably osteoarthritis.

[0124] The compositions of the present invention are used for the prevention and / or treatment of inflammatory diseases, preferably osteoarthritis. For preventive use, the compositions of the present invention can be administered to individuals suspected of having or at risk of having a disease described herein. For therapeutic use, the pharmaceutical compositions of the present invention can be administered to individuals, such as patients, who are already suffering from a disease described herein, in an amount sufficient to treat or at least partially prevent the disease or symptoms described herein. The effective amount will depend on the severity and course of the disease or symptoms, previous treatments, the individual's health and responsiveness to medications, and the diagnosis of a physician or veterinarian.

[0125] The composition can be formulated into any formulation selected from the group consisting of: sterile aqueous solutions, non-aqueous solvents, suspensions, tablets, pills, powders, granules, capsules, solutions for internal use, emulsions, syrups, lyophilizates, and suppositories, and can also be prepared into various parenteral or oral formulations. Regarding formulations, diluents or excipients commonly used in the art, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, can be used. Orally administered solid formulations can include tablets, pills, powders, granules, capsules, and the like, and can be prepared by mixing at least one compound with at least one excipient such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Orally administered liquid formulations can be suspensions, internal formulations, emulsions, syrups, or the like, and can include various excipients such as wetting agents, sweeteners, flavorings, and preservatives in addition to simple, common diluents such as water and liquid paraffin. Preparations for parenteral administration can include sterile aqueous solutions, non-aqueous solvents, suspending agents, emulsions, lyophilized agents, suppositories, etc. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate or the like. The base materials of suppositories can include Witepsol, Macrogol, Tween 61, cocoa butter, laurin butter, glycerin gelatin, etc.

[0126] In the present invention, the composition of the present invention can be formulated into a sterile aqueous solution, non-aqueous solvent, or suspension for parenteral administration, and specifically can be prepared into an injection. The pharmaceutical composition of the present invention may also include suitable carriers, excipients, or diluents commonly used in the art. In this regard, the content of the injectable composition of the present invention contained in the pharmaceutical composition as an active ingredient is not particularly limited.

[0127] The compositions of the present invention may be administered to an individual in a pharmaceutically effective amount.

[0128] In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment without causing side effects, and the level of the effective amount can be determined according to the type of individual, the severity of the disease, the age and sex of the individual, the type of disease, the activity of the drug, sensitivity to the drug, administration time, administration route, excretion rate, treatment period, factors including the inclusion of combined or concurrently used drugs, and other factors well known in the medical field.

[0129] The compositions of the present invention can be used as a separate therapeutic agent or in combination with other therapeutic agents, can be used sequentially or simultaneously with conventional therapeutic agents, and can be used in a single dose or multiple doses. Importantly, taking all of the above factors into account, the minimum amount that can achieve the maximum effect without causing side effects is used, which can be easily determined by those skilled in the art. The appropriate dosage of the compositions of the present invention can vary according to the patient's condition and weight, the severity of the disease, the formulation of the drug, the route of administration, and the administration period, and can be applied once a day or multiple times a day.

[0130] In the present invention, the composition of the present invention may be administered in combination with the functional substance described above.

[0131] The composition of the present invention can be applied to any individual who needs to prevent or treat osteoarthritis without limitation. The method of administration can be any method commonly used in the art without limitation. The composition can be applied to the joint cavity (e.g., cartilage), subcutaneously, intraperitoneally, intrapulmonaryly, and intranasally. For local treatment purposes, the composition can be applied by a suitable method, including intralesional administration, i.e., application to the joint cavity (e.g., cartilage). For example, administration can be achieved by administration (injection) into the joint cavity, but is not limited thereto.

[0132] The term "subject" herein refers to any animal suffering from or at risk of developing osteoarthritis. By administering the composition of the present invention to an individual suspected of suffering from osteoarthritis, effective treatment can be provided to the individual. The individual is not particularly limited and may be an animal such as a monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, or the like, or a bird, but the embodiments are not limited thereto.

[0133] In the present invention, the term "administering" refers to introducing the radioactive composition of the present invention into an individual suspected of having osteoarthritis by any suitable method, and the administration route can be various parenteral routes, as long as the composition reaches the target tissue. The composition of the present invention can be administered in a pharmaceutically effective amount, and the pharmaceutically effective amount is as described above.

[0134] The terms used in the embodiments are only used to illustrate specific example embodiments and are not used to limit the example embodiments. Unless otherwise specified in the content, the singular forms "a", "an", and "the" are intended to also include the plural forms. In this specification, the terms "include / comprise" and / or "include / comprises" are used to express the presence of the stated features, numbers, steps, operations, elements, and / or parts, but do not exclude the possibility of the presence or additional presence of one or more other features, numbers, steps, operations, elements, parts, and / or their combinations.

[0135] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the ordinary meanings understood by persons of ordinary skill in the art to which they pertain. Commonly used terms, as defined in dictionaries, should be understood as having the meanings inherent in the relevant technical context and should not be interpreted as idealizing or formalizing the meanings of terms not explicitly defined in this specification.

[0136] Below, the exemplary embodiments will be described in detail with reference to the accompanying drawings. However, various changes can be made to the exemplary embodiments. In this article, the present disclosure is not limited to the exemplary embodiments. The exemplary embodiments should be understood to include all changes made within the concept and technical scope of the present disclosure, and their equivalents and even their substitutes. In the process of describing the exemplary embodiments, when it is determined that the specific description of the relevant well-known structure or function will cause unnecessary confusion to the present disclosure, its specific description will be omitted.

[0137] Modes for Carrying Out the Invention

[0138] The following Examples 1 to 9 disclose TCA-loaded poly(organophosphazene). Figure 1 shown.

[0139] Example 1. Experimental Method A

[0140] Example 1-1. Ingredients

[0141] Hexachlorocyclotriphosphazene was purchased from Sigma-Aldrich (USA) and purified by sublimation at 55°C under vacuum (approximately 0.1 mmHg). Poly(dichlorophosphazene) was prepared as described by Bromberg L. et al. [Bromberg L., Temchenko M. Self-assembly in aqueous solutions of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)-b-poly(vinyl alcohol). Langmuir. 1999; 15: 8633–8639.]. Methoxypolyethylene glycol (Sigma-Aldrich, USA) with a molecular weight of 750 Da was substituted with α-amino-ω-methoxypolyethylene glycol (AMPEG) on the poly(organophosphazene) backbone. L-Isoleucine ethyl ester hydrochloride (IleOEt·HCl) (A&Z Food Additives, Hangzhou, China), aminoethanol (Sigma-Aldrich, USA), glutaric anhydride (Alfa Aesar, USA), and 4-(dimethylamino)pyridine (DMAP) (Alfa Aesar, USA) were purchased. Tetrahydrofuran (THF) and trimethylamine (TEA) were dried by reflux under dry nitrogen with sodium metal and barium oxide, respectively. Triamcinolone acetonide (TCA) was purchased from TCI (Japan). All other reagents were purchased from commercial suppliers.

[0142] Example 1-2. Synthesis of poly(organophosphazene)

[0143] All reactions were carried out in a dry nitrogen atmosphere using a Schlenk line.

[0144] 1-2-1. Synthesis of Precursor Polymer (PP, Aminoethanol Poly(Organophosphazene))

[0145] The precursor polymer (PP) was prepared as follows. In the first step, IleOEt·HCl (10.8 g, 55.22 mmol) was suspended in anhydrous THF (200 mL) and the reaction flask was cooled. In the second step, poly(dichlorophosphazene) (10.00 g, 8.63 mmol) was dissolved in anhydrous THF (200 mL) and slowly added to a reaction flask containing IleOEt·HCl (23.64 g, 12.08 mmol) dissolved in anhydrous THA and TEA. The reaction mixture was stirred in a dry ice bath for 12 hours and then stirred at 45°C for 24 hours. In the third step, AEtOH (1.42 g, 2.33 mmol) and AMPEG750 (13.59 g, 18.12 mmol) were dissolved in anhydrous THF and added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 hours and further stirred at 45°C for 24 hours. The reaction mixture was filtered and poured into n-hexane to obtain a precipitate, which was then reprecipitated twice in the same solvent system. The polymer product was further purified by dialysis using a Spectra / Por dialysis membrane (MWCO: 10-12 kDa) against methanol for 4 days at room temperature and against distilled water for 4 days at 4°C. The dialyzate was freeze-dried to obtain PP. The yield was 73.7%. NMR data are shown below.

[0146] 1 H NMR(CDCl3), δ(ppm):0.8–1.0(s,6H),1.1–1.3(b.3H),1.3–1.6(b,2H),1.6 –1.9(b,1H),2.8–3.3(b,2H),3.4–3.8(b,73H),3.9(s,1H),4.0–4.3(b,3H).

[0147] 1-2-2. Synthesis of Carboxylic Acid-Terminated Poly(Organophosphazene) (CP)

[0148] To replace the hydroxyl termini with carboxylic acid termini, PP (10 g, 12.25 mmol) was dissolved in anhydrous THF. The reaction mixture was stirred at room temperature for 12 hours and then further heated to 40-45 ° C for 24 hours. The final product, CP, was dialyzed with methanol at room temperature for 3 days and with water at 4 ° C for 3 days. Purified CP was obtained by freeze drying. The newly appeared peak at 2.1-2.32 ppm (b, 4H, CH2) represents CP. 1 The generated glutaric acid terminal was absent in H NMR. The yield was 96%, and the NMR data are as follows.

[0149] 1 H NMR (CDCl3), δ (ppm): 1H NMR (300MHz, CDCl3, δ), d (ppm): 0.8–1.0 (s, 6H, CH3), 1.1–1.3 (b, 3H, CH3), 1.3–1.6 (b, 2H, CH2), 1.6–1.9 (b, 1 H,CH),2.1–2.32(b,4H,CH2),2.8–3.3(b,2H,CH2),3.4–3.8(b,62H,CH2),3.9(s,1H,CH),4.0–4.3(b,3H,CH3).

[0150] Example 1-3. Confirmation of CP characteristics

[0151] The structure of the synthesized CP was determined by 1 H NMR (Varian Gem-ini-300 spectrometer, which uses CDCl3 in Fourier transform mode at 300 MHz) was used to estimate. The surface charge of the CP in aqueous solution was measured using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The molecular weight (MW) of the CP was determined using a gel permeation chromatography system (Tosoh, EcoSECHLC-8320GPC) with two Styragel columns (TSKgel SupermultiporeHZ-M and TSKgel SuperHZ-2500) in series at a flow rate of 0.35 mL / min at 25°C and a refractive index detector. THF containing 0.1 wt% tetrabutylammonium bromide was used as the mobile phase. Polystyrene (MW: 162; 580; 1920; 3090; 9590; 27,810; 70,500; 133,500; 290,300; 729,500; 1,074,000; 2,703,000) was used as a standard.

[0152] Example 1-4. In vivo compatibility experiment

[0153] 1-4-1. CP cytotoxicity assay

[0154] Mouse fibroblast cell line (NIH3T3) was used for CP cytotoxicity experiment. 4Cells were seeded at a density of 100 μg / well in a 96-well plate (SPL, South Korea) and allowed to attach overnight in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a humidified atmosphere of 5% CO₂. After 24 hours, the medium was replaced with fresh DMEM and treated with various concentrations of CP (0 mg / mL to 10 mg / mL in DMEM, n=6). After 24, 48, and 72 hours, cells were treated with MTT solution (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide), the medium was aspirated, and the cells were incubated for 3 hours. The precipitate was dissolved in 100 μl of DMSO, and the absorbance of the resulting purple solution was measured using a Spectra MAX 340 (BIO-RAD, Hercules, CA) at an excitation wavelength of 570 nm. The cell viability (%) was calculated as [ab]test / [ab]control×100%.

[0155] 1-4-2. Confirmation of the mutagenicity of CP

[0156] The mutagenicity of CP is assessed with several auxotrophic strains of Salmonella typhimurium. Determine mutagenicity (Ames test) by assessing the number of back-mutation mutant colonies. T98 and T100 tests use sterile liquid culture medium (2.5% Oxoid Nutrient Broth No.2). After cultivating for 10 hours in an oscillating water bath (37 ℃, 200rpm), 0.1mL cultured bacteria is mixed with 2mL top agar, 0.1mL CP solution and S9 fraction. The mixture is then carefully poured into the culture dish containing the 20mL minimum glucose agar plate prepared previously and cultivated until solidified. Count the colony number after cultivating for 48 hours at 37 ℃. Phosphate buffered saline (PBS) was used as a negative control group, and 2-nitrofluorene, benzo(a)pyrene, and sodium azide were used as positive controls specific for each bacterium, T98 and T100. All negative and positive controls were performed in the same manner. Minimal glucose agar plates were prepared using Bacto agar (Difco), Vogel-Bonner medium E, and 2% glucose.

[0157] Examples 1-5. Preparation of Self-Assembled Polymer Nanoparticles (PNs) and TCA-Encapsulated Polymer Nanoparticles (TePNs)

[0158] The synthesized CP was dissolved in a PBS solution at 4°C using a magnetic stirrer (preparing a 12 wt% CP solution and diluting 10 times). The prepared CP solution was observed using dynamic light scattering (DLS) based on a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) and a transmission electron microscope (TEM, CM30 electron microscope, Philips, California). The size and shape of the self-assembled PNs were observed. TCA-encapsulated polymer nanoparticles (TePNs) were prepared using three concentrations of TCA (0.3, 1.0, and 1.5 mg / mL). TCA was added directly to the PNs solution and gently mixed. The mixed solution was incubated at room temperature for 30 minutes to induce TCA encapsulation. The properties of TePNs were verified by DLS and TEM.

[0159] Example 1-6. Temperature-sensitive sol-gel transition behavior of TePN solution

[0160] The viscosity of PN aqueous solutions (12 wt %) was measured using a Brookfield RVDV-III+ viscometer at a fixed shear rate of 0.1 s-1 between 5 and 75°C. A set spindle speed of 0.2 rpm and a heating rate of 0.33°C / min were used for the measurements. Rheological measurements were performed using PN solutions (12 wt % PN in PBS) and TePN solutions (0.3, 1.0 and 1.5 mg / mL of TCA were added to the prepared 12 wt % PN solution) (MSC 102, Anton Paar, Germany). Storage modulus (G') and loss modulus (G") measurements were performed at an oscillation frequency of 1 Hz, an oscillation strain rate of 5%, temperatures of 4°C and 60°C, and a spacing length of 0.3 mm.

[0161] Example 1-7. In vivo decomposition study of TePN hydrogel

[0162] TePN solution (1.5 mg / mL TCA, 200 μL) was subcutaneously injected into the back of mice (6-week-old male Balb / c nude mice, Orient Bio, South Korea) using a 31-gauge needle. Localized TePN hydrogel formation was confirmed immediately after injection. The residual TePN hydrogel weight was measured on the scheduled day.

[0163] Example 1-8. In vitro release of TCA from TePN

[0164] TePN solution was prepared as a 12 wt% PN solution containing 0.3, 1.0 and 1.5 mg / mL TCA, respectively. 0.3 mL of the prepared TePN solution was added to a test tube and the temperature was raised to 37°C to confirm the formation of a hydrogel. 6 mL of PBS solution was gently added to the hydrogel and the test tube was gently shaken (50 rpm) and cultured in a 37°C water bath (KMC-12055W1, Vision, South Korea). The PBS solution was replaced with a fresh PBS solution at each time point. Water / acetonitrile (50 / 50, v / v%) was used as the eluent and the TCA release was determined by high performance liquid chromatography (HPLC, Agilent) and the established standard sample was calculated using UV detection at 240 nm.

[0165] Example 1-9. In vivo anti-OA effect of TePN hydrogel system

[0166] 1-9-1. Intra-articular injection of TePN in rats with osteoarthritis (OA)

[0167] Osteoarthritis (OA) was induced by intraarticular injection of sodium iodoacetate (MIA, Sigma-Aldrich, USA) (0.5 mg / 50 μl) into Sprague Dawley (SD) rats (6 weeks old, male, Orient Bio, South Korea) (n=6 per group). One week later, 0.3 mL of TePN solution was injected into the joints of OA-induced rats. Untreated MIA-induced OA rat model was used as a control group. Rats were sacrificed 8 weeks after TePN injection for further analysis.

[0168] 1-9-2. Intra-articular structures and X-ray and micro-computed tomography (μ-CT) scans

[0169] Eight weeks after injection, the rats were sacrificed and the knee joints were dissected from the femur and tibia. The isolated knee specimens were scanned using X-rays (In-Vivo Series, DXS PRO, Carestream, USA) and μ-CT (Aibira CT System, Carestream, USA). The distance between damaged cartilage was measured using μ-CT images and Image J software.

[0170] 1-9-3. Histological analysis

[0171] All collected tissues were embedded in paraffin and sectioned using a microtome (8 μm thickness). Tissue sections were deparaffinized, rehydrated, and stained for histological evaluation. Following staining with hematoxylin and eosin (H&E) and safranin-O, the histological appearance of each knee joint was observed using a light microscope (Nikkon, E400, Japan).

[0172] 1-9-4. RNA isolation and real-time polymerase chain reaction

[0173] Blood samples were collected at each time point (after OA induction, 1 week, 4 weeks, and 8 weeks after TePN hydrogel treatment). RNA was isolated using an RNA blood kit (QIAamp, QIAGEN) according to the manufacturer's instructions. cDNA was synthesized using Accupower CycleScriptRT Pre Mix (dT20) (BIO-RAD) according to the manufacturer's protocol. Real-time polymerase chain reaction (PCR) amplification and detection were performed using an ABI7300 real-time thermal cycler (Applied Biosystems, Foster City, CA, USA). The gene expression of proinflammatory cytokines such as metalloproteinase-3 (MMP-3), MMP-13, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and anti-inflammatory cytokines such as IL-4, IL-10, and IL-13 was studied respectively. The sequences of each genetic marker are shown in Table 1 below.

[0174]

Table 1

[0175]

[0176] Example 2. Synthesis and properties of poly(organophosphazene)

[0177] A poly(organophosphazene) with side chains primarily containing the hydrophobic l-isoleucine ethyl ester (IleOEt) and the hydrophilic α-amino-ω-methoxy poly(ethylene glycol) (AMPEG) was synthesized and used as a temperature-sensitive and in situ gelling drug reservoir system. Furthermore, by introducing a carboxylic acid terminus, the poly(organophosphazene) mimics the extracellular matrix of natural cartilage, which is primarily composed of chondroitin sulfate and hyaluronic acid, and interacts with various cytokines associated with cartilage homeostasis and regeneration, as shown in the chemical formula below.

[0178]

[0179] The molar ratio of the substituted side chains was calculated using NMR spectroscopy. The concentrations of IleOEt, AMPEG, and carboxylic acid in the final polymer were 70%, 10.5%, and 13.5%, respectively. The substitution ratio of each polymer complex was calculated by integrating the specific peaks of the six protons of IleOEt at 0.8-1.0 ppm, the four protons of carboxylic acid at 2.1-2.32 ppm, and the 62 protons of AMPEG at 3.4-3.8 ppm. 1 HNMR data were used to evaluate ( Figure 2 The charge of the final polymer was measured by zeta potential to be -4.0 ± 1.6, which is characterized by the negative charge of the cartilage extracellular matrix. GPC analysis using polystyrene standards showed a molecular weight of 50,085 Da.

[0180] In addition, the biocompatibility of poly(organophosphazene) was evaluated as follows. The Ames test is a test that evaluates the mutagenic potential of a test chemical in relation to its carcinogenic potential. For the mutagenicity test, Salmonella typhimurium TA98 or TA100 was treated with 200 μg of polymer. The polymer-treated group showed almost the same number of revertant mutant colonies as the negative control group ( Figure 3 These nontoxic and mutagenic properties demonstrate the safety of the synthesized polymer, supporting its use as a viable material for in vivo applications. In particular, for anti-OA treatment, TCA carriers must possess high biocompatibility to prevent the promotion of immune responses at the lesion site. For these reasons, the biocompatibility of the synthesized poly(organophosphazene) is suitable for use as an anti-osteoarthritis (OA) drug carrier.

[0181] Cytotoxicity and mutagenicity testing were performed to assess the toxicity of the resulting new drug or in vivo-usable substance. The toxicity of the synthesized poly(organophosphazene) was investigated using the MTT assay and the Ames test. The cytotoxicity of the polymers was tested in NIH 3T3 cells (polymer concentrations in the culture medium ranged from 0 mg / mL to 30 mg / mL). After 24 hours of treatment, cell viability of 90-95% was observed at all polymer concentrations. Figure 3 b). Even after the polymers with the same composition were treated with CP for 48 and 72 hours, no cytotoxicity was observed. Figure 4 This indicates that CP is a cytocompatible substance.

[0182] Example 3. Characteristics of self-assembled PN and TCA encapsulation

[0183] The side chains of the polymer are filled with hydrophobic and hydrophilic parts, thus having balanced amphiphilicity. The polymer exists in the form of spherical nanoparticles in an aqueous environment to minimize the exposure area of the hydrophobic IleOEt units of the polymer to water molecules. In addition, hydrophobic drugs such as TCA interact with the hydrophobic core of PN ( Figure 5 a). Self-assembled PN and TePN were studied by TEM and size distribution. Circular PN ( Figure 5 b), which is approximately 140 nm in size at 25°C (120 mg / mL PN in phosphate buffered saline (PBS) solution), with an error range of ≤4 nm. TCA, a low water-soluble drug, exhibits irregular nano- and micron-sized crystals (crystallin) ranging from 232.9 nm to 3343 nm (DLS and TEM data error range ≥1450 nm) in PBS solution (1.5 mg / mL TCA in PBS) ( Figure 5 c and Figure 6 However, after mixing and incubation with PN-dispersed PBS solution, micro-sized TCA aggregates disappeared from the size distribution. Only particles of approximately 140 nm were measured in a mixed solution of TCA and PN (120 mg / mL PN and 1.5 mg / mL TCA in PBS solution), with an error range of ≤4 nm ( Figure 5 This result indicates that PN nanoparticles successfully encapsulated TCA. The average size and polydispersity index (PDI) of these particles are shown in Figure d. Figure 5 e and Figure 7 The size change and its significant reduction indicate that TCA can be well encapsulated into the hydrophobic core of PN via hydrophobic interactions.

[0184] Example 4. Confirmation of the temperature-sensitive sol-gel transition behavior of TePN solution

[0185] In PN aqueous solutions above a certain concentration, significant viscosity changes and phase transitions will occur with temperature changes. This phase transition is due to the fact that as the temperature rises, the hydrogen bonds between the hydrophilic PEG chains of PN and water molecules weaken, while the hydrophobic interactions between PN increase. In this study, a 12wt% PN solution with the maximum storage modulus was prepared, which has good injectability, sufficient viscosity and can be gel-like at body temperature. Figure 8As shown in a, the viscosity of the PN solution at 32°C is close to 0 Pa·s. The viscosity value continues to increase at 32°C, reaching 518.75 Pa·s at 37°C. Similarly, both the storage modulus and the loss modulus increase at 32°C, and in the rheological study, the storage modulus (G') is greater than the loss modulus (G") between 32°C and 44°C. At 37°C, the G' and G" values are 1284.3 and 765 Pa, respectively, indicating a gel state ( Figure 8 b).

[0186] In addition, if Figure 8 As shown in Figure c, because the physical properties of drug delivery hydrogels significantly affect the drug release pattern or duration, the sol-gel transition behavior was monitored after loading with TCA. The storage modulus and loss modulus of TePN solutions were measured after loading with three different doses of TCA. The storage modulus and loss modulus of all groups showed similar changes with temperature. These results demonstrate that the loaded TCA was uniformly encapsulated within the PN and had little effect on the sol-gel transition behavior, and it is predicted that the hydrogels would exhibit similar behavior even with different drug doses.

[0187] Example 5. In vivo decomposition of TePN hydrogel

[0188] Since biodegradation is a fundamental requirement for biomaterials and drug carriers, the rate of hydrogel decomposition and general symptoms surrounding the tissues where PN hydrogels were locally present were monitored. The prepared PN solution was injected subcutaneously into mice using a 31-gauge needle. PN hydrogel formation was confirmed directly after injection, and the remaining PN hydrogel ( Figure 9 The amount of PN hydrogel residue decreased over time until day 42 ( Figure 9 b and Figure 10 These biodegradable properties, the mild conditions used to form the PN hydrogel, and the long residence time at the injection site indicate that the developed PN hydrogel system is a long-term drug reservoir suitable for use as a drug delivery vehicle.

[0189] Example 6. In vitro confirmation of the sustained release properties of TCA in TePN

[0190] Steroidal anti-inflammatory drugs such as TCA have a short residence time after administration into the joint cavity and are quickly excreted from the body. Therefore, their long-term anti-inflammatory effects are limited. Taking advantage of the fact that strongly interacting drugs are often highly dependent on the decomposition properties of the material, the TePN hydrogel system can control the TCA release pattern and duration by controlling the dissociation and decomposition rates of the PN hydrogel. The TCA release behavior was tested using freshly prepared TePN solutions containing three different concentrations of TCA. After the TePN hydrogel was formed at 37°C, each TePN hydrogel was immersed in PBS buffer. The PN group showed rapid swelling in less than 1 day. In contrast, most of the hydrogels containing TCA showed swelling after 1 week, indicating that the hydrophobic interaction between TCA and the hydrogel improved the stability of the polymer structure in aqueous solution ( Figure 11 After 4 weeks all gels started to lose mass ( Figure 12 This is closely related to the 42-day TCA release pattern of the hydrogel ( Figure 12 b). Initial TCA release was confirmed to be inhibited within the first 24 hours in the three TePN hydrogel groups containing TCA concentrations of 0.3, 1.0, and 1.5 mg / mL, with observed values of 14.45%, 9.55%, and 9.72% of the total TCA load, respectively. These results contrast sharply with the half-life of TCA without a delivery vehicle, which was only 1.27 hours when administered directly to humans. In each TePN hydrogel group, approximately half of the TCA load was released by day 7, day 14, and day 16 at 0.3 mg / mL, 1.0 mg / mL, and 1.5 mg / mL, respectively. Release continued until the TePN hydrogel completely disintegrated. Furthermore, the TePN hydrogel group with the lowest TCA concentration exhibited a faster release pattern than the other two groups. Low TCA concentrations can lead to loss of the hydrophobic network, rapid swelling, and rapid dissolution. In the TePN hydrogels containing 1.0 mg / mL and 1.5 mg / mL TCA, the TCA release pattern was linear over time. The disintegration and TCA release times of the TePN hydrogels were nearly similar. The suppressed initial release and sustained TCA release indicate that TCA strongly interacted with the polymer and was well encapsulated by the PN.

[0191] Example 7. Confirmation of the long-term anti-inflammatory and cartilage degeneration prevention effects of a single injection of TePN hydrogel in an OA rat model

[0192] The long-term anti-inflammatory and cartilage degeneration inhibitory effects of a single injection of the TePN hydrogel reservoir system were confirmed. A sodium iodoacetate (MIA)-induced OA rat model was prepared and treated with the TePN hydrogel system. To simulate the early stages of OA, TePN was injected one week after the OA model was established. In addition to the TePN hydrogel group, a direct injection of TCA solution and a saline solution group were set up as control groups. 300μl of each solution of saline, TePN, and TCA was prepared and injected into the injured area of the rat.

[0193] After 8 weeks of treatment, the rats were sacrificed and the knee joints were dissected. The results showed that only the TePN-treated group showed a significantly reduced inflammatory response compared with the other groups ( Figure 13 ). In addition, X-ray and μ-CT images were taken to conduct morphological studies, e.g. Figure 14 As shown in a. The saline-treated group showed advanced osteoarthritis with severe cartilage defects and cartilage destruction. However, each TePN-treated group showed good morphological characteristics, indicating that the sustained-release TCA well suppressed the early inflammation of OA. In addition, the anti-OA effect of all TePN hydrogel groups was significantly improved compared with the TCA solution group. The damaged state of the cartilage in the direct IA injection of TCA solution group failed to treat inflammation and prevent cartilage degeneration in the long term. The cartilage pathological condition in the TCA solution group was similar to or worse than that in the saline solution group. This is due to the side effects of direct IA injection of TCA on chondrocyte toxicity and local treatment with high-dose TCA, which may inhibit fibroblast growth and collagen synthesis by reducing the production of hydroxyproline, resulting in reduced structural stability of the connective tissue around the knee joint. For statistical evaluation, the distance between the cartilage and ... Figure 14 b). Most left and right DIC distances increased similarly in the saline and TCA solution-treated groups. A statistically significant difference was observed only in the left cartilage distance between the saline and high-concentration TCA solution (1.5 mg / mL) groups. Severe cartilage damage may be due to two factors: 1) toxicity from direct, short-term exposure to high-dose TCA; and 2) insufficient sustained anti-inflammatory effects due to the rapid disappearance of TCA.

[0194] In addition, histological staining confirmed that the TePN hydrogel group effectively prevented cartilage degeneration, while the direct injection of TCA solution group failed to treat OA ( Figure 15). H&E and Safranin O staining showed the morphological characteristics of each experimental group. The cartilage in the saline and TCA solution-treated groups became thinner in shape, lost tissue with chondrocytes, and the characteristic red staining of glycosaminoglycans that can be seen in healthy cartilage disappeared. These results indicate that treatment with saline and TCA solution has little effect on the onset and progression of early OA. However, the cartilage morphology in the TePN hydrogel group was similar to that of normal cartilage, and the cellular characteristics of chondrocytes were well maintained. This experiment confirmed that the continuous release of TCA through a one-time injection of the TePN hydrogel system can effectively prevent further inflammation and the progression of OA.

[0195] Example 8. Confirmation of the effect of continuous TCA release using the TePN hydrogel system on pro-inflammatory gene inhibition and anti-inflammatory gene activation

[0196] Changes in inflammatory gene expression were assessed during the experiment. Pro-inflammatory and anti-inflammatory gene expression levels were evaluated from calibrated blood samples. One week after MIA-induced osteoarthritis, the gene expression levels of the proinflammatory cytokines MMP-3, MMP-13, IL-6, and TNF-α increased by 5.60, 4.25, 4.80, and 5.35 times, respectively, compared to the control group. Figure 16 (a) Distinct gene expression patterns were observed in each experimental group at 1, 4, and 8 weeks after injection of TePN or TCA solution. Gene expression levels of proinflammatory cytokines decreased over time in the TePN hydrogel group, but continued to increase in the TCA solution group. Because the expression of proinflammatory cytokines and MMPs contributes to cartilage degeneration, effectively reducing these levels could successfully prevent cartilage degeneration. After 8 weeks, the levels of MMP-3, MMP-13, IL-6, and TNF-α cytokine genes in the TePN hydrogel group, which received the highest TCA concentration, decreased by 1.62-fold, 1.67-fold, 1.49-fold, and 1.62-fold, respectively, compared to the saline-treated group. In contrast, the levels of each proinflammatory cytokine gene continued to increase over time in the TCA solution group. Because directly administered TCA rapidly dissipates, the anti-inflammatory effect of a single IA injection of TCA solution is insufficient to prevent the breakdown of the extracellular matrix and cartilage collagen.

[0197] At the same time, anti-inflammatory cytokine genes such as IL-4, IL-10 and IL-13 were monitored ( Figure 16b). This gene expression is known to be associated with the stimulation of the survival, proliferation, and differentiation of immune cells such as macrophages and specific T cells (Th2). In addition, the expression levels of these cytokines can be used as a criterion for inhibiting the synthesis of proinflammatory cytokines such as interferon-γ, interleukins, and TNF-α. One week after the onset of MIA-induced osteoarthritis, the gene expression levels of IL-4, IL-10, and IL-13 in all treatment groups were almost similar to those in the normal state. No significant differences were seen between the treatment groups until four weeks after the induction of inflammation. However, after eight weeks, the expression level of each gene in the TePN hydrogel-treated group increased significantly. In addition, the magnitude of the increase in gene expression increased according to the loaded TCA concentration. After eight weeks, the IL-4, IL-10, and IL-13 gene expression levels in the TePN hydrogel with the highest TCA concentration were 1.91-fold, 2.25-fold, and 1.92-fold higher than those in the saline-treated group, respectively. These results demonstrate the efficiency of sustained TCA release and the long-term anti-inflammatory effect of a single injection. There were no abnormal changes in body weight ( Figure 17 PN hydrogel treatment without TCA showed no changes in the expression levels of pro-inflammatory and anti-inflammatory genes. In other words, the carrier PN hydrogel system does not affect the systemic inflammatory response.

[0198] Example 9. Optimization of release characteristics according to the IleOEt / AMPEG molar ratio

[0199] The hydrophobicity of PN and the associated drug loading capacity and sustained release ability vary depending on the molar ratio of IleOEt / AMPEG. Therefore, we attempted to determine the polymer composition with the best release characteristics by adjusting the molar ratio of IleOEt / AMPEG.

[0200] Poly[(ethyl isoleucinate) 1.47 (Aminomethoxypolyethylene glycol 750) 0.24 (Ethanol) 0.21 (acid) 0.08 Phosphazene] n (hereinafter referred to as PPZ 43), poly[(isoleucine ethyl ester) 1.37 (Aminomethoxypolyethylene glycol 750) 0.25 (Ethanol) 0.27 (acid) 0.11 Phosphazene] n (hereinafter referred to as PPZ 46) and poly[(isoleucine ethyl ester) 1.44 (Aminomethoxypolyethylene glycol 750) 0.30 (Ethanol) 0.08 (acid) 0.18 Phosphazene] n (hereinafter referred to as PPZ 24) was prepared as follows. The NMR data of the prepared PPZ are shown in FIG. Figure 18 shown.

[0201] PPZ 43 was prepared as follows. The precursor polymer (PP) was prepared as follows. In the first step, IleOEt·HCl (24.65 g, 112.98 mmol) was suspended in anhydrous THF (200 mL) and the reaction flask was cooled. In the second step, poly(dichlorophosphazene) (10.00 g, 8.63 mmol) was dissolved in anhydrous THF (200 mL) and slowly added to a reaction flask containing IleOEt·HCl (24.65 g, 112.98 mmol) dissolved in anhydrous THA and TEA. The reaction mixture was stirred in a dry ice bath for 12 hours and then stirred at 45°C for 24 hours. In the third step, AEtOH (1.05 g, 17.26 mmol) and AMPEG750 (22 g, 29.34 mmol) were dissolved in anhydrous THF and added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 hours and further stirred at 45°C for 24 hours. The reaction mixture was filtered and poured into n-hexane to obtain a precipitate, which was then reprecipitated twice in the same solvent system. The polymer product was further purified by dialysis using a dialysis membrane (Spectra / Por, MWCO: 10-12 kDa) against methanol at room temperature for 4 days and against distilled water at 4°C for 4 days. The dialyzate was freeze-dried to obtain PP.

[0202] In order to replace the hydroxyl end with the carboxylic acid end, PP (10 g, 12.25 mmol) was dissolved in anhydrous THF. The reaction mixture to which glutaric anhydride (2.80 g, 24.5 mmol) and dimethylaminopyridine (2.99 g, 24.5 mmol) were added was stirred at room temperature for 12 hours and then further heated to 40-45 ° C for 24 hours. The final product CP was dialyzed with methanol at room temperature for 3 days and with water at 4 ° C for 3 days using a dialysis membrane. Purified CP was obtained by freeze drying. The newly appeared peak at 2.1-2.32 ppm (b, 4H, CH2) represents CP 1 The resulting glutaric acid termini were absent in the H NMR.

[0203] PPZ 46 was prepared as follows. The precursor polymer (PP) was prepared as follows. In the first step, IleOEt·HCl (23.98 g, 122.53 mmol) was suspended in anhydrous THF (200 mL) and the reaction flask was cooled. In the second step, poly(dichlorophosphazene) (10.00 g, 8.63 mmol) was dissolved in anhydrous THF (200 mL) and slowly added to a reaction flask containing IleOEt·HCl (23.98 g, 122.53 mmol) dissolved in anhydrous THA and TEA. The reaction mixture was stirred in a dry ice bath for 12 hours and then stirred at 45°C for 24 hours. In the third step, AEtOH (1.32 g, 21.57 mmol) and AMPEG750 (21.36 g, 28.48 mmol) were dissolved in anhydrous THF and added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 hours and further stirred at 45°C for 24 hours. The reaction mixture was filtered and poured into n-hexane to obtain a precipitate, which was then reprecipitated twice in the same solvent system. The polymer product was further purified by dialysis using a dialysis membrane (Spectra / Por, MWCO: 10-12 kDa) against methanol at room temperature for 4 days and against distilled water at 4°C for 4 days. The dialyzate was freeze-dried to obtain PP.

[0204] In order to replace the hydroxyl end with the carboxylic acid end, PP (10 g, 12.25 mmol) was dissolved in anhydrous THF. The reaction mixture to which glutaric anhydride (2.80 g, 24.5 mmol) and dimethylaminopyridine (2.99 g, 24.5 mmol) were added was stirred at room temperature for 12 hours and then further heated to 40-45 ° C for 24 hours. The final product CP was dialyzed with methanol at room temperature for 3 days and with water at 4 ° C for 3 days using a dialysis membrane. Purified CP was obtained by freeze drying. The newly appeared peak at 2.1-2.32 ppm (b, 4H, CH2) represents CP 1 The resulting glutaric acid termini were absent in the H NMR.

[0205] PPZ 24 was prepared as follows. The precursor polymer (PP) was prepared as follows. In the first step, IleOEt·HCl (26.43 g, 134.61 mmol) was suspended in anhydrous THF (200 mL) and the reaction flask was cooled. In the second step, poly(dichlorophosphazene) (10.00 g, 8.63 mmol) was dissolved in anhydrous THF (200 mL) and slowly added to a reaction flask containing IleOEt·HCl (26.43 g, 134.61 mmol) dissolved in anhydrous THA and TEA. The reaction mixture was stirred in a dry ice bath for 12 hours and then stirred at 45°C for 24 hours. In the third step, AEtOH (1.05 g, 17.26 mmol) and AMPEG750 (15.53 g, 20.71 mmol) were dissolved in anhydrous THF and added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 hours and further stirred at 45°C for 24 hours. The reaction mixture was filtered and poured into n-hexane to obtain a precipitate, which was then reprecipitated twice in the same solvent system. The polymer product was further purified by dialysis using a dialysis membrane (Spectra / Por, MWCO: 10-12 kDa) against methanol at room temperature for 4 days and against distilled water at 4°C for 4 days. The dialyzate was freeze-dried to obtain PP.

[0206] In order to replace the hydroxyl end with the carboxylic acid end, PP (10 g, 12.25 mmol) was dissolved in anhydrous THF. The reaction mixture to which glutaric anhydride (2.80 g, 24.5 mmol) and dimethylaminopyridine (2.99 g, 24.5 mmol) were added was stirred at room temperature for 12 hours and then further heated to 40-45 ° C for 24 hours. The final product CP was dialyzed with methanol at room temperature for 3 days and with water at 4 ° C for 3 days using a dialysis membrane. Purified CP was obtained by freeze drying. The newly appeared peak at 2.1-2.32 ppm (b, 4H, CH2) represents CP 1 The resulting glutaric acid termini were absent in the H NMR.

[0207] The T values of the prepared PPZ 43, 46, and 24 were measured. max And displayed in Figure 19 As shown in the figure, the hydrophobicity becomes stronger with the increase of IleOEt / AMPEG molar ratio. max The value decreases. Figure 19 (b) shows the cumulative release of each compound loaded with TCA over time, expressed in mg and %. It was confirmed that the stronger the hydrophobicity, the slower the release of TCA, a hydrophobic drug.

[0208] Reference Example 6 and Figure 12The TCA release curve shown in Figure 1 shows that the TCA release rate from TePN 1.5 (TCA-encapsulated PN loaded with TCA at a concentration of 1.5 mg / ml) is 0.011 to 0.018 mg / day (11-18 μg / day). The in vivo dosage of TePN is approximately 200-300 μl, which slowly decomposes and releases TCA over 30 days. Therefore, it was planned to calculate the amount of TCA released and exposed in vivo over a day and apply it to in vitro experiments. To this end, the 1.5 mg / ml TCA contained in TePN 1.5 was diluted 20-fold to 160-fold, with loading concentrations of 9.375, 18.75, 37.5, and 75 μg / ml, respectively.

[0209] The complex loaded with TCA and PPZ was treated in chondrocytes in an induced inflammatory environment, and the expression of inflammation-related factors was measured by PCR. To induce an inflammatory environment, an in vitro arthritis model was formed by treating human chondrocytes with 10 ng / ml IL-1β. The model was treated with the TCA-PPZ complex at different concentrations, and the relative expression levels of MMP3, MMP13, IL-6, and TNF-α were measured. The results are shown in Figure 2. Figure 20 As shown in the figure. As can be seen from the figure, after treatment with IL-1β, MMP3, MMP13 and IL-6 (inflammatory cytokines) increased rapidly, and the inflammatory environment was well induced. At this time, when treated with 9.375 to 37.5ug / ml TCA-PPZ complex, it was confirmed that the expression of MMP3, MMP13 and IL-6 decreased sharply. However, when the highest concentration of 75ug / ml was applied, the lower effect was not obvious, and the inflammation relief effect was further lower than the concentration of 9.375 to 37.5ug / ml. In other words, high doses of TCA will have a negative impact. It can be seen that a drug delivery system that slowly releases a constant concentration of drug is more conducive to the treatment of OA.

[0210] The following experiments were performed to determine the optimal IleOEt / AMPEG molar ratio. In the in vitro release experiment, the release amount of 200 μl of TCA-PPZ complex was confirmed as shown in Table 2.

[0211]

Table 2

[0212] TCA release (ug) 43GA 46GA 24GA 1 day 19.32 58 146.8 2 days 10.28 34.16 3 days 5.2 17.06 4 days 4.4 9.71

[0213] Based on this, it is assumed that the estimated amount of TCA released when treated with 5 ml of TCA-PPZ solution is shown in Table 3. In the in vitro OA model, chondrocytes are mainly treated with IL-1β. Since 10 ml of culture medium is often used, it is assumed that 5 ml of TCA-PPZ hydrogel, which is equivalent to half the volume of the culture medium, is used for treatment.

[0214]

Table 3

[0215] TCA release (ug, estimated) 43GA 46GA 24GA 1 day 483 1450 3670 2 days 257 854 3 days 130 426.5 4 days 110 242.8

[0216] Based on the predictions in Table 3 above, the following calculations were performed to confirm the optimal IleOEt / AMPEG molar ratio. First, the total culture volume was 10 ml, and the estimated TCA release in Table 3 was divided by 10 to predict the TCA release concentration in μg / ml. Figure 21 Shows TCA release concentrations by date. Figure 21 , within the range of 9.375ug / ml to 37.5ug / ml for the optimal expression levels of MMP3, MMP13 and IL-6, the polymer combination range contained from day 1 to day 4 was 43GA, and the IleOEt / AMPEG molar ratio was approximately 6.125, showing the best sustained-release properties within this molar ratio range.

[0217] The preparation of poly(organophosphazene) for loading celecoxib (CXB) and its effects are disclosed in Example 10 below. Figure 22 A schematic diagram showing the treatment of Achilles tendonitis (AT) by celecoxib-loaded poly(organophosphazene).

[0218] Example 10. Experimental Method B

[0219] Example 10-1. Ingredients

[0220] Hexachlorocyclotriphosphazene was purchased from Sigma-Aldrich (USA) and recrystallized under vacuum at 60°C. Poly(dichlorophosphazene) was synthesized as described in Example 2. L-Isoleucine ethyl ester hydrochloride (IleOEt·HCl) was purchased from A&Z Food Additives, Hangzhou, China, and dried under vacuum at 55°C for 10 days. Methoxypolyethylene glycol (750 Da) was purchased from Sigma-Aldrich (USA) and replaced with α-amino-ω-methoxy-poly(ethylene glycol) (AMPEG). Ethanolamine (Sigma-Aldrich, USA), glutaric anhydride (Alfa Aesar, USA), and 4-(dimethylamino)pyridine (DMAP, Alfa Aesar, USA) were prepared. THF was dried using sodium metal (Aldrich, USA) and benzophenone (Daejeong, Korea). TEA was dried using barium oxide (Daejeong, Korea). Celecoxib (CXB) was purchased from EDQA (Europe).

[0221] Example 10-2. Synthesis of PNP

[0222] The reactions were all carried out in a dry nitrogen atmosphere using a standard double-row vacuum ventilation system (Schlenk line). IleOEt·HCl (10.38 g, 53.06 mmol) was dissolved in anhydrous THF. The reactor was stirred in a dry ice bath and 50 mL of dry TEA was added. Poly(dichlorophosphazene) (5.00 g, 43.14 mmol) was dissolved in dry THF and added to the reactor. The reaction temperature was set to 50°C for 24 hours, and ethanolamine (0.91 g, 15.1 mmol) and AMPEG (13.59 g, 18.12 mmol) were added to allow the polymer to react for a further 48 hours. The polymer solution was filtered and the solvent was removed using a rotary evaporator. The polymer was precipitated by pouring into n-hexane (Daejeong, South Korea) twice and purified using methanol for 4 days using a dialysis membrane (Spectra / Por, MWCO: 10-12 kDa) and further purified with distilled water for 4 days. Use syringe filter (Syringe filter) with 0.45 μ m hole to filter the polymer solution of purification and freeze-dry to obtain the poly (organophosphazene) of bonded aminoethanol.Dry THF is added in aminoethanol-poly (organophosphazene) (10g, 158.93mmol) to combine carboxylic acid group.Glutaric anhydride (5.98g, 52.41mmol) and DMAP (6.40g, 52.41mmol) are dissolved in anhydrous THF and added in aminoethanol PPZ solution.Then the solution is stirred and cultivated at 45 ℃ for 24 hours.Then use dialysis membrane that polymer is utilized methanol dialyzed 4 days, and before freeze-drying, use distilled water dialyzed 4 days.By above-mentioned preparation process, obtain the polymer consisting of (IleOEt), (AMPEG750), (EtOH) and (Acid).

[0223] Example 10-3. Confirmation of PNP characteristics

[0224] 1 The H NMR results are as follows:

[0225] 1 H NMR(CDCl3), δ(ppm):0.8–1.0(s,6H),1.1–1.3(b,3H),1.3–1.6(b,2H),1.6–1 .9(b,1H),2.1–2.3(b,4H),2.8–3.3(b,2H),3.4–3.8(b,62H),3.9(s,1H),and 4.0–4.3(b,3H)

[0226] The molecular weight of the polymer was determined using a gel permeation chromatography system (EcoSEC HLC-8320GPC, Tosoh) and a refractive index detector. Two Styragel columns (TSKgel SupermultiporeHZ-M and TSKgel SuperHZ-2500) were connected and a flow rate of 0.35 mL min-1 was used at 40°C. THF and 0.1% (w / v) tetrabutylammonium bromide were used as mobile phases. Polystyrene (MW: 162; 580; 1920; 3090; 9590; 27810; 70500; 133500; 290300; 729500; 1074000; and 2703000Da) was used as standards. Fourier transform infrared spectroscopy (FTIR) was performed using an FTIR spectrometer (Nicolet i20 FTIR spectrometer, Thermo Fisher Scientific) to characterize the presence of CXB and PNP in PCNP. The sample was dissolved in chloroform at 1 wt% and coated on a KBr circular absorption cell (Sigma-Aldrich, USA). The coated sample was analyzed after drying the chloroform and FTIR spectra were obtained by scanning 32 times in the wave number range of 4000 to 500 cm-1.

[0227] Example 10-4. Cell viability test

[0228] NIH3T3 fibroblast cells and serially diluted PNP hydrogels (0 to 10 mg / mL) were used. Cells were plated at 2×10 4 Cells were seeded at a density of 100 μg / well in 96-well cell culture plates (SPL, South Korea) and cultured for 24 hours at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The medium was replaced with FBS-free DMEM containing various concentrations of PNP, CXB, and PCNP. After 24 hours, MTT solution (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) was added and incubated for 2 hours at 37°C in a CO2 incubator. Dimethylsulfoxide (200 μl, Daejung, South Korea) was added, and the absorbance of the solution was measured at 570 nm using a Spectra MAX 340 spectrophotometer (BIO-RAD, Hercules, CA). The absorbance of the experimental and control groups was used to calculate the NIH3T3 cell survival rate according to the following formula.

[0229] Cell survival rate (%) = absorbance (experimental group) / absorbance (control group)

[0230] Example 10-5. Rheological test

[0231] A rheometer (Anton Parr, AT) was used to determine the temperature-sensitive sol-gel transition of PPZ. The concentration of the PNP and PCNP hydrogel solutions was 10 wt%. The modulus was measured at the correct temperature. The rheometer used 25.0 mm parallel plates and a 0.5 mm zero gap. The modulus was measured under oscillatory stress at a frequency of 0.8 Hz, 10% strain, and various temperatures. Sweep frequency tests were also performed at 37°C and 10% shear strain. All modulus data were analyzed using Anton Parr software from Rheocompass.

[0232] Example 10-6. Acquisition of cryo-SEM images

[0233] For cryo-SEM experiments, a Quanta 3D FEG (FEI, The Netherlands) equipped with an Alto 2500 cryotransfer system (Gatan, UK) was used. PNP and PCNP samples were quickly immersed in liquid nitrogen slurry for 0.5 seconds. After a few seconds, the freezer was emptied and the samples were transferred to a cryo-storage chamber at approximately -190°C that was evacuated to 10 -5 mbar. Metal deposition was performed by plasma sputtering with a current of 3 mA for 60 s. The metal-coated sample was transferred to a chamber that was previously evacuated to 10 -5 mbar pressure and precooled to −190° C. The cryo-SEM images were acquired using a 5-keV beam at 11.8 pA.

[0234] Example 10-7. Dynamic Light Scattering (DLS)

[0235] The particle sizes of PNP, CXB, and PCNP were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The nanoparticles were dissolved in phosphate-buffered saline (Well Gene, South Korea) at a concentration of 0.1 wt% and the particle sizes were measured at 5°C, 25°C, 37°C, and 45°C for two cycles.

[0236] Zeta potential was evaluated by preparing 0.5 wt% PNP and PCNP (4 mg CXB) at room temperature. The measurements were performed using a DLS (Malvern Instruments Ltd., Malvern, UK).

[0237] Example 10-8. In vitro and in vivo hydrogel decomposition

[0238] To confirm in vitro swelling and disintegration, 200 μl of 10 wt% PNP hydrogel was weighed into the insert and incubated in a 37°C oven for 5 minutes to induce complete gelation. The hydrogel pieces were then immersed in PBS at 37°C. The mass change of the hydrogel was measured at defined time points.

[0239] To measure the swelling and decomposition of PNP, PNP was dissolved in PBS at 10 wt% and stirred at 4°C. Next, 200 μl of the polymer solution was slowly injected subcutaneously into the back of each mouse (CrljOri:CD1(ICR), 6-week-old, male, OrientBio, South Korea). The injected PNP was extracted and the remaining amount was measured at the designated time.

[0240] Example 10-9. In vitro release of CXB from PCNP

[0241] PCNP hydrogels were prepared by mixing 10 wt% PNP with CXB. PNCP (200 μl) was then placed in a Millicell (diameter: 12 mm, Millipore, USA) and placed in a 37°C oven for 5 minutes. The PCNPs were then immersed in a 15 mL conical tube containing 6 mL of PBS and incubated in a shaker (KMC-12–55W1, Vision, KR) at 37°C and 50 rpm. The PBS was allowed to swell at certain times. Released CXB was measured by high-performance liquid chromatography (Agilent Technologies) using formaldehyde (75%) and water (25%) as the mobile phase.

[0242] Kinetic models are used to fit the experimental data. These models include the zero-order model Level 1 model Higuchi Model and the Ritger-Peppas model The left side of each equation represents the percentage of drug released at time t. k and n represent the kinetic constant and diffusion exponent, respectively.

[0243] Example 10-10. In vivo tendon regeneration test

[0244] All experiments using mice were conducted in accordance with the relevant legal and institutional guidelines of the Institutional Animal Care Committee (IACUC) of Korea University College of Medicine and were approved by the IACUC (approval number KOREA-2018-0049). Collagenase (Thermo Fisher Inc., USA, 50 μL) was injected into 9-week-old mice (DooYeol Biotec, Seoul, South Korea). After 1 week, CXB solution dissolved in DMSO at a concentration of 1 or 4 mg / (100 μL) was injected to treat the defective tendon. PCNP (CXB 1 mg) and PCNP (CXB 4 mg) hydrogels were prepared by stirring 1 mg or 4 mg of CXB in 100 μL of 10 wt% PNP hydrogel at 4°C. The PCNP hydrogel was then injected into the defective tendon model.

[0245] Examples 10-11. Real-time polymerase chain reaction (RT-PCR)

[0246] Blood samples were collected 4 weeks after CXB solution or PCNP was injected into the defective tendon. RNA blood kit (QIAGEN, QIAamp) was used for RNA isolation. cDNA was synthesized using Accupower's CycleScript RT Premix (dT20) (BIO-RAD). Real-time polymerase chain reaction (RT-PCR) was performed using an ABI7300 real-time thermal cycler (Applied Biosystems, USA), and pro-inflammatory cytokines (COX-2, IL-1, IL-6, MMP-3, MMP-13 and TNF-α) and anti-inflammatory cytokines (IL-4 and IL-10) were detected. Genetic marker sequences are shown in Table 4 below.

[0247]

Table 4

[0248]

[0249] Examples 10-12. Histological processing and analysis

[0250] Defective tendons treated with PCNP or CXB solution were extracted and washed with DPBS. Each tendon was immersed in 4% paraformaldehyde for 24 hours to fix the tissue. The tendons were then rinsed and sectioned in paraffin (5 μm sections). Paraffin sections were placed in a 60°C oven and dewaxed in xylene containing 100%, 90%, 80%, and 70% ethanol. Sections were stained with H&E and MT. n = 3 per group.

[0251] Example 10-13. Analysis of Hydroxyproline Content

[0252] To demonstrate the alleviating effect of PCNP and CXB solution treatment on AT, hydroxyproline analysis was used to evaluate the collagen content of rat tendon tissue. To hydrolyze tendon tissue, 5 mg of tendon tissue was immersed in 6N HCl solution and heated at 120°C for 3 hours. After hydrolysis, the tendon tissue was centrifuged at 3000 rpm for 3 minutes, and the supernatant was collected and transferred to a 96-well plate. The 96-well plate was then incubated in a 60°C oven for 12 hours. Chloramine T reagent (6 μL) was mixed with oxidation buffer (94 μL), and the mixture was added to each sample or standard solution. The resulting solution was slowly transferred to a 96-well plate, and p-dimethylaminobenzaldehyde reagent (100 μL) was added to each sample or standard and incubated at 60°C for 90 minutes. The plate was quantified using a Flash multi-function reader (Varioskan TM , Thermo Scientific, USA) and the hydroxyproline content was measured at an optical density of 450 nm.

[0253] Examples 10-14. In vivo mechanical testing

[0254] To confirm the presence of AT, in vivo mechanical testing was performed using selected rat calcaneal tendons. Each sample was loaded into an Instron mechanical tester (AG-10KNX, Shimadzu, Japan) using a specially designed apparatus and then pulled in the sagittal direction at a crosshead speed of 5 mm / min and a preload of 1 N to measure tensile strength and tendon stiffness.

[0255] Examples 10-15. Statistical data analysis

[0256] All quantitative results are expressed as mean and standard deviation. Graphs were created using Origin software. Statistical analysis was performed using the Student's t-test and is expressed as *P < 0.05, **P < 0.01.

[0257] Example 11. Synthesis and characterization of PCNP hydrogel

[0258] Temperature-sensitive PPZ nanoparticle (PNP) hydrogels were designed for in situ gelation and hydrophobic drug encapsulation. PNP was synthesized by substituting hydrophobic IleOEt and hydrophilic AMPEG onto the PPZ backbone. Figure 23 As shown, the synthesized PNP structure is 1 H NMR spectroscopy confirmed that carboxylic acid groups such as chondroitin sulfate and hyaluronic acid are substituted with negatively charged functional groups in the polymer backbone, thereby mimicking the natural ECM and inducing interaction with cytokines during tissue regeneration. 1H NMR was used for quantification. The peaks at 2.0 and 2.5 indicated that the acidic groups were successfully conjugated to the PNP hydrogel backbone. The molecular weight of PNP was 62920 Da using GPC, and polystyrene was used as the standard material ( Figure 23 c). Use 1 The H NMR spectrum showed the presence of IleOEt, AMPEG, EtOH, and carboxylic acid groups in 71%, 17%, 1%, and 11% ratios, respectively. The high hydrophobic IleOEt content induced hydrophobic gelation and tight hydrophobic interactions with CXB. Figure 24 As shown in Figure 2, FTIR spectra of PCNP, PNP, and CXB confirmed the CXB loading of PCNP. The aromatic C=C peak at 1530–1630 cm-1 observed in the PCNP data indicated CXB loading.

[0259] Typically, CXB needs to be dissolved in an organic solvent or chemically modified to achieve medical purposes. However, the present invention can successfully dissolve CXB in PNP simply by mixing it in a solution state without the need for solvents or chemical modifications. Figure 25 As shown in Figure 2, the gel state is maintained based on the temperature-sensitive gelling properties of PNP. PCNP was prepared by stirring CXB with a 10 wt% PNP solution at 4°C, thereby encapsulating CXB in PNP. The temperature-dependent gelation of PCNP was confirmed by injecting the polymer solution into water at 4°C and 37°C. Figure 26 As shown, unlike the polymer solution at 4°C, a gel-like 3D structure was observed when the 37°C group was injected into the aqueous solution. This means that the sol-gel transition is likely to gel within seconds after injection into the body, so a stable 3D structure can be formed without dispersion. The storage modulus and loss modulus of the PNP and PCNP hydrogels were monitored while precisely controlling the temperature between 5°C and 60°C to determine the temperature-dependent quantitative rheological characteristics of the gel. The sol-gel transition of the PNP and PCNP hydrogels occurs within a certain temperature range (20-25°C) and remains in a gel state at body temperature (37°C). The storage modulus begins to increase with increasing temperature, and the highest value is observed at approximately 35°C to 38°C. As Figure 27 shown.

[0260] The energy storage and loss coefficients of PCNPs are affected by the CXB concentration ( Figure 27b to d). The maximum storage modulus of the PNP hydrogel at 35.1°C was 454.3 Pa. However, the maximum storage modulus of the PCNP (CXB 1 mg) and PCNP (CXB 4 mg) hydrogels was 655.0 Pa at 38.1°C and 994.3 Pa at 37.6°C. No significant differences were observed in the temperature and maximum storage modulus between the PNP and PCNP hydrogel groups. However, the maximum storage modulus value increased after the addition of CXB. This indicates that the hydrophobic interaction between the PNP hydrogel and CXB molecules increased the modulus due to the role of CXB as a hydrophobic crosslinker. These data indicate a successful interaction between the PNP and CXB molecules.

[0261] Example 12. Composite between CXB and PNP

[0262] The hydrophobic CXB was found to be uniformly dissolved and well distributed in the PNP solution. The PCNPs were formed by simple stirring at low temperatures, a clear advantage of this system. Due to the hydrophobic nature of CXB, toxic solvents or surfactants are typically required to improve its solubility in clinical trials. However, the fact that CXB can be easily incorporated into the PNPs at low temperatures without the need for toxic solvents or surfactants is a significant advantage.

[0263] It was observed that amphiphilic PNP hydrogel networks formed at a given concentration and body temperature through hydrophobic interactions between polymer chains. However, the amphiphilic nature of PNPs means that the polymer structure can self-assemble through single chain folding to form polymer micelle particles at low concentrations (0.5 wt%). The morphologies of PNPs and PCNPs were characterized in both the gel state and the diluted state ( Figure 28 a), the results showed that PNP and PCNP hydrogels exhibited porous structures with pore sizes of 6.2 μm and 4.9 μm, respectively. The surface structure of PCNP was rougher than that of PNP. However, no CXB aggregation was observed, indicating that the hydrophobic CXB was well integrated into the PCNP hydrogel matrix. After 20-fold dilution, both PNP and PCNP formed micelles by self-assembly, and the particle structure was observed. Various particle sizes were measured. However, the particles in the PCNP group were smaller than those in the PNP group. The hydrogel and particle morphology of the PNP and PCNP hydrogels indicated that CXB was successfully encapsulated in PNP, and the PCNP morphology was affected by the encapsulated CXB.

[0264] To further confirm the hydrophobic interaction between PNP and CXB, the changes in the size of polymer nanoparticles before and after mixing were studied by adding different concentrations of CXBs ( Figure 28b). This experiment was conducted under precise temperature conditions to observe the particle size differences caused by hydrophobic interactions between PNP and CXB molecules. As the temperature increased from 4°C to 45°C, the PNP particle size was observed to gradually decrease from 240.0 nm to 126.2 nm. This is a result of the correlation between temperature and hydrophobicity. The hydrophobic moieties interact closely at high temperatures, resulting in polymer nanoparticles with a contracted shape. PNP and PCNP particles maintained similar sizes regardless of temperature. This demonstrates that the hydrogel network possesses stable temperature-sensitive and reversible physical properties due to non-covalent interactions, offering multiple advantages, including facile drug delivery and injectability. Upon incorporation of CXB into PNP, the particle size decreased with increasing CXB concentration at all temperatures. This suggests that the CXB molecules within the PNP act as hydrophobic crosslinkers, allowing the PCNP to form a more contracted structure. Therefore, the interaction between CXB and the PNP hydrogel demonstrates that CXB can be encapsulated into PNP at varying concentrations without surfactant, potentially enhancing therapeutic efficacy and minimizing side effects.

[0265] like Figure 28 As shown in Figure 3, the zeta potential and particle size were studied together. Due to the carboxyl groups of the PNP backbone, the zeta potential of PNP was measured to be -19.9 mV. However, after incorporating CXB into PNP, the zeta potential of PCNP (CXB 1 mg) and PCNP (CXB 4 mg) decreased significantly to -13.1 and -9.5 mV, respectively. This is because the hydrophobic CXB, which is smaller than the PNP particle size, is encapsulated in the core of the PCNP micelle structure ( Figure 28 b). This indicates that the hydrophilic PEG on the PCNP surface covers the negative charges, so the hydrophobic interaction successfully binds CXB to PNP to form PCNP with small pores and particle size.

[0266] Example 13. Mass loss of PCNP hydrogel in vitro and in vivo and sustained release of CXB from the hydrogel

[0267] To predict the maintenance level of PCNP hydrogel after injection into the body, the mass loss of the hydrogel was measured and the release of CXB from the hydrogel was analyzed to determine whether the PCNP hydrogel could retain and release CXB for a long time. The results are shown in Figure 2. Figure 29 The in vitro disintegration data showed the swelling and disintegration pattern in an aqueous environment. The PNP hydrogel swelled to 147% within 3 days and then slowly dissolved within 5 weeks ( Figure 30 ).like Figure 29As shown in Figure (a), the CXB release rate from the PCNP hydrogel remained nearly constant over 29 days, even under high swelling conditions, due to the stable interaction between the PNP hydrogel and CXB. The higher CXB release rate in the 1 mg CXB group was likely due to increased hydrophobic interactions between CXB and PNP at higher CXB concentrations, resulting in a more compact structure.

[0268] PCNPs biodegraded via hydrolysis, resulting in a sustained release of CXB over 28 days. The observed correlation between the degradation and release profiles indicated that CXB release was associated with the dissolution of the PCNP hydrogel. Furthermore, PCNP swelling observed early in the experiment (~7 days) did not result in a sudden release of CXB, suggesting that release was related to dissolution rather than swelling of the PCNP hydrogel.

[0269] CXB release data were analyzed by fitting various kinetic models, including zero-order model, first-order model, Higuchi model and Ritger-Peppas model (Table 5 below). Comparison of correlation coefficients (R2) showed that the Ritger-Peppas model was most suitable for both PCNP (CXB 1 mg) and PCNP (CXB 4 mg). When the Ritger-Peppas model was used, a mixed release mechanism including swelling, erosion and polymer porosity may be involved. Since CXB is released by swelling and decomposition of PCNP, this kinetic model is applicable to the case of simultaneous use with PCNP. In addition, the diffusion mechanism can be explained by the diffusion index (n). The diffusion index (n) of PCNP (CXB 1 mg) and PCNP (CXB 4 mg) was calculated to be 0.62 and 0.57, respectively, both resulting in non-Fickian diffusion.

[0270]

Table 5

[0271]

[0272] To measure the biodegradation rate in vivo, the PNP hydrogel was subcutaneously injected into the back of mice. Unlike the dispersion of the polymer solution observed at 4°C, the PNP hydrogel rapidly gelled at body temperature due to its temperature-sensitive gelling properties ( Figure 26 Furthermore, the PNP hydrogels maintained a stable modulus in the angular frequency range of 1 to 100 rad / s at 37 °C ( Figure 31 This indicates that the PNP hydrogel is suitable for injection and maintains a gel structure in vivo. The mass of the injected PNP hydrogel increased to 111% on the second day in the in vivo environment and then continued to decrease for more than 28 days ( Figure 29 b). This indicates that the PNP hydrogel in vivo gradually decomposes after the initial water uptake of the PNP hydrogel network. Figure 32The results showed that the size of the PCNP hydrogel gradually decreased in vivo. These results indicate that CXB is slowly released after injection of PCNP hydrogel in vivo and that the PCNP hydrogel remains at the injection site for approximately 1 month.

[0273] Example 14. Inhibitory Proinflammatory Cytokines and Anti-inflammatory Effects of PCNP Hydrogel on Defective Tendon

[0274] Prior to in vivo experiments, the in vitro cytotoxicity of PNP hydrogels was investigated to confirm the side effects ( Figure 33 Even at high concentrations of PNP (10 mg mL-1), cell viability remained above 70%. The mass loss data from in vivo experiments showed that the 200 μg PNP hydrogel injected decomposed within 30 days, indicating that approximately 6.6 μg PNP was released from the injected hydrogel structure per day ( Figure 29 ). Therefore, PNP hydrogel was confirmed to be a biocompatible drug carrier with no side effects. In addition, PNP hydrogel decomposes into non-toxic substances such as phosphate, poly (ethylene glycol) and ammonia through hydrolysis.

[0275] The cytotoxicity of CXB and PCNP is as follows Figure 33 b and Figure 33 Figure c shows the CXB group exhibiting high cytotoxicity at 625 μg / ml, while the PCNP group exhibited no significant cytotoxicity at 40,000 μg / ml CXB. This is because the low solubility of CXB leads to aggregation, exposing cells to high concentrations of highly cytotoxic drugs. PNPs can deliver drugs without aggregation or cytotoxicity, making them suitable carriers for CXB.

[0276] PCNP can effectively encapsulate different volumes of CXB, which can effectively control the therapeutic dose for the successful treatment of many inflammatory diseases. In the present invention, PCNP (CXB 1 mg) and PCNP (CXB 4 mg) as injection doses were used to evaluate the therapeutic effect of AT.

[0277] CXB reduces inflammation and pain by increasing the expression of anti-inflammatory cytokines such as IL-4 and IL-10. CXB treatment also reduces the expression of pro-inflammatory cytokines (TNF-α, IL-1, IL-6, COX-2, MMP-3, and MMP-13) in cartilage and synovium. These anti-inflammatory and pro-inflammatory cytokines are directly related to tendon healing. Pro-inflammatory cytokines affect ECM homeostasis and tendon remodeling, but can also promote tenocyte apoptosis. IL-4 increases the proliferation of tenocytes involved in tendon healing, while IL-10 affects cell proliferation and survival through the STAT3 signaling pathway.

[0278] To analyze the pro-inflammatory cytokine suppression and anti-inflammatory effects of the developed carrier, PCNP hydrogels were injected into the defective Achilles tendon model, and the levels of anti-inflammatory and pro-inflammatory cytokines were determined using RT-PCR ( Figure 34 The levels of anti-inflammatory cytokines (IL-4 and IL-10) in the blood were measured after 4 weeks of treatment ( Figure 34 a and b). Compared with the control group, IL-4 expression increased by 1.2 times in the group treated once with CXB, and increased by 1.3 times in the group treated three times. Compared with the control group, IL-4 expression increased by 1.5 times and 1.7 times in the PCNP (1 mg and 4 mg CXB) injection groups, respectively, and IL-10 showed a similar pattern of increasing by 1.3 times and 1.5 times, respectively. The PCNP (1 and 4 mg CXB) groups increased by 1.8 times and 2.2 times, respectively. Compared with the control group, there was no significant change in the expression of anti-inflammatory cytokines in the untreated group, which indicates that no anti-inflammatory effect would be produced without treatment. The anti-inflammatory cytokine gene expression level was higher in the CXB solution-treated group, and the expression of anti-inflammatory cytokines was higher after injection of PCNP hydrogel. This shows that CXB injection caused an anti-inflammatory effect, and PCNP injection further enhanced the anti-inflammatory effect due to the sustained release of CXB locally in the defective Achilles tendon.

[0279] The expression of proinflammatory cytokines (COX-2, IL-1, IL-6, MMP-3, MMP13, and TNF-α) was further investigated and shown to be Figure 34 c to Figure 34 h. Compared with the control group, the expression of COX-2, IL-1, IL-6, MMP-3, MMP13 and TNF-α in the untreated group with severe inflammation was 7.6-, 6.4-, 7.1-, 7.1-, 8.1- and 7.8-fold, respectively. Compared with the control group, the expression of proinflammatory cytokines COX-2, IL-1, IL-6, MMP-3, MMP-13 and TNF-α was 6.3-, 5.4-, 6.0-, 5.9-, 7.2- and 6.7-fold, respectively, which were significantly lower than the values observed in the untreated group. In the PCNP (CXB 4 mg) hydrogel injection group, the levels of proinflammatory cytokines increased by 5.6-, 4.7-, 5.2-, 5.1-, 6.6- and 5.8-fold compared with the control group. The expression levels of most proinflammatory cytokines in all PCNP injection groups were lower than those in the CXB solution injection group. The pro-inflammatory and anti-inflammatory cytokine expression data indicated that a single local injection of PCNP hydrogel produced a higher anti-inflammatory effect than multiple injections of CXB solution due to the slow release of CXB into the defect area.

[0280] Example 15. In vivo regeneration of defective Achilles tendon

[0281] To analyze the effect of local sustained release of CXB from PCNP hydrogel on tendon regeneration in vivo, defective Achilles tendons were prepared using collagenase, and the same concentration groups were used to study the regeneration of defective Achilles tendons ( Figure 35 H&E and MT staining data showed that ECM arrangement was destroyed and collagen was decomposed in the collagenase 50 group, indicating that the tendon was completely damaged by collagenase ( Figure 35 b and Figure 35 h). The damaged tendon partially regenerated 4 weeks after injection of CXB solution ( Figure 35 c and d). MT staining data clearly showed that more collagen was regenerated in the group that received 3 injections of CXB solution compared to the group that received only 1 injection of CXB ( Figure 35 i and j). This indicates that the time the defective tendon is exposed to CXB has a positive effect on tendon regeneration. H&E staining results showed that the tendon in the PCNP hydrogel-treated group achieved tissue alignment and almost complete recovery ( Figure 35 e and Figure 35 f). MT staining results showed that collagen regeneration was improved in the PCNP hydrogel group compared with the group injected with CXB solution. The CXB concentration of PCNP hydrogel also affected tendon regeneration ( Figure 35 e to Figure 35 These results suggest that continuous exposure of defective tendons to CXB is required to induce long-term regeneration of defective tendons.

[0282] In vivo regeneration and functional recovery of tendon structures using hydroxyproline assays, mechanical stiffness, and strength testing ( Figure 36 ). Collagen is one of the main components of tendon, providing rigid mechanical properties. Collagen is mainly composed of hydroxyproline. Therefore, the amount of hydroxyproline in the tissue represents the amount of collagen. It can be seen that the hydroxyproline content of 35.5μg / mg normal tendon tissue was significantly reduced to 11.3μg / mg in the untreated group ( Figure 36 A) Hydroxyproline content increased in the CXB solution (injected once and three times weekly) groups, reaching 15.3 μg / mg and 17.2 μg / mg, respectively. Further increases were observed in the PCNP hydrogel injection groups (CXB 1 mg and 4 mg), reaching 21.4 μg / mg and 24.4 μg / mg, respectively. Hydroxyproline content was proportional to the number of CXB solution injections and the CXB concentration in the PCNP hydrogel. PCNP hydrogel provided longer CXB exposure time to the Achilles tendon defect than CXB solution, and the different concentrations of encapsulated CXB may directly affect the therapeutic effect.

[0283] The stiffness and tensile strength of the treated tendons were measured, and mechanical and functional regeneration were evaluated by comparison with normal tendons ( Figure 36b and c). In the untreated group, the normal tendon stiffness decreased significantly from 36.3N / mm to 13.7N / mm. The groups treated with CXB solution (injected once and 3 times per week) had higher stiffness (16.4 and 18.6N / mm), while the stiffness of the PCNP hydrogel-treated group increased to 24.5 and 26.4N / mm (CXB 1mg and 4mg), which is similar to the normal tendon value. The tendon regeneration capacity of the CXB solution-treated group was improved, and this effect was more obvious in the PCNP hydrogel-treated group. The tensile strength data were similar to the stiffness results. The tensile strength of the normal tendon in the untreated group decreased from 33.7MPa to 11.4MPa. It was observed that the tensile strength of the CXB solution-treated group (injected once and 3 times per week) was 12.5 and 14.7MPa, respectively, and the PCNP hydrogel-treated group (CXB 1mg and 4mg) recovered to 66% and 71% of the normal Achilles tendon tissue, respectively. This indicates that PCNP hydrogel induced tendon ECM regeneration and restoration of tendon mechanical function.

[0284] Although this disclosure includes specific examples, it will be apparent to those skilled in the art that various changes in form and details may be made in these examples without departing from the spirit and scope of the claims and their equivalents. Suitable results can be obtained if the described techniques are performed in a different order, and / or if components in the described systems, architectures, devices, or circuits are combined in a different manner, and / or replaced or substituted with other components or their equivalents.

[0285] Accordingly, other implementations are within the scope of the following claims.

[0286] This invention was completed with the support of the following projects: Technology development of intelligent solutions for disaster safety (Project No.: 2E31600; Project time: 20220101 to 20221231), Technology development of local drug delivery platforms for the treatment of general muscle main chain diseases (Project No.: 1711056093, Project time: 20170401 to 20171231), and Development of hydrogel engineering technology for neural network reconstruction (Project No.: 1711134231, Project time: 20210701 to 20211231).

Claims

1. A composition for preventing or treating inflammatory diseases, characterized in that: Include: Polyphosphazene polymers; and A therapeutically effective amount of a drug for controlling and treating inflammation, The polyphosphazene polymer comprises the following components on the phosphorus atoms of the polyphosphazene main chain represented by the following chemical formula 1: The first part is an amino acid ester represented by the following chemical formula 2; A second part, which is polyethylene glycol represented by the following Chemical Formula 3; and The third part, which contains aminoethanol and organic acid, [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] In the chemical formula, R1 is C 1-6 Alkyl, C 1-6 Alkenyl or C 6-10 Aryl-C 1-6 alkyl; R2 is hydrogen, methyl, isopropyl, 1-methylpropyl, 2-methylpropyl, thiomethyl, methylthioethyl, benzyl, hydroxybenzyl or 2-indolylmethyl; R3 is C 1-6 alkyl; n is an integer from 3 to 100,000, p is an integer from 1 to 20, The molar ratio of a:b is 5.5:1 to 7.5:1, a is 70 mol% to 80 mol%.

2. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The organic acid in the third portion includes glutaric acid.

3. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The R1 is methyl, ethyl, propyl, butyl, benzyl or 2-propenyl.

4. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The R3 is a methyl group.

5. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The drug for treating inflammatory diseases is hydrophobic.

6. The composition for preventing or treating inflammatory diseases according to claim 5, characterized in that The hydrophobic drug for treating inflammatory diseases is at least any one selected from the group consisting of triamcinolone acetonide, methylprednisolone, dexamethasone, celecoxib, ibuprofen, naproxen, indomethacin, ketoprofen, etodolac, meloxicam, rofecoxib, etoricoxib, valdecoxib, lumiracoxib and diclofenac.

7. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The polymer slowly releases the drug for treating inflammatory diseases.

8. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The slow release is to release the drug at a drug concentration within the therapeutically effective amount range.

9. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The polyphosphazene polymer is dissolved in a solvent at a concentration of 1 wt % to 50 wt %.

10. The composition for preventing or treating inflammatory diseases according to claim 9, characterized in that The solvent is at least one selected from the group consisting of water, buffer solution, acidic solution, alkaline solution, saline solution, physiological saline, water for injection, cell culture medium and glucose saline.

11. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The polymer exhibits a sol-gel transition behavior in the range of 5° C. to 70° C. and forms a hydrogel at a predetermined set temperature.

12. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The polymer gels when applied to a living body or an in vitro environment and loses temperature sensitivity at a predetermined set temperature, thereby maintaining a gel state regardless of temperature.

13. The composition for preventing or treating inflammatory diseases according to claim 1, characterized in that The inflammatory disease is any one selected from the group consisting of osteoarthritis, degenerative arthritis, rheumatoid arthritis, osteoporosis and Achilles tendinitis.

Images