Compositions for the treatment of joint diseases and their preparation methods

Abstract

This invention relates to a composition for the treatment of joint diseases and a method for preparing the same, and more specifically, to a composition comprising a crosslinked hyaluronic acid hydrogel obtained by a crosslinking reaction of hyaluronic acid or a pharmaceutically acceptable salt thereof with an alkylene diamine crosslinking agent and a method for preparing the same. The composition is characterized by a storage modulus (G', 2.5 Hz, 25°C) of 250-850 Pa and a degree of crosslinking of the crosslinked hyaluronic acid (CrD; number of crosslinking agents bound at both ends / number of hyaluronic acid units) of less than 5 mol%; when the composition is administered to a patient once over a period of more than 3 months in injectable form, it can show an improvement in joint disease symptoms. The composition has a low degree of crosslinking to allow for flexible adjustment of the dosing interval, while maintaining high biocompatibility, biodegradability, and clinically significant storage modulus, thus reducing the burden on patients caused by frequent dosing, and can be expected to provide excellent clinical results with improvement in joint disease symptoms for more than 3 months.

Description

Technical Field

[0001] This invention relates to a composition for the treatment of joint diseases and a method for preparing the same, and more specifically, to a cross-linked hyaluronic acid hydrogel composition and a method for preparing the same, obtained by a cross-linking reaction of hyaluronic acid or a pharmaceutically acceptable salt thereof with an alkylene diamine cross-linking agent. The composition maintains high biocompatibility and clinically significant storage modulus even at low degrees of cross-linking, thereby reducing the burden on patients caused by frequent administration, enabling flexible dosing intervals, and can be expected to improve joint disease symptoms for more than 3 months. Background Technology

[0002] With an aging population, the prevalence of joint diseases is increasing globally. Among them, osteoarthritis, the most common joint disease, is a condition that causes joint pain and degeneration, leading to functional impairment. Hyaluronic acid (HA), a biomolecule that constitutes synovial fluid, has a structure composed of linearly linked disaccharide repeating units of β-N-acetyl-D-glucosamine and β-D-glucuronic acid. It is known that when injected into the joint, it acts as a lubricant and shock absorber, thereby reducing joint pain and improving joint mobility.

[0003] Hyaluronic acid preparations used for the treatment of joint diseases are typically developed as intra-articular injections. However, unmodified hyaluronic acid is rapidly broken down and excreted from the body by hyaluronidase within a few days, resulting in low bioavailability. Therefore, unmodified hyaluronic acid preparations are developed as 3-5 doses administered weekly over a period of 6 months. However, this approach carries the risk of synovial membrane damage due to frequent use and the inconvenience of requiring frequent patient visits.

[0004] To address this issue, single-application formulations are being actively researched. These formulations utilize multiple cross-linking agents to develop hydrogels that cross-link hyaluronic acid, improving bioavailability and maintaining efficacy with only one application over six months. For example, Korean Patent No. 10-2275105 discloses a high-density network structure of cross-linked hyaluronic acid with 1,4-butanediol diglycidyl ether (BDDE) and its preparation method. Korean Patent No. 10-2400586 discloses a method for preparing a powdered hyaluronic acid cross-link suitable for mass production using BDDE and divinylsulfone (DVS) and its hydrogel.

[0005] In recent years, based on the discovery that the recognition site of hyaluronidase is the carboxyl group (-COOH group) of hyaluronic acid, cross-linking agents that use this site as the target reactive group for cross-linking reactions have been proposed to improve biocompatibility. Among these, alkylene diamine cross-linking agents, such as hexamethylenediamine, are known to have lower genotoxicity risks compared to existing cross-linking agents such as BDDE or DVS, and have been reported to have the advantage of easier removal of residual cross-linking agents, thus improving biocompatibility. For example, Korean Patent Nos. 10-0674177 and 10-1062320 disclose cross-linked hyaluronic acid prepared using such alkyl diamines and alkylene diamine cross-linking agents, their medical applications, and preparation methods.

[0006] Existing reports indicate that the type of cross-linking agent, its binding site with hyaluronic acid, and the amount of cross-linking agent residue may affect biocompatibility, and even clinical efficacy and safety. However, in the formulation and industrial application of hyaluronic acid cross-linked products using specific cross-linking agents, sufficient information and preparation methods regarding properties that can guarantee clinical efficacy, particularly the physical properties of the composition such as the degree of cross-linking (CrD) and viscoelasticity, have not yet been fully established or proposed.

[0007] Hyaluronic acid-based treatments for joint diseases are primarily developed as intra-articular injections. In the case of such invasive injections, the high frequency of repeated administration, such as current 3- or 5-dose regimens, can increase the physical and psychological burden on patients, not only exacerbating pain but also potentially increasing the risk of infection.

[0008] On the other hand, while single-dose formulations that maintain their effectiveness with once-every-six-month treatments offer the advantage of avoiding repeated administration, insurance policies typically include a lull period before the next treatment date during which medical expenses are not reimbursed. Considering the characteristics of joint diseases and the widely recognized necessity of combining medications with oral medications and physical therapy, and taking into account patients' preferences regarding the frequency of hospital visits and the financial burden of non-insurance coverage, the availability of multiple options for medication intervals is a more preferable option for patients.

[0009] Therefore, there is a need to develop a composition for the treatment of joint diseases based on cross-linked hyaluronic acid with high bioavailability and high biocompatibility, which can flexibly adjust the application interval according to the patient's needs and maintain clinical efficacy for several months, as well as its preparation method.

[0010] [Existing Technical Documents] [Patent Literature] Korean Patent No. 10-2400586 (published on June 17, 2020) Korean Patent No. 10-2275105 (published on March 18, 2015) Korean Patent No. 10-1400907 (published on January 18, 2012) Korean Patent No. 10-1062320 (published on February 5, 2009) Korean Patent No. 10-0674177 (published November 14, 2001) / International Application PCT / EP1999 / 008481 (WO2000 / 027887; published May 18, 2000) Korean Patent Publication No. 10-2013-0028012 (published on March 18, 2013) Summary of the Invention

[0011] (a) Technical problems to be solved The technical problem of the present invention is to provide a composition for the treatment of joint diseases. Compared with existing hyaluronic acid crosslinkers used for the treatment of joint diseases, the composition has a low degree of crosslinking (CrD) that allows for flexible adjustment of the application interval, while maintaining high biocompatibility, biosustainability, and a storage modulus (G') that can exert clinical efficacy. This reduces the burden on patients caused by frequent application and can provide improvement in the clinical symptoms of joint diseases for a long period of more than 3 months.

[0012] Another technical problem of the present invention is to provide the aforementioned joint disease treatment composition, a syringe kit for filling the composition with a single clinically effective volume that can exert clinical efficacy, and a method for preparing the same.

[0013] (II) Technical Solution To address the aforementioned technical problems, this invention provides a composition for treating joint diseases and a method for preparing the same. The composition comprises a cross-linked hyaluronic acid hydrogel obtained by a cross-linking reaction of hyaluronic acid or a pharmaceutically acceptable salt thereof with an alkylene diamine cross-linking agent. The storage modulus (G', 2.5 Hz, 25°C) of the composition is 250-850 Pa. The degree of cross-linking (CrD) of the cross-linked hyaluronic acid is less than 5 mol%. The composition is administered to the patient once in injectable form over a period of more than 3 months.

[0014] Hyaluronic acid formulations are proposed as viscoelastic-based visco-supplementation preparations, with higher molecular weights or viscoelasticity believed to correlate with better pain relief and other efficacy. Storage modulus or elasticity (G') and loss modulus or viscosity (G'') are considered. However, considering the diverse molecular weights and viscoelasticity ranges of approved products, it is highly likely that while a correlation exists between viscoelasticity and efficacy, this correlation is not a simple linear relationship. This aligns with our animal studies and clinical trial results, which indicate that, depending on composition, the correlation between viscoelasticity and efficacy is not a simple direct proportionality (Experimental Example 7; Table 7 and...). Figure 2 In addition, the literature also reports that, in addition to the viscoelasticity of hyaluronic acid, various physical properties and the interaction between hyaluronic acid and other components in synovial fluid may also affect efficacy (Balazs EA et al., Arthritis Rheum. 1967;10(4):357–376; Korean Patent No. 10-2400586).

[0015] In this invention, while defining the range of storage modulus G' for clinical efficacy, factors leading to a decrease in storage modulus G' in existing preparation methods are eliminated, and the preparation process is improved. Therefore, this invention novelly proposes a composition and its preparation method that still possesses a storage modulus (G') capable of exerting effective clinical effects in the human body even when the degree of crosslinking is less than 5 mol%.

[0016] In addition, depending on the "degree of crosslinking" (CrD; there are also instances where it is mistranslated as "crosslinking rate," but this specification uses "degree of crosslinking" consistently), the storage modulus (G') and residual amount of crosslinking agent in the final composition may vary, which may affect clinical efficacy and biocompatibility. The "degree of crosslinking (CrD)" refers to the molar ratio of crosslinking agent molecules that undergo crosslinking in the disaccharide units of hyaluronic acid (Kenne et al., CarbohydratePolymers 2013;91:410-418). It can be expressed as the product of the "degree of modification" (MoD; defined as the molar ratio of crosslinked hyaluronic acid relative to the total number of moles of hyaluronic acid disaccharide; sometimes translated as "crosslinking modification rate" or mistranslated as "degree of crosslinking" in some literature; this specification uses "degree of modification" consistently) and the "crosslinking ratio" (CrR; defined as the ratio of crosslinking agent molecules that undergo crosslinking relative to the total number of crosslinking agent molecules bound to hyaluronic acid; sometimes translated as "crosslinking ratio" or "effective crosslinking rate" in some literature; this specification uses "crosslinking ratio" consistently).

[0017] For crosslinked hydrogels crosslinked with alkylene diamines, it has been disclosed that when prepared by existing preparation methods, they typically exhibit a crosslinking degree of 5 mol% to 20 mol%. For example, embodiments of Korean Patent No. 10-1400907 and Korean Patent No. 10-0674177 disclose that, although there may be differences due to variations in reaction conditions such as the molecular weight of hyaluronic acid, the molar number of crosslinking agents relative to hyaluronic acid, initial concentration and pH, washing and swelling conditions, and the methods used in the residual crosslinking agent removal step (e.g., ethanol precipitation and the use of dialysis membranes), when prepared within the range of hyaluronic acid content and crosslinking agent molar percentage commonly used in the art, after the removal steps of residual crosslinking agents and unreacted substances, the composition exhibits a crosslinking degree of 5 mol% or more, mainly exhibiting a crosslinking degree of 6 mol% to 14 mol%.

[0018] Hyaluronidase, acting as a degrading enzyme, breaks down hyaluronic acid by recognizing its carboxyl groups (-COOH groups). Typically, when the degree of cross-linking is less than 5 mol%, the proportion of unreacted carboxyl groups that did not participate in cross-linking increases. This makes hyaluronic acid easily degraded by hyaluronidase, leading to difficulties in achieving target levels of biocompatibility. Furthermore, hydrogels with low cross-linking degrees exhibit decreased viscoelasticity after subsequent sterilization or heat treatment, making it difficult to achieve a clinically effective storage modulus (G'), thus hindering the expectation of clinical efficacy. On the other hand, when the degree of cross-linking exceeds 35 mol%, the swelling properties of the hydrogel decrease, potentially leading to viscoelasticity issues that may not demonstrate the degree of joint protection it is beneficial to. Additionally, the increased content of residual cross-linking agents after in vivo degradation increases the likelihood of side effects such as inflammatory reactions, potentially reducing biocompatibility. Current manufacturing processes use dialysis membranes to remove residual cross-linking agents, and the fine grinding required for dialysis may lead to a decrease in storage modulus. For hydrogels with a moderate degree of crosslinking, ethanol precipitation is sometimes used instead of dialysis to remove crosslinking promoters soluble in organic solvents such as HOBt, removing residual crosslinking agents while precipitating hydrogel particles. However, this process is known to cause a decrease in the storage modulus (G'). Therefore, if residual crosslinking agents can be adequately removed without dialysis or ethanol precipitation while maintaining the storage modulus (G'), it would not only enable various formulation adjustments such as application interval adjustments, but also reduce the initial amount of hyaluronic acid required. Furthermore, by shortening the process steps and reducing quality management costs, it would help lower production costs.

[0019] Therefore, in order to solve the above-mentioned technical problems, this invention provides a method for preparing a low-crosslinking composition that removes residual crosslinking agents to less than 2 ppm (the detection limit) without using dialysis or ethanol precipitation, and a composition that enables the application of this method. It has been confirmed that this method can maintain a clinically effective storage modulus (G') without reducing the storage modulus (G').

[0020] The present invention will now be described in more detail.

[0021] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art related to this invention. Furthermore, while preferred methods or embodiments are described in this specification, similar or equivalent content is also included within the scope of this invention. The entire contents of all publications referenced in this specification are incorporated herein by reference.

[0022] According to one aspect of the present invention, a composition for treating joint diseases and a method for preparing the same are provided. The composition for treating joint diseases comprises a crosslinked hyaluronic acid hydrogel obtained by a crosslinking reaction of hyaluronic acid or a pharmaceutically acceptable salt thereof with an alkylene diamine crosslinking agent represented by Chemical Formula 1 or Chemical Formula 2; the storage modulus (G', 2.5 Hz, 25°C) of the composition for treating joint diseases is 250-850 Pa; the degree of crosslinking of the crosslinked hyaluronic acid (CrD; number of crosslinking agents bound at both ends / number of hyaluronic acid units) is less than 5 mol%; the composition is administered to a patient once in injectable form over a period of more than 3 months. [Chemical Formula 1] [HA] x -C(O)-NH-R1-NH-C(O)-[HA] y [Chemical Formula 2] [HA] z -C(O)-NH-R1-NH2 In chemical formulas 1 and 2, HA is hyaluronic acid with one carboxyl group removed or a pharmaceutically acceptable salt thereof; R1 is unsubstituted or substituted C3-C 10 Alkylene; x, y, and z are each independent integers from 100,000 to 5,000,000.

[0023] The hyaluronic acid in this invention is a biopolymer composed of linearly linked disaccharide repeating units of β-N-acetyl-D-glucosamine and β-D-glucuronic acid, and can be hyaluronic acid or its pharmaceutically acceptable salts. The molecular weight of the hyaluronic acid or its pharmaceutically acceptable salts can be from 100,000 to 5,000,000 Daltons (Da), and the intrinsic viscosity can be 1.0 m. 3 / kg to 4.0m 3 / kg, but not limited to this.

[0024] According to one embodiment of the present invention, the pharmaceutically acceptable salt of hyaluronic acid can be an inorganic acid salt such as sodium, potassium, calcium, magnesium, zinc, or cobalt salts, or an organic salt such as tetrabutylammonium salts, but is not limited thereto. According to one embodiment of the present invention, the pharmaceutically acceptable salt of hyaluronic acid can be a sodium salt. According to one embodiment of the present invention, the hyaluronic acid or its pharmaceutically acceptable salt can be a product isolated from microorganisms, synthesized, or commercially purchased, but is not limited thereto.

[0025] According to one embodiment of the present invention, R1 can be an unsubstituted or C4-C6 alkylene group substituted with hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy groups. According to one embodiment of the present invention, R1 can be an unsubstituted C4-C6 alkylene group. According to one embodiment of the present invention, the alkylene diamine crosslinking agent can be hexamethylenediamine (HMDA). According to one embodiment of the present invention, the alkylene diamine crosslinking agent can be applied to the reaction in the form of hexamethylenediamine dihydrochloride (1,6-hexamethylenediamine dihydrochloride).

[0026] The crosslinking reaction in this invention refers to the reaction in which the carboxyl group of hyaluronic acid or its pharmaceutically acceptable salt combines with an alkylene diamine crosslinking agent. One or both ends of the alkylene diamine crosslinking agent can crosslink with hyaluronic acid or its pharmaceutically acceptable salt, and each hyaluronic acid or crosslinking unit can be randomly continuous.

[0027] According to a specific embodiment of the present invention, the storage modulus (G', in Pascals, Pa) of the composition, under analytical conditions of 2.5 Hz and 25°C, can be from 250 Pa to 850 Pa (250-850 Pa), preferably from 342 Pa to 721 Pa (342-721 Pa). In this case, the composition can be a therapeutic composition for joint diseases consisting solely of cross-linked hyaluronic acid, a mixture of unmodified hyaluronic acid and cross-linked hyaluronic acid, or a mixture containing a pharmaceutically acceptable carrier. It is a finished pharmaceutical product, filled into a syringe or the like and subjected to heat treatment or sterilization, ready for administration to a patient. The storage modulus (G') refers to the storage modulus of the finished pharmaceutical product at the time of manufacture or the estimated storage modulus (G') inferred for clinical application to a patient.

[0028] According to a specific embodiment of the present invention, the degree of crosslinking (CrD) of the crosslinked hyaluronic acid in the composition can be greater than 0 mol% and less than 5 mol%, preferably greater than 0 mol% and less than 1 mol%. In this invention, "degree of crosslinking (CrD)" refers to the molar ratio (mol%) of hyaluronic acid in the crosslinked product that is crosslinked at both ends of a hyaluronic acid unit, defined by the following formula (Kenne et al., Carbohydrate Polymers 2013;91:410–418): According to one embodiment of the invention, the composition may be a mixture of cross-linked hyaluronic acid hydrogel diluted to a suitable concentration with the same phosphate buffer solution (pH 6.0 to 8.0) used in washing the hydrogel and then mixed with unmodified hyaluronic acid. According to another embodiment of the invention, the composition may be a composition characterized by adjusting the cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid to a baseline content and mixing them at a weight ratio (w / w) of 70:30 to 99:1. Preferably, the composition may be a composition characterized by a baseline content of 2 w / w% of cross-linked hyaluronic acid to unmodified hyaluronic acid and a weight ratio of 90:10 of cross-linked hyaluronic acid hydrogel to unmodified hyaluronic acid.

[0029] According to one specific embodiment of the present invention, the joint disease can be osteoarthritis, rheumatoid arthritis, or psoriatic arthritis, but is not limited thereto. The injectable containing the composition can be used in combination with nonsteroidal anti-inflammatory drugs, immunosuppressants, antirheumatic drugs, biological agents, and physical therapy. In one specific embodiment of the present invention, intra-articular administration of the composition reduces the release of MMP-3 replaced by human VEGF165a in the synovial fluid of beagle joints, and it is proposed that these substances are not only important therapeutic targets for osteoarthritis, but also for human rheumatoid arthritis (Experimental Example 8; Pulik L et al., Reumatologia 2023; 61(3):191–201; Kim J et al, Exp. Mol. Med. 2020; 52: 843–853). The above-mentioned joint diseases can occur in the knee, spine, shoulder, arm, leg, finger joints, etc., but are not limited thereto. According to one embodiment of the present invention, the osteoarthritis may be knee osteoarthritis. According to one embodiment of the present invention, the treatment may be for the improvement of symptoms, cure, or inhibition of progression, preferably for the improvement of joint pain or joint function, but is not limited thereto.

[0030] According to another aspect of the invention, a kit comprising a syringe filled with the composition can be provided, preferably a kit comprising a pre-filled syringe, which takes into account the specificity of treatment methods for joint diseases such as intra-articular injection, in order to reduce the risk of infection during injection, and in combination with the limited volume of the human joint cavity, to fill the effective volume of a single clinical injection at a dose of 2 mL to 6 mL. According to literature reports, the average volume of synovial fluid in human joints is 6.7 ± 2.3 mL (Heilmann H et al., Z Orthop Unfall. 1996;134(2):144–8). In addition, a clinical trial involving 253 patients has been reported to demonstrate that intra-articular injection of 6 mL of cross-linked hyaluronic acid preparation, administered twice at a rate of once every 6 months, has a tolerable safety profile up to 26 weeks (Chevalier Xet al., Ann Rheum Dis. 2010;69(1):113-9). Furthermore, the maximum known volume of currently approved intra-articular injectables is 6 mL (Synvisc One®, Genzyme), which also supports the rationality of the proposed maximum effective volume (6 mL) for a single clinical injection. When cross-linked hyaluronic acid is applied to the human body, especially for intra-articular injection, the limited joint cavity volume and the high storage modulus (G') of cross-linked hyaluronic acid may result in high extrusion force during injection. Therefore, the design and filling volume of the pre-filled syringe must consider not only the initial storage modulus (G'0) of cross-linked hyaluronic acid and the effective volume for clinical efficacy in the synovial fluid after injection, but also the appropriate extrusion pressure during injection and changes in storage modulus (G') during heat treatment or sterilization. Therefore, special design is required to meet the convenience of the operator and patient during injection, as well as the formulation characteristics.

[0031] According to another aspect of the present invention, a method for preparing the composition or a syringe kit filled with the composition is provided, the method comprising the following steps: i) Dissolve hyaluronic acid or a pharmaceutically acceptable salt thereof in a solvent; ii) Mix an alkaline aqueous solution into the solution, wherein the alkaline aqueous solution contains a dissolved alkylene diamine crosslinking agent, a peptide bond promoter, and a carboxyl activator, and then filter the solution; iii) Crosslinked hyaluronic acid is prepared by carrying out a crosslinking reaction for more than 10 hours under constant temperature conditions (30℃ to 50℃); iv) The cross-linked hyaluronic acid is pulverized, washed, and swollen to prepare a hydrogel; v) Homogenize the hydrogel particles to prepare a mixed hydrogel composition, either directly or by mixing. vi) Fill the hydrogel composition into a storage container or syringe and perform heat treatment or sterilization.

[0032] According to one embodiment of the present invention, the solvent is water, i.e., distilled water or water for injection. According to one embodiment of the present invention, the concentration of hyaluronic acid or its pharmaceutically acceptable salt in the solution of step i) can be from 1 w / w% to 20 w / w, preferably from 5 w / w% to 10 w / w%. According to one embodiment of the present invention, in step ii), the alkylene diamine crosslinking agent can be mixed in the form of 3.5 mol% to 80 mol% of the hyaluronic acid units, preferably from 10 mol% to 30 mol%. The alkaline aqueous solution can be an aqueous solution of lithium hydroxide, sodium hydroxide, or potassium hydroxide, but is not limited thereto. Sodium hydroxide is preferred.

[0033] According to a specific embodiment of the present invention, in step iii), to overcome the shortcomings of existing high-viscosity dedicated stirrers that fail to uniformly mix the crosslinking agent and hyaluronic acid, leading to excessive reaction, a constant-temperature stirring tank and a revolution-rotation planetary centrifugal mixer can be used. When the crosslinking reaction overreacts, not only is it difficult to control uniform quality, but depending on the conditions, foreign matter in the form of a film that is difficult to completely remove may also be generated in subsequent preparation processes, potentially leading to a serious quality reduction, such as the inability to ship the finished injectable drug. The revolution-rotation planetary centrifugal mixer provides strong uniform mixing capability by generating centrifugal force through clockwise revolution at low temperatures and counterclockwise rotation at a fixed ratio (0.3 to 0.6) of the revolution speed, thus suppressing excessive crosslinking reaction. According to a specific embodiment of the present invention, by repeatedly running the revolution-rotation planetary centrifugal mixer at a revolution speed of 300 rpm for a total of 60 minutes or more for uniform mixing, excessive crosslinking reaction can be suppressed. According to a specific embodiment of the present invention, the crosslinking reaction in step iii) can be carried out at a constant temperature of 30°C to 50°C for more than 10 hours.

[0034] According to one embodiment of the present invention, a peptide bond promoter may be further mixed in step ii). According to one embodiment of the present invention, the peptide bond promoter may be selected from N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, 1-hydroxy-7-azabenzotriazole, sulfo-N-hydroxysulfosuccinimide, and mixtures thereof. According to one embodiment of the present invention, the amount of the peptide bond promoter may be from 10 mol% to 100 mol% of the hyaluronic acid repeating unit.

[0035] According to one specific embodiment of the present invention, a carboxyl activator may be further mixed in step ii). According to one specific embodiment of the present invention, the carboxyl activator may be a 1-alkyl-3-(3-dimethylaminopropyl)carbodiimide or other 1-alkyl-3-(3-dimethylaminopropyl)carbodiimide; a 1-ethyl-3-(3-(trimethylammonio)propyl)carbodiimide or other 1-alkyl-3-(3-(trimethylammonio)propyl)carbodiimide; or a 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide or other 1-cycloalkyl-3-(2-morpholinoethyl)carbodiimide, but is not limited thereto. According to one specific embodiment of the present invention, the amount of the carboxyl activator can be from 10 mol% to 100 mol% of the hyaluronic acid repeating unit.

[0036] According to one specific embodiment of the present invention, in step iv), the washing solution used to remove unreacted residual crosslinking agent from the crosslinked hyaluronic acid and for refluxing can be a phosphate buffer solution, but is not limited thereto. According to one specific embodiment of the present invention, the phosphate buffer solution can be prepared using sodium chloride (NaCl), anhydrous disodium hydrogen phosphate (Na2HPO4), and sodium dihydrogen phosphate monobasic dihydrate (NaH2PO4·2H2O), and filtered using a 0.22 μm filter membrane. According to one specific embodiment of the present invention, the pH of the phosphate buffer solution can be 6.0 to 8.0. According to one specific embodiment of the present invention, in step iv), the time required for washing and swelling reaction can be 9 hours or more, preferably 13 hours or more. According to one specific embodiment of the present invention, in step iv), in order to remove unreacted residual crosslinking agent, the washing solution can be changed multiple times, and the mesh used during washing can have a pore size that prevents the pulverized crosslinked hyaluronic acid from being lost while facilitating the inflow and outflow of the buffer solution.

[0037] According to a specific embodiment of the present invention, in step vi), after washing and swelling are completed, the washing solution is removed, the hydrogel is dehydrated, and the composition is homogenized into particles. The homogenization of the composition can be performed using a sieve with a pore size greater than 100 μm and less than 500 μm, preferably greater than 180 μm and less than 500 μm. Smaller particle sizes lead to a decrease in storage modulus (G'), which may affect efficacy. Larger particle sizes require higher extrusion pressure during injection (Shizomo AAM et al., J. Appl. Polym. Sci., 2013;128: 2180-2185). Excessive extrusion pressure increases the probability of intraoperative injection leakage, causing significant inconvenience to the operator. Therefore, considering the use of syringes and needles commonly used in injections, the maximum particle size should not exceed 500 μm.

[0038] According to one embodiment of the present invention, in step vi), the composition is filled into the syringe, and the effective volume for a single clinical injection can be 2 mL to 6 mL, preferably 3 mL to 5 mL. According to one embodiment of the present invention, when filling a mixture of cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid in a weight ratio of 90:10, the effective volume for a single clinical injection in clinical trials of knee osteoarthritis is 3 mL. According to one embodiment of the present invention, when only cross-linked hyaluronic acid hydrogel is filled in knee osteoarthritis, the effective volume for a single clinical injection is 5 mL. Pre-filled syringes for intra-articular injection, prepared according to aseptic principles, require the entire effective volume needed for a single injection to be filled into the pre-filled syringe at once to avoid infection and contamination during injection. The syringe is then autoclaved and sterilized, and a disposable sterile needle is attached for further sterilization. Considering the decrease in storage modulus (G') caused by autoclaving, the initial storage modulus (G') needs to be precisely planned.

[0039] In order to improve process efficiency and yield, the preparation method of the joint disease treatment composition of the present invention may modify some of the above-mentioned processes or include other preparation processes.

[0040] (III) Beneficial Effects The joint disease treatment composition based on cross-linked hyaluronic acid, cross-linked with an alkylene diamine cross-linking agent according to the present invention, exhibits superior biosustainability compared to unmodified hyaluronic acid and superior biocompatibility compared to other cross-linking agents. The present invention provides a joint disease treatment composition, a syringe kit filled with the composition, and a method for preparing the same, and supports treatment methods based on the composition. The joint disease treatment composition, through the introduction of a novel preparation method that maintains a certain level of storage modulus (G') even at low cross-linking degrees, maintains high biosustainability, biocompatibility, and clinically significant storage modulus even at low cross-linking degrees, reducing the burden on patients due to frequent application and enabling flexible application intervals. Furthermore, it can demonstrate significant clinical symptom improvement for more than 3 months in patients with joint diseases such as osteoarthritis, thus possessing industrial potential in the pharmaceutical industry and other sectors. Attached Figure Description

[0041] Figure 1 This is an example of a measurement used to calculate the degree of crosslinking of crosslinked hyaluronic acid crosslinked with hexamethylenediamine according to a specific embodiment of the present invention. 1 A schematic diagram of the H NMR analysis results.

[0042] Figure 2In Experiment 7 of the present invention, Examples 22 to 27 and Comparative Example 5 were applied to a chemical substance (MIA)-induced arthritis model in SD rats, and the degree of improvement of the knee joint tissue evaluation score (Mankin score) on day 28 was compared and illustrated in the figure.

[0043] Figure 3 This is Experimental Example 8 of the present invention, in which Examples 28 to 30 and Comparative Example 5 were applied to a beagle dog osteoarthritis model, and the reduction effects of MMP-3 and VEGF165a inflammatory factors in the synovial fluid of the knee joint were compared and illustrated in the figure. (#P>0.05 compared to (vs.) solvent control group) Figure 4 In Experimental Example 9 of the present invention, after administering Examples 31 to 32 and a placebo once into the joint cavity of patients with knee osteoarthritis, the degree of improvement in weight-bearing pain (measured at 100 mm VAS) and WOMAC score relative to baseline and the percentage of patients taking the rescue medication were compared over 24 weeks and the graph is shown.

[0044] Figure 5 This is a graph showing the differences between the groups in Experimental Example 10 of the present invention, where Example 33 and Comparative Example were administered to patients with knee osteoarthritis via intra-articular injection, and the degree of improvement in joint line tenderness at week 12 after the first administration and the amount of improvement in weight-bearing pain at week 12 after the second administration were compared. Detailed Implementation

[0045] The present invention will now be described in more detail through examples. However, these examples are merely illustrative to aid in understanding the invention, and the scope of the invention is not limited thereto.

[0046] Analytical methods (1) Crosslinking degree analysis: 2,4,6-trinitrobenzenesulfonic acid (TNBS) test Theoretically, if the total crosslinking agent content and the residual crosslinking agent content are known, the amount of crosslinking agent bound at only one end during crosslinking can be calculated. The amount of crosslinking agent bound at both ends can then be calculated using the difference. The amount of the crosslinking agent alkylene diamine bound at only one end, i.e., the amount of unbound primary amine ends in the alkylene diamine that are not bound to hyaluronic acid, can be quantified by measuring the absorbance after reacting with 2,4,6-trinitrobenzenesulfonic acid (TNBS, TNBSA).

[0047] The amount of crosslinking agent bonded at both ends = (total amount of crosslinking agent - amount of crosslinking agent bonded at one end - amount of residual crosslinking agent) Degree of crosslinking (CrD) = Number of crosslinking agents bonded at both ends / Number of hyaluronic acid units Mix approximately 1 mL of the sample with 4 mL of hyaluronidase (50 U / mL) solution, stir at 8 g and react for 8 hours at 37°C, then add 35 mL of 0.1 M sodium bicarbonate buffer (pH 8.0) to dilute to 1:8. The TNBS reaction test was performed according to the manufacturer's instructions provided for TNBS (Thermo Scientific, USA; Product No. 28997). Mix 0.5 mL of diluted sample or standard with 0.25 mL of 0.01 w / v TNBS solution, stir and react for 2 hours at 37°C, then add 0.25 mL of 10% sodium dodecyl sulfate (SDS; Sigma-Aldrich, USA; Product No. 4509) solution and 0.125 mL of 1N hydrochloric acid (HCl) to terminate the reaction. The absorbance was measured at 335 nm using a UV spectrophotometer (Infinite M200; Tecan GmbH, Austria). The TNBS solution was prepared at 5 w / v using 0.1 M sodium bicarbonate buffer and diluted to 0.01 w / v before use. For the standard curve, HMDA∙2HCl was dissolved in 0.1 M sodium bicarbonate solution, and at least five concentrations were set and measured within the range of 4–24 µg / mL.

[0048] (2) Crosslinking degree analysis: Nuclear magnetic resonance spectroscopy (NMR) For the crosslinking degree of HMDA crosslinked hyaluronic acid hydrogels prepared by a novel method without a drying-hydration step, crosslinking products with low crosslinking degree were analyzed for more accurate crosslinking degree analysis. 1 The analysis was performed using H-NMR spectroscopy. For the amount of hyaluronic acid units, the relative amounts of crosslinking agent (crosslinked at only one end and crosslinked at both ends) can be calculated from the integral values ​​of the N-acetyl (-OCH3) peak on the hyaluronic acid unit, using the integral values ​​of the non-overlapping alkylene (-CH2) peaks. Furthermore, for the total amount of crosslinking agent, its relative amount is calculated from the integral values ​​of the alkylene (-CH2) peaks, and the degree of crosslinking (CrD) is obtained from their ratio. For example, representative HMDA crosslinked hyaluronic acid... 1 H-NMR spectrum as shown Figure 1 As shown. Figure 1The upper left side shows the chemical formulas of HMDA with only one end crosslinked and HMDA with both ends crosslinked. The carbon sites of HMDA with only one end crosslinked are labeled a, b, c, d, e, and f, while the carbon sites of HMDA with both ends crosslinked are labeled 1, 2, and 3. In the spectrum, the relative amount of hyaluronic acid units is calculated by dividing the integral value of the N-OCH3 base peak shown at 1.8 ppm by 3. The amount of HMDA with only one end crosslinked is calculated by dividing the integral value of the e-position -CH2 near 1.45 ppm by 2. The amount of crosslinking agent with both ends crosslinked is calculated by dividing the integral value of the 2-position -CH2 near 1.35 ppm. The peak near 1.35 ppm is an overlap peak of the b-position -CH2 peak of HMDA with only one end crosslinked and the 2-position -CH2 peak of HMDA with both ends crosslinked. Therefore, the e-position -CH2 signal value, which is the same as the b-position -CH2 signal value, is subtracted from the total peak integral value and then divided by 2. Similarly, subtract the integral values ​​corresponding to d and c from the integral values ​​corresponding to the resonance line at 1.1-1.2 ppm to calculate the integral value corresponding to the 3-position hydrogen of the crosslinking agent that is bonded at both ends. Average the calculated integral values ​​corresponding to the 2-position and 3-position hydrogens and divide by the number of hydrogen atoms (4) of the crosslinking agent that is bonded at only one end to calculate the value proportional to the relative amount of crosslinking agent bonded at both ends. The resonance line near 3 ppm overlaps with the resonance line corresponding to hyaluronic acid and therefore was not used.

[0049] Degree of crosslinking (CrD) = Number of crosslinking agents bonded at both ends / Number of hyaluronic acid units As an acid-based pretreatment method for NMR analysis, a trifluoroacetic acid (TFA)-heating method was used to decompose the unit bonds of the cross-linked hyaluronic acid composition and improve solubility. In this case, to maintain the binding between the cross-linking agent and hyaluronic acid and to only sever the bonds between repeating units of hyaluronic acid, the TFA treatment time was optimized to 30 minutes to 1 hour. 0.5 mL of the cross-linked hyaluronic acid sample was dissolved in 4.5 mL of 6.667 M TFA to achieve a final TFA concentration of 6 M, and then incubated in a water bath at 90 °C for 30 minutes. The solution was then diluted with approximately three times its volume of distilled water to a TFA concentration below 1.5 M, and the solution was thoroughly frozen in an ultra-low temperature freezer at -70 °C for at least 2 hours, followed by freeze-drying to remove the solvent. The freeze-dried sample was dissolved in more than 700 µL of D₂O solvent, loaded into an NMR tube, and subjected to NMR at 25 °C with 300 scans (more than 50), a rotation frequency of 20, and a relaxation time (delay) of 2 seconds. 1The analysis was performed using 1H NMR (AVANCE III 400 MHz NMR, BRUKER Corp., USA; Magnet System Ascend400'54).

[0050] (3) Energy storage modulus analysis The storage modulus or elasticity (G') and loss modulus or viscosity (G'') of the sample were measured using a rotational rheometer (TA Instruments Ltd., AR-2000ex, USA) (Ghosh et al., Biomacromolecules 2005;6:2857–2865). The apparatus was set to 25°C and calibrated using a 40 mm diameter, 2° aluminum cone plate measuring geometry. A sufficient amount of sample was loaded into the center between the upper and lower geometry of the plate, and adjusted so that the sample did not adhere to the sides or top of the geometry. The geometry was lowered to the set interval, and after confirming that the area under the geometry was filled with sample, any remaining sample outside the geometry was removed. The strain of the geometric component was set to 1%, and shear strain was periodically applied to the specimen during frequency oscillations of 0.1-10 Hz. The modulus value at 2.53 Hz was defined as the viscoelastic value. The damping factor (tanδ) is a numerical value characterizing whether the properties of a substance are closer to those of a solid or a liquid, calculated using G'' / G'. The closer tanδ is to 1, the closer it is to a liquid state with low elasticity and high viscosity; the closer tanδ is to 0, the closer it is to a solid state with high elasticity and low viscosity.

[0051] (4) Analysis of residual crosslinking agent To detect residual HMDA in the sample, approximately 8 g of the sample was accurately weighed and diluted to 10 mL with hyaluronidase solution (6800 units / mL phosphate-buffered saline (PBS); hyaluronidase from bovine testis, type IS). The sample was then decomposed at 40°C for 3 hours. Approximately 160 mg of HMDA standard was accurately weighed, dissolved in water, and diluted to 100 mL. This solution was then diluted 1:100, and an appropriate amount was added to 2 mL of hyaluronidase solution. The solution was then diluted to 10 mL with PBS to prepare a standard solution for the calibration curve. To each 10 mL sample, 2.5 mL of 3% ammonia solution, 7.5 mL of 5 mol / L sodium hydroxide solution, and 0.1 mL of internal standard solution were added. 0.5 mL of ethyl chloroformate was added, and the mixture was shaken at approximately 1000 rpm for 3 minutes to initiate the reaction. The internal standard solution was prepared by diluting a 4 v / v solution of 1,3-diaminopropane at a ratio of 1:80. After the reaction, 2.5 mL of toluene was added, and the mixture was shaken at approximately 1000 rpm, centrifuged, and the supernatant was collected. The amount of HMDA in the supernatant was measured by gas chromatography (GC), and the ratio of the peak area of ​​HMDA to that of the internal standard was calculated. A calibration curve was plotted based on the peak area ratio of the standard solution, and the residual HMDA was quantified using the calibration curve.

[0052] The analytical conditions for gas chromatography are as follows.

[0053] (5) Extrusion pressure measurement test The extrusion pressure of the specimen was analyzed using a universal testing machine (EZ-S-500N, Shimadzu Corp., Japan). A syringe containing the specimen was placed below the load cell of the apparatus, along with a 21G needle. The load cell was adjusted to a distance from contact with the syringe tip, and the syringe tip was pressed through the load cell at a speed of 5 mm / min, measuring the applied force. A load cell with a permissible value higher than the pressure range to be measured was selected, and a flat push rod was installed at the syringe tip to ensure uniform force applied to the syringe handle. The pressure generated when the specimen flows into the needle during the initial stage of pressure measurement is lower than the pressure during syringe injection. Force can be continued even without a specimen when the injection is almost complete at the end of the measurement. Therefore, the average extrusion pressure value measured when the middle portion of the syringe filler fluid is discharged between the initial and final measurements is defined as the extrusion pressure.

[0054] Example 1: Preparation of HMDA crosslinked hyaluronic acid hydrogel of the present invention 60 g of sodium hyaluronate with an average molecular weight of 1.0 MDa to 2.0 MDa was completely dissolved in 760 g of distilled water. 6.29 g of hexamethylenediamine dihydrochloride (HMDA∙2HCl) as a crosslinking agent, 8.42 g of N-hydroxybenzotriazole hydrate (HOBt∙H2O) as a peptide bond promoter, and 11.9 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) as a carboxyl activator were completely dissolved and mixed into the sodium hyaluronate solution. The mixture was placed in a thermostatic stirring tank and stirred three times at a revolution-rotation centrifugal stirrer (OST-CM-18000-C; Ostar Corp., South Korea) for a total of at least 60 minutes to ensure uniform mixing of the sodium hyaluronate solution and the crosslinking reaction solution. After mixing, the thermostatic stirring tank was placed in a thermostatic chamber and subjected to a crosslinking reaction at 40°C for at least 10 hours. The reacted HMDA crosslinked hyaluronic acid hydrogel was then pulverized, and the pulverized composition was placed in a washing machine (OST-FWM-040; Ostar Corp., South Korea) for washing and swelling for at least 9 hours. The washing solution was prepared as follows: 1060 g of sodium chloride (NaCl), 137.5 g of anhydrous disodium hydrogen phosphate (Na₂HPO₄), and 45 g of sodium dihydrogen phosphate dihydrate (NaH₂PO₄∙2H₂O) were dissolved in 125 L of water for injection. The solution was then filtered through a 0.22 μm filter to prepare a phosphate buffer solution (pH 6.0–8.0). When the residual crosslinking agent (HMDA) was removed to below the detection limit of 2 ppm, the washing solution was removed, and the hydrogel was dehydrated at 3 rpm for 30 minutes. The hydrogel was then homogenized using a milling apparatus (OST-PM-60; OstarCorp., South Korea) equipped with a 300 µm sieve, and then heat-treated at 122 °C or autoclaved.

[0055] Example 2: Preparation of a 3mL pre-filled syringe filled with HMDA cross-linked hyaluronic acid hydrogel In Example 2, the HMDA crosslinked hyaluronic acid hydrogel prepared in Example 1 was diluted with a solution having the same composition as the buffer solution used during washing, so that the effective ingredient, based on sodium hyaluronate, reached 20 mg / mL. Then, 3 mL of the solution was filled into a 3 mL syringe and autoclaved.

[0056] Examples 3 and 4: Preparation of 5mL pre-filled syringes filled with HMDA cross-linked hyaluronic acid hydrogel In Examples 3 and 4, the HMDA crosslinked hyaluronic acid hydrogel prepared in Example 1 was diluted with a solution having the same composition as the buffer solution used during washing, so that the effective ingredient, based on sodium hyaluronate, reached 20 mg / mL. Then, 5 mL of the solution was filled into a 5 mL syringe and autoclaved.

[0057] Examples 5 to 10: Preparation of HMDA-crosslinked hyaluronic acid hydrogels for comparing crosslinking reaction times Examples 5 to 10 were prepared using the same method as described in Example 1, except that the crosslinking reaction time was set to 0.66 hours, 2 hours, 4 hours, 10 hours, 22 hours, and 48 hours, respectively, and the storage modulus was measured.

[0058] Examples 11 to 15: Preparation of HMDA crosslinked hyaluronic acid hydrogels for comparing washing process times Examples 11 to 15 were prepared by the same method as described in Example 1, except that the washing process time was set to 1 hour, 5 hours, 7 hours, 20 hours, and 48 hours.

[0059] Examples 16 to 19: Preparation of 5 mL pre-filled syringes for comparing the pore size of particle homogenization sieves filled with HMDA crosslinked hyaluronic acid hydrogel Examples 16 to 19 were prepared by the same method as described in Example 3, except that the sieve pore size during particle homogenization was set to 400µm, 300µm, 180µm, and 100µm, respectively.

[0060] Examples 20 and 21: Preparation of cross-linked hyaluronic acid hydrogel pre-filled syringes for animal experiments, cross-linked with hexamethylenediamine (HMDA), for confirming extrusion pressure. In Examples 20 and 21, the HMDA crosslinked hyaluronic acid hydrogel prepared in Example 1 was diluted with a solution having the same composition as the buffer solution used during washing, so that the effective ingredient reached 20 mg / mL based on sodium hyaluronate. Then, 3 mL and 5 mL of the solution were filled into 3 mL and 5 mL syringes and autoclaved.

[0061] Examples 22 to 30: Preparation of pre-filled syringes containing a mixture of cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid for animal efficacy evaluation experiments In Examples 22 to 30, the HMDA crosslinked hyaluronic acid hydrogel prepared in Example 1 was diluted with a solution having the same composition as the buffer solution used during washing, so that the active ingredient, based on sodium hyaluronate, reached 20 mg / mL. Then, it was mixed with 20 mg / mL of unmodified hyaluronic acid at constant ratios as shown in Tables 7 and 8 to prepare a composition for the treatment of joint diseases. The mixture was then filled into a 3 mL pre-filled syringe and autoclaved.

[0062] Examples 31 to 33: Preparation of HMDA-crosslinked hyaluronic acid hydrogel pre-filled syringes for clinical trials In Examples 31 to 33, the HMDA crosslinked hyaluronic acid hydrogel prepared in Example 1 was diluted with a solution having the same composition as the buffer solution used during washing, so that the effective ingredient, based on sodium hyaluronate, reached 20 mg / mL. Then, it was filled into a pre-filled clinical syringe specially designed to achieve low extrusion pressure during injection and ease of use, with a single clinical injection effective volume of 3 mL (Example 31) or 5 mL (Examples 32 and 33), and then sterilized by autoclaving.

[0063] Examples 34 and 35: Preparation of pre-filled syringes containing a mixture of HMDA-crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid for clinical trials In Examples 34 and 35, the HMDA crosslinked hyaluronic acid hydrogel prepared in Example 1 was diluted with a solution having the same composition as the buffer solution used during washing, so that the effective ingredient, based on sodium hyaluronate, reached 20 mg / mL. Then, it was mixed with 20 mg / mL of unmodified hyaluronic acid at weight ratios of 90:10 and 20:80 to prepare a composition for the treatment of joint diseases. The composition was filled into a pre-filled clinical syringe specially designed to achieve low extrusion pressure and ease of use during injection, with a single effective injection volume of 3 mL, and then autoclaved.

[0064] Comparative Examples 1 to 4: Preparation of HMDA-crosslinked hyaluronic acid hydrogels based on existing preparation methods for comparison In Comparative Examples 1 to 3, 24 g of sodium hyaluronate with a molecular weight of 1.0 MDa was completely dissolved in 660 g of distilled water. In Comparative Example 4, it was completely dissolved in distilled water at twice its weight ratio. To facilitate the cross-linking reaction with the carboxyl groups of hyaluronic acid, a cross-linking agent, hexamethylenediamine dihydrochloride, was added. To adjust the degree of cross-linking, the amount added was 10-20 mol% of the repeating unit of sodium hyaluronate (20 mol% corresponds to 1.39 g dissolved in 5 g of distilled water). The solution was filtered through a 0.22 μm filter and then added. A 0.25 N NaOH aqueous solution filtered through a 0.22 μm filter was added to adjust the pH to 6.0 to 6.5. 8.08 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 11.47 g of 1-hydroxybenzotriazole (HOBt) were added to distilled water and completely dissolved, then filtered through a 0.22 μm filter and added to the above mixture. In a high-viscosity stirrer, the mixture was stirred at 30 rpm at 45°C for 30 minutes, and then left to stand at 45°C for at least 12 hours without stirring to allow for cross-linking. The cross-linked hydrogel was initially pulverized and then passed through a sieve with a pore size of 180 µm for particle homogenization. 80% ethanol filtered through a 0.22 μm filter was added, and hydrogel powder was obtained by precipitation. 100 times the volume of 1.3% NaCl solution was added and stirred for 1 hour. 80% ethanol was added again to obtain a precipitate. The obtained hydrogel precipitate was placed in 100% ethanol for 10 minutes, and then dried under reduced pressure at 40°C for 12 hours to confirm that residual EDC, HOBt, and HMDA had been removed to below the detection limit of 2 ppm. The dried product was then recovered. The dried product was swollen with water for injection at a concentration of 20 mg / ml, and then the hydrated cross-linked hyaluronic acid hydrogel was autoclaved at 121°C for 15 minutes.

[0065] Comparative Examples 5 and 6: BDDE cross-linked hyaluronic acid hydrogel pre-filled syringes used for comparison Comparative Examples 5 and 6 used commercially available BDDE cross-linked sodium hyaluronate gel injections, namely Synovian injection from LG Chem Corporation (LG Chem, Korea). ® Injection; 3g per 3.0mL pre-filled syringe; This product is also marketed as Hyruan ONE by Yifan Pharmaceuticals (China). ® Listed (also available as HyruanONE ®Hyalone (injection, 3.03g per pre-filled syringe, from Yifan Pharmaceutical (China) and Shin Poong Pharm. Co., Ltd. (Korea), is also available.

[0066] Experimental Example 1: Measurement of the crosslinking degree (CrD) of HMDA-crosslinked hyaluronic acid in this invention The degree of crosslinking of Example 2 and Comparative Example 1 was measured according to the methods described in analytical methods (1) to (2), and the results are shown in Table 1 below. The experimental results confirmed that for Comparative Example 1, which was prepared by a conventional preparation method after undergoing the drying-hydration step according to the present invention and removing residual crosslinking agent by ethanol precipitation, the degree of crosslinking could be analyzed by TNBS analysis and NMR analysis. On the other hand, for the crosslinked hyaluronic acid composition of the present invention, such as Example 2, when its degree of crosslinking was low to about 0 mol% to about 2 mol%, it could not be accurately measured by TNBS analysis, so the degree of crosslinking was measured by NMR analysis.

[0067] [Table 1] Experimental Example 2: Measurement of the physical properties of the HMDA crosslinked hyaluronic acid hydrogel of the present invention When observing the knee joint, a representative joint, it has been reported that the average values ​​of the storage modulus (G'; elasticity) and loss modulus (G''; viscosity) of the synovial fluid in healthy adults aged 21-27 are 117±13 Pa and 45±8 Pa (both at 2.5 Hz) (Stitik et al., Future Rheumatol. 2008;3(3):215–222). In contrast, the storage modulus (G', 2.5 Hz) of synovial fluid in normal elderly individuals aged 52–78 years was reduced to 19 ± 3 Pa, particularly in adult patients aged 21–45 years with osteoarthritis or those undergoing moderate to severe total knee arthroplasty, where the storage modulus was 8.5 ± 0.5 Pa and 1.9 ± 0.5 Pa (both at 2.5 Hz), respectively, showing a significant decrease (Stitik et al., Future Rheumatol. 2008;3(3):215–222; Mazzucco D et al., J Orthop Res 2002;20(6):1157–1163). Therefore, hyaluronic acid-based compositions, when administered via intra-articular injection, have shown promise in the treatment of joint diseases, particularly in improving joint pain and function, by promoting joint lubrication and restoring the reduced storage modulus of synovial fluid to levels seen in younger adults. Therefore, the target value of the final storage modulus in the synovial fluid after injection of the composition of the present invention is set as 117 Pa, which is the storage modulus of synovial fluid in young adults. The minimum value of the reduced storage modulus of synovial fluid before injection is estimated to be 1.9 Pa. Considering the average amount of synovial fluid in human bone and joints (6.7 ± 2.3 mL) and the expected volume of the therapeutic composition (2-6 mL), and taking into account the dilution factor caused by the synovial fluid, the lower limit of the target storage modulus of the therapeutic composition is calculated to be 232.1 Pa by the following formula.

[0068] Lower limit of target storage modulus of compositions for treating joint diseases =[117 (target value) × 2 (expected minimum dilution factor)] - 1.9 (inferred initial synovial fluid storage modulus of the patient) =232.1 (Pa) Furthermore, as a therapeutic composition, in order to meet clinical requirements for biocompatibility, the amount of residual crosslinking agent needs to be controlled below 2 ppm.

[0069] According to the methods described in analytical methods (2) to (4), the degree of crosslinking, storage modulus, and residual crosslinking agent (residual HMDA) of Examples 3 to 4 and Comparative Examples 1 to 4 were measured, and the results are shown in Table 2 below. The experimental results confirmed that, using existing preparation methods including ethanol precipitation, in order to prepare a composition with a residual crosslinking agent of less than 2 ppm, the degree of crosslinking needs to reach more than 5 mol% at the same hyaluronic acid concentration (20 mg / ml) in order to prepare a therapeutic composition with a target storage modulus of more than 232.1 Pa. On the other hand, the composition according to the present invention can still achieve the target storage modulus even when the degree of crosslinking is less than this value, especially at a low degree of crosslinking of 1 mol%, and a composition with a high storage modulus can be obtained without increasing the hyaluronic acid concentration (Comparative Example 4 compared to Examples 3 to 4).

[0070] [Table 2] Experimental Example 3: Based on the change in storage modulus during cross-linking reaction time The storage modulus of Examples 5 to 10 was measured according to the method described in the analytical method (3), and the results are shown in Table 3 below. The experimental results show that the longer the crosslinking reaction time, the greater the increase in storage modulus, but it tends to saturate after 10 hours.

[0071] [Table 3] Experimental Example 4: Changes in Residual Crosslinking Agent (HMDA) Based on Washing Time The residual HMDA in Examples 11 to 15 was measured according to the method described in analytical method (4), and the results are shown in Table 4 below. The experimental results confirmed that a washing process of more than 20 hours using a phosphate buffer solution (pH 6.0 to 8.0) is required to control the residual HMDA below 2 ppm. To infer a more accurate time, when fitting all data to a linear regression curve of logarithmic concentration, it was inferred that a washing process of more than 13.12 hours was required. When fitting data around 7-20 hours, it was inferred that a washing process of more than 8.82 hours was required, i.e., a washing process of more than 9 hours was required.

[0072] [Table 4] Experimental Example 5: Based on the change in storage modulus of particle homogenization sieve pore size The storage modulus of Examples 16 to 19 was measured according to the method described in the analytical method (3), and the results are shown in Table 5 below. Experimental results show that when the cross-linked hyaluronic acid hydrogel particles were homogenized, a sharp decrease in storage modulus was confirmed when the sieve pore size was below 180 µm.

[0073] [Table 5] Experimental Example 6: Comparison of extrusion pressures during injection of the HMDA crosslinked hyaluronic acid hydrogel of the present invention The extrusion pressures during injection of Examples 20, 21, and Comparative Example 3 were measured according to the method described in analytical method (5), and the results are shown in Table 6 below. The experimental results show that although the extrusion pressure increases with the filling volume, it also shows a significantly lower extrusion pressure at the same hyaluronic acid concentration (20 mg / ml) compared to the commercially available BDDE crosslinked hyaluronic acid product (Comparative Example 3).

[0074] [Table 6] Experiment Example 7: Evaluation of the therapeutic efficacy of joint disease treatment in a rodent arthritis model The physical properties of Examples 22 to 27 and Comparative Example 5 were measured according to the methods described in analytical methods (2) to (4), and the results are shown in Table 7. The results of evaluating the therapeutic efficacy of Examples 22 to 27 and Comparative Example 5 on arthritis using a rat (rat) model induced by the chemical monosodium iodoacetate (MIA), commonly used as an arthritis model, are shown in Table 7. Figure 2 middle.

[0075] The hair around the right knee of rats (Sprague-Dawley rats; Daehan-Bio, South Korea; 7-8 weeks old) was completely shaved. Then, using a Hamilton syringe fitted with a 21-30G needle, 50 µL of MIA (I2512, Sigma-Aldrich, USA) solution was injected into the right knee joint cavity to induce osteoarthritis (Udo M et al., Osteoarthritis and cartilage 2016 24:1284–1291). The MIA was dissolved in 0.9% physiological saline at a concentration of 50 mg / mL and filtered through a 0.22 µm filter. Seven days after the MIA injection, arthritis was confirmed to be stably induced. 50 µL of the solvent (physiological saline), the composition of Examples 22 to 27, and Comparative Example 5 was then injected into the knee joint cavity of selected animals. During the 28-day period, joint swelling, body weight, and blood inflammatory markers were evaluated. On day 28 after MIA injection (day 21 after sample administration), a necropsy was performed, and tissue samples were collected from the knee joint. The tissues were stained, and the state of the tissues and chondrocytes was evaluated. The Mankin's score was calculated. The Mankin's score is the most basic histopathological observation for evaluating osteoarthritis. It is based on the degree of articular cartilage surface damage, staining characteristics, changes in chondrocyte count, and the formation of clones induced by osteoarthritis. A higher score indicates a higher degree of osteoarthritis. Statistical analysis between groups was performed using SPSS ver2 (IBM, USA). Normality was tested using the Shapiro-Wilk test. If normality was found, ANOVA was used for intergroup comparisons; otherwise, the Kruskal-Wallis test / Man-Whitney test was used.

[0076] The experimental results showed that when HMDA crosslinked hyaluronic acid hydrogel was mixed with unmodified hyaluronic acid in various mixing ratios from 90:10 to 20:80, the lower the ratio of crosslinked hyaluronic acid, the lower the storage modulus (G'), i.e., elasticity, and the higher the tanδ (G'' / G'). On the other hand, the loss modulus (G''), i.e. viscosity, first increased and showed the highest value when the ratio of crosslinked hyaluronic acid hydrogel to unmodified hyaluronic acid was 50:50, and then decreased again.

[0077] The efficacy of Examples 22 to 27 and Comparative Example 5 in a rat arthritis model was evaluated using the Mankin score, a joint site assessment score evaluating tissue and chondrocyte morphology and structure. The results showed that, compared to the Sham control group (without induced arthritis), the solvent control group (with arthritis induced by MIA) exhibited significantly increased tissue damage. When a mixture of cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid was applied intra-articularly, a trend towards reduced tissue damage was observed with increasing storage modulus (Example 22, 90:10 mixture of cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid). Furthermore, even with a lower storage modulus, a trend towards reduced tissue damage was observed with increasing unmodified hyaluronic acid ratio (e.g., above 70%). Compared to the solvent control group, the Example 27 application group, i.e., the composition of cross-linked hyaluronic acid hydrogel to unmodified hyaluronic acid at a ratio of 20:80, showed a significant reduction in tissue damage even with a lower storage modulus.

[0078] [Table 7] P<0.01 compared to the solvent control group Experiment Example 8: Evaluation of the therapeutic efficacy of joint disease treatment in a non-rodental osteoarthritis model In human joint diseases, weight load has a significant impact on the exacerbation of symptoms such as pain. Therefore, compared to rodents, medium-sized non-rodent models may be more useful for evaluating treatment efficacy. The results of evaluating the treatment efficacy of osteoarthritis in Examples 28 to 30 and Comparative Example 5 using a surgically induced beagle dog osteoarthritis model are shown in Table 8. Figure 3 middle.

[0079] Beagles (Covance, USA; 12 months old) were anesthetized with 50 mg / kg zoletil 50 (Virbac, France) and 2.5 mg / kg xylazine (Bayer Ag, Germany). The bilateral knee joints were dehaired, disinfected with povidone and 70% ethanol, and the skin was incised. Blunt dissection was performed on the surrounding tissues to expose the articular surface of the right femur. The cartilage was then damaged via medial meniscectomy and anterior cruciate ligament transection. The Sham control group did not undergo meniscectomy or anterior cruciate ligament transection; the wounds were simply sutured with 4-0 nylon sutures. During the 7-day period following osteoarthritis induction, the antibiotic cephradine (30 mg / kg) and the analgesic tramadol (3 mg / kg) were administered. One week after cartilage injury induction, osteoarthritis was induced by artificial exercise (30 minutes / session, once daily) for 3 weeks. When the average walking assessment score reached 2.5-3.0 or higher, the animals were randomly assigned to groups and the test samples were administered. The animals were anesthetized, and then, using a C-arm machine (C-arm, Arcadis Varic, Siemens Co.), 1 mL of solvent (physiological saline) at a volume of 20 mg / mL was administered into the joint cavity of both knees in a single dose. The compositions of Examples 28, 28 to 30, and Comparative Example 5 were administered. Pain during walking was assessed weekly for a total of 12 weeks. At week 12, an autopsy was performed to confirm the Mankin's score (the histological evaluation of the joint area). Following the manufacturer's instructions, the levels of MMP-3 and VEGF165a in the synovial fluid were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. Statistical analysis was performed as in Experiment 6. MMP-3 is known to be a degradative enzyme produced by synovial cells and chondrocytes in osteoarthritis or rheumatoid arthritis, playing an important role in the destruction of cartilage matrix and connective tissue such as collagen (Pulik L et al., Reumatologia 2023;61(3):191–201). VEGF 165a also plays a role in osteoarthritis or rheumatoid arthritis, acting not only as an angiogenesis factor but also as an inflammatory factor, participating in abnormal cell growth or exacerbating joint inflammation (Kim J et al., Exp. Mol. Med. 2020; 52: 843–853).

[0080] The efficacy of the beagle osteoarthritis models in Examples 28 to 30 and Comparative Example 5 was evaluated through gait score assessment, histopathological evaluation, and analysis of the effects on the secretion of inflammatory factors in the synovial fluid. The results showed that in the non-rodent osteoarthritis model, a higher storage modulus correlated with better symptom improvement (Table 8). Specifically, in Example 30, the application of a mixture of HMDA crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid at a weight ratio of 90:10 resulted in the fastest achievement of the target gait improvement score (2 points) (28 days) compared to other composition application groups, and maintained at 2 points until week 12. Furthermore, at week 9, a significant improvement in gait score was observed compared to the solvent control group. In the histopathological evaluation at week 12, compared to the solvent control group, all composition application groups showed improved cartilage structure scores, and in the mixture of HMDA crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid, a higher storage modulus tended to indicate a greater degree of improvement in cartilage structure scores. Specifically, in Example 30, when HMDA-crosslinked hyaluronic acid hydrogel was mixed with unmodified hyaluronic acid at a weight ratio of 90:10, the target walking improvement score (2 points) was achieved fastest (day 28) compared to other composition application groups, and remained at 2 points until week 12. Furthermore, compared to the solvent control group, which experienced increased secretion of inflammatory factors induced by osteoarthritis, the release of MMP-3 and VEGF 165a in the synovial fluid was statistically significantly inhibited, demonstrating potential therapeutic efficacy for joint diseases. Figure 3 ).

[0081] [Table 8] P<0.01 compared to the solvent control group Although this result exists in a medium-sized beagle dog arthritis model under body weight load, it differs from the U-shaped correlation between storage modulus and efficacy observed in the small animal rat arthritis model in Experiment 7. However, the sample size was small, and the commercially available BDDE cross-linked hyaluronic acid injection used as a control group has demonstrated clinical efficacy. Therefore, it can be inferred that when the weight ratio of cross-linked hyaluronic acid hydrogel to unmodified hyaluronic acid is above 70:30 and the storage modulus is high, it is considered that the effect is more likely to be observed.

[0082] Experimental Example 9: Clinical efficacy evaluation of the HMDA cross-linked hyaluronic acid hydrogel of the present invention in patients with knee osteoarthritis (Phase 1 / 2 clinical trial) A phase 1 / 2 clinical trial was conducted on 20 patients with knee osteoarthritis to evaluate the clinical efficacy (efficacy) of Examples 31 and 32, and the results are presented in [the table / document / etc.]. Figure 4 middle.

[0083] Clinical trial design This study investigated the efficacy of a randomized, double-blind, placebo-controlled, dose-escalation clinical trial administered once over a 6-month period to adult patients with mild to moderate knee osteoarthritis. The trial drug (Examples 31 to 32), used as the investigational drug in the joint disease treatment composition of this invention, or an equivalent dose of placebo, was administered intra-articularly to the knee joint. The investigational drug and placebo used are as follows: - Test drug 1: Test substance (Example 31, 20 mg / ml, 3 mL intra-articular injection composition) - Placebo 1: 3 mL of injectable saline solution without the test substance - Test drug 2: Test substance (Example 32, 20 mg / ml, 5 mL intra-articular injection composition) - Placebo 2: 5 mL of normal saline solution for injection without the test substance Subjects meeting the following inclusion and exclusion criteria were randomly assigned to cohort A and cohort B, receiving two different doses of investigational drug 1 and investigational drug 2, respectively, for a 6-month follow-up observation. Efficacy was evaluated at the time of administration and at 6, 12, and 24 weeks post-administration using the following assessment methods. Each cohort consisted of 10 subjects, with 8 in the investigational drug group and 2 in the placebo group. One subject in the investigational drug group and one subject in the placebo group dropped out of each cohort, leaving 8 subjects in each of cohorts A and B (7 in the investigational drug group and 1 in the placebo group) for efficacy evaluation. As described in Example 9, a maximum daily dose of 4g of the rescue drug (500mg of acetaminophen) was permitted.

[0084] Main selection and exclusion criteria Patients who met all of the following primary inclusion criteria 1 through 4 were included.

[0085] (Main selection criteria) 1. Adult men and women aged 40 and above 2. Osteoarthritis patients with KL grade 1-3 within 6 months of screening or at the time of screening. 3. During the screening and consultation, patients should be diagnosed with unilateral or bilateral knee osteoarthritis according to the clinical diagnostic criteria of the American College of Rheumatology (ACR), experience knee pain, and meet three or more of the following conditions. ① Age > 50 years old ② Morning stiffness <30 minutes ③ There is a friction sound during the activity. ④ Bone tenderness ⑤ Bony enlargement ⑥ No palpable warmth of synovium upon palpation. 4. Weight-bearing pain (WBP) - 100mm: VAS measurement result of 40mm or more. (Main exclusion criteria) Patients who met any of the following primary exclusion criteria 1 to 10 were excluded.

[0086] 1. BMI ≥ 35 kg / m² 2 2. Individuals with severe hip osteoarthritis or osteoarthritis of other joints whose screening time point would significantly affect the evaluation of knee osteoarthritis. 3. Patients with diseases that may affect the evaluation of efficacy and safety. 4. Individuals with joint infections or skin diseases who are unsuitable for injectable administration. 5. Those whose patellofemoral joint space has completely disappeared. 6. Screening participants who have received the following treatments within 14 days of their initial consultation (but participants who have completed a 14-day wash-out period are eligible for enrollment). -Including anti-inflammatory and analgesic drugs such as glucosamine, chondroitin sulfate, and nonsteroidal anti-inflammatory drugs (NSAIDs), traditional Chinese medicine preparations, or physical therapy, etc. 7. Patients who have received systemic steroid medications at the application site within the past 3 months. 8. Individuals who have received intra-articular injections of hyaluronic acid or intra-articular corticosteroids at the application site within the past 6 months. 9. Those who have undergone surgery within the past 6 months or plan to undergo surgery within the past 10 months. 10. Screening patients who present with moderate or severe joint effusion via the patellar tap test. Effectiveness evaluation methods The primary endpoints for evaluating effectiveness included improvement in weight-bearing pain (WBP) relative to baseline as a pain indicator, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) which comprehensively assesses pain, physical function, and stiffness, and indicators related to rescue medicine use, all evaluated using the 100mm visual analogue scale (VAS). For the VAS, regarding pain levels associated with knee osteoarthritis, patients marked their subjective pain levels on a 100mm line, from 0mm (no pain) to 100mm (unbearable pain). Researchers verified the patient's markings and recorded them numerically on an evaluation sheet. The improvement was quantified based on the difference between the length of the patient's markings before application (pre-application baseline) and the length of the markings at each evaluation point after application. The WOMAC assessment followed established methods in the field, using a questionnaire where patients self-reported pain severity (5 items), stiffness severity (2 items), and difficulty with daily activities (17 items) over the past 48 hours. Each item was graded using a 5-point Likert scale (0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe). The scores (maximum 96 points) of the three items were summed and recorded (Bellamy N et al., J. Rheumatol. 1988–1940; Ehrich EW et al., J. Rheumatol. 2000;27(11):2635–2641). Rescue medication use evaluated the percentage of patients who took acetaminophen as a rescue medication during the clinical trial.

[0087] The experimental results showed that when a single intra-articular application of the joint disease treatment composition of the present invention, test drug 1 (Example 31), test drug 2 (Example 32), or placebo was administered into the knee joint cavity, patients receiving 5 mL of test drug (Example 32) showed a trend of improvement in WOMAC and weight-bearing pain compared to those receiving placebo. Figure 5 On the other hand, compared to the placebo, no improvement was observed in weight-bearing pain as measured by VAS or in stiffness, function, and other symptoms as measured by WOMAC with 3 mL of the investigational drug (Example 31). However, compared to the placebo, which did not reduce the use of acetaminophen as a salvage drug, the proportion of patients taking salvage drugs was reduced in the investigational drug 1 group to a similar degree as in the investigational drug 2 group. Therefore, the effect of the placebo may be due to the use of analgesics, and this should be taken into account when interpreting it. A dose of 5 mL was subsequently selected for clinical use.

[0088] Experimental Example 10: Clinical efficacy evaluation of the HMDA cross-linked hyaluronic acid hydrogel of the present invention in patients with knee osteoarthritis (Phase 3 clinical trial) The clinical efficacy of Examples 33 and Comparative Example 5 was evaluated in a therapeutic confirmatory phase 3 clinical trial involving 223 patients with knee osteoarthritis, using the methods described below. The results are shown in Table 9 and Figure 5 middle.

[0089] Clinical trial design In adult patients with mild to moderate knee osteoarthritis, a multicenter, randomized, double-blind, active-controlled, non-inferiority clinical trial was conducted. The efficacy of the clinical trial drug (Example 33) or active control drug (Comparative Example 5), used as the treatment composition for joint diseases of the present invention, was analyzed by administering it intra-articularly at 6-month intervals, including single administration and repeated administration. Follow-up was conducted up to 3 months after re-administration. The investigational drug and placebo used are as follows: - Test drug: Test substance (Example 33, 20 mg / ml, 5 mL intra-articular injection composition) -Active control drug: Comparative Example 5 (BDDE cross-linked hyaluronic acid cross-linked compound, commercially available product for single-dose intra-articular injection) 3 mL of intra-articular injection composition. The investigational drug or active control was administered intra-articularly to the knee joint of the subjects a total of two times at 24-week intervals. Subjects meeting the following inclusion and exclusion criteria were randomly assigned to two groups to receive either the investigational drug or the active control. Efficacy was evaluated at evaluation points at weeks 2, 6, 12, 24 (re-administration), and 36 after the first administration, using the methods described below. As described in Experimental Example 9, a maximum daily dose of 4g of the rescue drug (500mg of acetaminophen) was permitted.

[0090] Main selection and exclusion criteria The inclusion and exclusion criteria described in Experiment Example 9 are basically the same.

[0091] Effectiveness evaluation methods The primary endpoint for evaluating effectiveness was the improvement in weight-bearing pain (WBP) relative to baseline, assessed using the 100mm visual analog scale (VAS). The 100mm VAS was performed using the same method described in Experimental Example 9. Secondary endpoints assessed the improvement in joint-line tenderness on pressure. Physical assessment of joint-line tenderness was performed at each visit using a 4-point scale [0 = none, 1 = mild, 2 = moderate, 3 = severe]. A smaller difference between groups (less than 0) indicated a higher level of effectiveness.

[0092] The primary endpoint was the change in weight-bearing pain (WBP-100mm-VAS) at 12 weeks relative to baseline. To test the non-inferiority of the experimental group in the change in WBP at 12 weeks relative to baseline compared to the active control group, an analysis of covariance was performed with baseline WBP as a covariate. The least squares mean (LS Mean) and standard error (SE), the difference in least squares means between groups (LS Mean Difference, calculated as the difference between the least squares mean of the experimental group and the least squares mean of the active control group), and the corresponding two-sided 95% confidence interval (CI) were calculated. A non-inferiority margin of -10mm was defined as a treatment effect in the experimental group being non-inferior to that in the active control group. In this case, the baseline value was defined as the value before administration.

[0093] The test results showed that when the test drug (Example 33), a therapeutic composition for joint diseases of the present invention, was administered intra-articularly to the knee joint cavity at 6-month intervals, the improvement in weight-bearing pain relative to baseline was measured at week 12. This significantly improved weight-bearing pain in patients with mild to moderate knee osteoarthritis (improvement in weight-bearing pain relative to baseline was 23.71 mm). Furthermore, compared to the currently commercially available active control group (Comparative Example 5), which has proven efficacy, the test drug demonstrated non-inferiority in improving weight-bearing pain in patients with mild to moderate knee osteoarthritis, thus confirming the pain improvement effect (corrected difference in weight-bearing pain between groups was -2.26, see Table 9 below for determination). In addition, compared to the active control group (Comparative Example 5), the test group showed a significant reduction in joint line tenderness, thus confirming its significant improvement effect on joint line tenderness in knee osteoarthritis (difference between groups was -0.17, P < 0.001). Figure 5 ).

[0094] In addition, after 6 months (24 weeks), the test drug (Example 33) or the active control drug (Comparative Example 5) was administered again, and the results of the improvement in weight-bearing pain relative to week 24 were reconfirmed at the 12-week time point. Compared with the active control drug, the test drug showed a better trend of improvement in weight-bearing pain, thus confirming that the re-administration showed a better pain improvement effect (the corrected difference in weight-bearing pain between groups was -4.75). Figure 5 ).

[0095] [Table 9] P<0.001 relative to baseline value Experimental Example 11: Clinical efficacy evaluation of the HMDA crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid mixture of the present invention in patients with knee osteoarthritis (Phase 1 / 2 clinical trial). A phase 1 / 2 clinical trial was conducted on 40 patients with knee osteoarthritis to evaluate the clinical efficacy of Examples 34 to 35 and Comparative Example 5, and the results are shown in Table 10.

[0096] Clinical trial design Adult patients with mild to moderate knee osteoarthritis were included in this study. The clinical trial drug (Examples 34 to 35), active control drug (Comparative Example 5), or placebo, used as the treatment composition for joint diseases of the present invention, was administered intra-articularly to the knee joint once and followed up for 6 months. The results were analyzed through a randomized, double-blind, placebo, and active control clinical trial. The investigational drug, placebo, and active control drug used are as follows: - Test drug 1: The test substance (Example 34; the baseline content of HMDA crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid is 2 w / w% (20 mg / ml), and the weight ratio is 90:10) is a 3 mL intra-articular injection composition. - Test drug 2: The test substance (Example 35; HMDA crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid at a baseline content of 2 w / w% (20 mg / ml), weight ratio of 20:80) 3 mL of an intra-articular injection composition. - Placebo: 3 mL of normal saline solution for injection without the test substance -Active control drug: Comparative Example 5 (BDDE cross-linked hyaluronic acid cross-linked compound, commercially available product for single-dose intra-articular injection) 3 mL of intra-articular injection composition. Subjects meeting the following inclusion and exclusion criteria were randomly assigned to four groups to receive intra-articular administration of investigational drug 1, investigational drug 2, placebo, or active control drug. Efficacy was evaluated at evaluation points at weeks 2, 6, 12, 16, 24, and 36 post-administration using the methods described below. As described in Experiment Example 9, a maximum daily dose of 4g of the rescue drug (500mg of acetaminophen) was permitted.

[0097] Main selection and exclusion criteria The inclusion and exclusion criteria described in Experiment Example 9 are basically the same.

[0098] Effectiveness evaluation methods The primary endpoints for evaluating effectiveness were weight-bearing pain and WOMAC measured using a 100 mm VAS. The 100 mm VAS and WOMAC were evaluated using the same methods described in Experimental Example 9. A smaller difference between groups (less than 0) indicated a higher effectiveness. Analysis of covariance was used to analyze differences between groups.

[0099] The test results showed that when test drug 1 (Example 34), the joint disease treatment composition of the present invention, was administered intra-articularly into the knee joint cavity once, it significantly improved weight-bearing pain and WOMAC scores in patients with mild to moderate knee osteoarthritis compared to commercially available BDDE cross-linked hyaluronic acid hydrogel injections, which served as the active control group (Comparative Example 5) (Tables 10, 11, and 12). In particular, the composition of the present invention, with a weight ratio of HMDA cross-linked hyaluronic acid hydrogel to unmodified hyaluronic acid of 90:10, showed a significant improvement in weight-bearing pain at week 6, compared to the active control group and the placebo group, despite a lower dose of the rescue drug than other groups, thus confirming a significantly superior early pain improvement effect (change from baseline of -35.10; Tables 10 and 12). Furthermore, the significantly lower doses of the rescue drug at weeks 12 and 16 also supported the significant pain improvement effect of this composition (Table 12). In comparison, while the effect of test drug 2 (Example 35; weight ratio 20:80) was lower than that of test drug 1, it showed a superior trend toward improvement in weight-bearing pain and WOMAC compared to the active control group (Tables 10 and 11). The dosage of WOMAC and the rescue drug also showed a decreasing trend compared to placebo (Tables 11 and 12). The effects of placebo, in particular, need to be interpreted in conjunction with the dosage of the rescue drug, including for each group.

[0100] [Table 10] P<0.05 compared to the active control group [Table 11] [Table 12] Experimental Example 12: Comparison of storage modulus and improvement in weight-bearing pain in the clinical trial drug of the HMDA-crosslinked hyaluronic acid hydrogel of the present invention The storage modulus (G') of the clinical trial medicines containing the compositions of Examples 31 to 35 was compared with the weight-bearing pain (100 mm VAS-WBP) values ​​used as efficacy evaluation indicators in the clinical trials of patients with knee osteoarthritis in Examples 9 to 11, and is shown in Table 13. The storage modulus range of the clinical trial medicine batches that have been clinically used and confirmed to be effective was observed to be 219-721 Pa. In particular, the storage modulus range that showed excellent pain improvement in clinical practice compared to commercially available products, i.e., the storage modulus range of the HMDA crosslinked hyaluronic acid hydrogel of the present invention that ensures efficacy, was estimated to be 342-721 Pa (2.5 Hz, 25°C).

[0101] [Table 13] p<0.001 relative to baseline value Reference Example 1: Clinical safety evaluation of the HMDA cross-linked hyaluronic acid hydrogel of the present invention in patients with knee osteoarthritis. Examples 32 to 34, based on two clinical trials in Examples 9 to 10, have provided experience in administering the clinical trial drug 213 times to 123 adult patients with knee osteoarthritis. No adverse drug reactions, serious adverse drug reactions, or deaths related to the clinical trial drug were reported, and the safety evaluation results showed no worrying risks in terms of safety and tolerability.

[0102] The present invention has been described above with a focus on preferred embodiments. Those skilled in the art will understand that the present invention can be implemented in variations without departing from its essential characteristics. Therefore, the embodiments disclosed above should be considered illustratively rather than restrictively. The scope of the present invention is defined by the claims, not by the foregoing description, and all differences from the equivalent scope of the claims should be interpreted as being included within the scope of the present invention.

Claims

1. A composition for treating joint diseases comprising a crosslinked hyaluronic acid hydrogel obtained by a crosslinking reaction of hyaluronic acid or a pharmaceutically acceptable salt thereof with an alkylene diamine crosslinking agent represented by the following chemical formula 1 or 2; The storage modulus G' of the composition for treating joint diseases is 250-850 Pa at 2.5 Hz and 25°C. The degree of crosslinking (CrD) of crosslinked hyaluronic acid, which is the ratio of the number of crosslinking agents bonded at both ends to the number of hyaluronic acid units, is less than 5 mol%. The joint disease treatment composition is administered to the patient once over a period of more than 3 months in the form of an injection: [Chemical Formula 1] [HA] x -C(O)-NH-R1-NH-C(O)-[HA] y [Chemical Formula 2] [HA] z -C(O)-NH-R1-NH2 In chemical formula 1 and chemical formula 2, HA is hyaluronic acid with one carboxyl group removed or a pharmaceutically acceptable salt thereof; R1 is unsubstituted or substituted C3-C 10 Alkylene; x, y, and z are each independent integers between 100,000 and 5,000,000.

2. The composition according to claim 1, characterized in that, The pharmaceutically acceptable salts of the hyaluronic acid are sodium, potassium, calcium, magnesium, zinc, cobalt, or tetrabutylammonium salts.

3. The composition according to claim 1, characterized in that, R1 is an unsubstituted or C4-C6 alkylene group substituted with hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy groups.

4. The composition according to claim 3, characterized in that, The alkylene diamine crosslinking agent is hexamethylenediamine.

5. The composition according to claim 1, characterized in that, The energy storage modulus G' of the composition at 2.5 Hz and 25 °C is 342-721 Pa.

6. The composition according to claim 1, characterized in that, The degree of cross-linking (CrD) of cross-linked hyaluronic acid is less than 1 mol%.

7. The composition according to claim 1, characterized in that, The weight ratio of the cross-linked hyaluronic acid hydrogel to unmodified hyaluronic acid is 70:30 to 99:

1.

8. The composition according to claim 7, characterized in that, The baseline content of the cross-linked hyaluronic acid hydrogel and the unmodified hyaluronic acid is 2 w / w, and the weight ratio is 90:

10.

9. The composition according to any one of claims 1 to 8, characterized in that, The joint disease mentioned is osteoarthritis.

10. The composition according to any one of claims 1 to 9, characterized in that, The treatment is for the improvement, cure, or inhibition of symptom progression.

11. The composition according to claim 10, characterized in that, The treatment aims to improve joint pain or joint function.

12. A kit comprising a pre-filled syringe filled with the composition of any one of claims 1 to 11, such that the clinically effective volume of the composition for a single injection is a dose of 2 mL to 6 mL.

13. A preparation method, characterized in that, The preparation method is the preparation method of the composition according to any one of claims 1 to 11 or the kit according to claim 12, and the preparation method includes the following steps: i) Dissolve hyaluronic acid or a pharmaceutically acceptable salt thereof in a solvent; ii) Mix an alkaline aqueous solution into the solution, wherein the alkaline aqueous solution contains a dissolved alkylene diamine crosslinking agent, a peptide bond promoter, and a carboxyl activator, and then filter the solution; iii) Crosslinked hyaluronic acid is prepared by carrying out a crosslinking reaction for more than 10 hours under constant temperature conditions, i.e., 30°C to 50°C; iv) The cross-linked hyaluronic acid is pulverized, washed, and swollen to prepare a hydrogel; v) Homogenize the hydrogel particles to prepare a mixed hydrogel composition, either directly or by mixing. vi) Fill the hydrogel composition into a storage container or syringe and perform heat treatment or sterilization.

14. The preparation method according to claim 13, characterized in that, The concentration of the alkylene diamine crosslinking agent is from 10 mol% to 30 mol% of hyaluronic acid or its pharmaceutically acceptable salt units.

15. The preparation method according to claim 13, characterized in that, In step ii), a constant-temperature stirring tank and a centrifugal stirrer with revolution and rotation are used to suppress excessive cross-linking reaction.

16. The preparation method according to claim 13, characterized in that, The revolution speed of the centrifugal agitator is above 300 rpm, and the total operation time is above 60 minutes.

17. The preparation method according to claim 13, characterized in that, The peptide bond promoter in step ii) is selected from N-hydroxysuccinimide, 1-hydroxybenzotriazole, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, 1-hydroxy-7-azobenzotriazole, sulfonyl-N-hydroxysulfosuccinimide and mixtures thereof.

18. The preparation method according to claim 13, characterized in that, The carboxyl activator in step ii) is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-(trimethylammonium)propyl)carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide and mixtures thereof.

19. The preparation method according to claim 13, characterized in that, In step iv), washing and swelling are performed for more than 9 hours using a phosphate buffer solution with a pH of 6.0 to 8.

0.

20. The preparation method according to claim 13, characterized in that, In step v), particle homogenization is performed using a sieve with a pore size greater than 180 μm and less than 500 μm.

21. The preparation method according to claim 13, characterized in that, The joint disease is knee osteoarthritis, and the composition consists only of cross-linked hyaluronic acid hydrogel, which is filled into a pre-filled syringe with a clinically effective volume of 5 mL per injection.

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