Composition for treating joint diseases and preparation method therefor

Abstract

The present invention relates to a composition for treating joint diseases and a preparation method therefor and, more specifically, to a composition comprising a cross-linked hyaluronic acid hydrogel obtained by a cross-linking reaction between hyaluronic acid or a pharmaceutically acceptable salt thereof and an alkylene diamine cross-linking agent, and a preparation method therefor. The composition has a storage modulus (G', 2.5 Hz, 25° C) of 250-850 Pa, has a degree of cross-linking (CrD; number of cross-linking agents bound to both sides / number of hyaluronic acid units) of less than 5 mol %, and exhibits an effect of alleviating symptoms of joint diseases for a period of 3 months or longer when administered once as an injectable formulation to a patient. The composition has a low degree of crosslinking that allows flexible adjustment of the dosing interval while maintaining high biocompatibility, in vivo persistence, and a clinically meaningful storage modulus, thereby reducing patient burden caused by frequent administration and being expected to provide an excellent clinical effect of alleviating joint disease symptoms for three months or longer.

Description

Composition for treating joint diseases and method for manufacturing the same

[0001] The present invention relates to a composition for treating joint diseases and a method for manufacturing the same, and more specifically, to a cross-linked hyaluronic acid hydrogel composition obtained by a cross-linking reaction of hyaluronic acid or a pharmaceutically acceptable salt thereof and an alkylenediamine cross-linking agent, which maintains high bioavailability and clinically significant storage modulus despite low degree of cross-linking, thereby reducing the burden on patients due to frequent administration and enabling flexible administration intervals, while simultaneously expecting an effect of improving joint disease symptoms for more than 3 months.

[0002] The prevalence of joint diseases is increasing worldwide due to the aging population, and osteoarthritis, the most common of these joint diseases, is a condition that causes joint pain and degeneration, leading to functional impairment. Hyaluronic acid (hyaluronate, HA), a biopolymer that makes up the synovial fluid of the joint, has a structure in which repeating units of disaccharides composed of β-N-acetyl-D-glucosamine and β-D-glucuronic acid are linearly connected. It is known to act as a lubricant and shock absorber when injected into the joint, thereby relieving joint pain and improving joint mobility.

[0003] Hyaluronic acid preparations used for the treatment of joint diseases are commonly developed as intra-articular injections. However, unmodified hyaluronic acid has limitations in terms of low bioavailability because it is broken down by the hyaluronidase enzyme within a few days and rapidly excreted from the body. Accordingly, unmodified hyaluronic acid preparations have been developed as 3- or 5-dose regimens administered at weekly intervals for a total of 3 to 5 times over a period of 6 months. However, these preparations may cause damage to the synovial membrane within the joint cavity due to frequent administration, and patients face the inconvenience of having to visit the clinic frequently.

[0004] To solve these problems, active research is being conducted on a single-dose therapy that improves bioavailability and sustains effects for six months with only a single administration by developing hydrogels cross-linked with hyaluronic acid using various cross-linking agents. For example, Korean Patent No. 10-2275105 discloses a cross-linked hyaluronic acid with a high-density network structure cross-linked with 1,4-butanediol diglycidyl ether (BDDE) and a method for manufacturing the same, and Korean Patent No. 10-2400586 discloses a powdered hyaluronic acid crosslink suitable for mass production and a method for manufacturing the hydrogel using BDDE and divinyl sulfone (DVS).

[0005] Recently, crosslinking agents have been proposed that enhance bioavailability by utilizing the carboxyl group (-COOH group) of hyaluronic acid as the recognition site of hyaluronidase, thereby utilizing it as a target reaction site for crosslinking reactions. Among these, alkylenediamine crosslinking agents such as hexamethylenediamine are known to have a lower risk of genotoxicity compared to existing crosslinking agents such as BDDE or DVS, and have been reported to have the advantage of improving biocompatibility by more easily removing residual crosslinking agents. For example, Korean Patents No. 10-0674177 and No. 10-1062320 disclose crosslinked hyaluronic acid using such alkyldiamine and alkylenediamine crosslinking agents, its medical uses, and methods for manufacturing the same.

[0006] As such, it has been reported that the type of crosslinking agent, the binding site with hyaluronic acid, and the crosslinking agent residue can affect biosustainability, biocompatibility, and furthermore, clinical efficacy and safety. However, regarding the formulation and industrial application of hyaluronic acid crosslinks using specific crosslinking agents, it is difficult to say that sufficient information and manufacturing methods have yet been adequately established or presented concerning the physical properties of the composition—particularly the degree of crosslinking (CrD) and viscoelasticity—that can guarantee clinical efficacy.

[0007] Hyaluronic acid-based treatments for joint diseases are primarily developed as intra-articular injections. In the case of these invasive injections, high-frequency repeated administration, such as current 3- or 5-dose regimens, places a physical and mental burden on patients and can increase the risk of infection as well as pain.

[0008] On the other hand, while single-dose regimens that improve upon these aspects and provide sustained efficacy with a single administration for six months have the advantage of avoiding repeated administration, insurance coverage often includes a waiting period during which medical expenses are not supported until the next administration. Considering that the need to combine oral medication or physical therapy is widely known due to the nature of joint diseases, and taking into account both the patient's preference regarding the frequency of hospital visits and the economic burden caused by the lack of insurance coverage, it would be more desirable for patients to have various options regarding the administration interval.

[0009] Therefore, there is a need to develop a composition for treating joint diseases and a method for manufacturing the same, based on cross-linked hyaluronic acid with high biosustainability and biocompatibility, which allows for flexible adjustment of the administration interval according to the patient's needs while continuously maintaining clinically effective effects for several months.

[0010] [Prior Art Literature]

[0011] [Patent Literature]

[0012] Korean Registered Patent No. 10-2400586 (Published June 17, 2020)

[0013] Korean Registered Patent No. 10-2275105 (Published on March 18, 2015)

[0014] Korean Registered Patent No. 10-1400907 (Published Jan. 18, 2012)

[0015] Korean Registered Patent No. 10-1062320 (Published on Feb. 5, 2009)

[0016] Korean Registered Patent No. 10-0674177 (Published Nov. 14, 2001) / International Application PCT / EP 1999 / 008481 (WO 2000 / 027887; Published May 18, 2000)

[0017] Korean Patent Publication No. 10-2013-0028012 (Published on March 18, 2013)

[0018] The present invention has a technical objective of providing a composition for treating joint diseases that can reduce the burden on patients caused by frequent administration and provide an effect of improving clinical symptoms of joint diseases for a long period of more than 3 months, while maintaining a storage modulus (G') that exhibits high biocompatibility, biosustainability, and clinically effective efficacy, while having a low degree of cross-linking (CrD) that allows for flexible adjustment of the administration interval compared to existing hyaluronic acid cross-links used for treating joint diseases.

[0019] Meanwhile, another technical objective of the present invention is to provide a method for manufacturing the same, including a composition for treating joint diseases and a syringe kit filled with the composition in a single-use clinically effective volume capable of exhibiting clinically effective efficacy.

[0020] To solve the above technical problem, the present invention provides a composition for treating joint disease and a method for preparing the same, comprising a cross-linked hyaluronic acid hydrogel obtained by a cross-linking reaction of hyaluronic acid or a pharmaceutically acceptable salt thereof and an alkylenediamine cross-linking agent; having a storage modulus (G', 2.5 Hz, 25℃) of 250-850 Pa; having a degree of cross-linking (CrD) of the cross-linked hyaluronic acid of less than 5 mol%; and being administered once as an injection to a patient over a period of 3 months or more.

[0021]

[0022] Hyaluronic acid preparations have been proposed as visco-supplementation agents based on viscoelasticity, and it has been believed that the greater the molecular weight or viscoelasticity, the more superior the efficacy, such as in pain relief. Storage modulus or elasticity (G') and loss modulus or viscosity (G"). However, considering the diverse molecular weights and viscoelastic ranges of previously approved products, it is estimated that while there is a correlation between viscoelasticity and efficacy, it is highly likely that this correlation is not a simple linear direct proportion. This is consistent with the results of our own animal studies and clinical trials, which confirmed that the correlation between viscoelasticity and efficacy is not simply direct proportional depending on the composition (Experimental Example 7; Table 7 and Fig. 2). Furthermore, the literature reports that, in addition to the viscoelasticity of hyaluronic acid, various physical properties and interactions between hyaluronic acid and other components in synovial fluid can affect efficacy (Balazs EA et al., Arthritis Rheum. 1967;10(4):357-376; Korean Patent No. 10-2400586).

[0023]

[0024] In the present invention, the range of storage modulus (G') exhibiting clinically effective efficacy is limited, and at the same time, factors causing a decrease in storage modulus (G') in existing manufacturing methods are eliminated and the manufacturing process is improved. Through this, a composition having a storage modulus (G') capable of exhibiting effective clinical effects in the human body despite having a crosslinking degree of less than 5 mol% and a method for manufacturing the same are newly presented.

[0025]

[0026] Meanwhile, differences in the storage modulus (G') and cross-linking agent residue of the final composition may occur depending on the 'degree of cross-linking' (CrD; although there have been cases where it was mistranslated as 'cross-linking rate,' this specification uses 'degree of cross-linking' consistently), and this may further affect clinical efficacy and biocompatibility. Here, 'degree of crosslinking' (CrD) refers to the molar ratio of crosslinking agent molecules per hyaluronic acid disaccharide monomer (Kenne et al., Carbohydrate Polymers 2013;91:410-418), and can be expressed as the product of 'degree of modification' (MoD; defined as the ratio of the number of moles of crosslinked hyaluronic acid to the total number of moles of hyaluronic acid disaccharides, and while some literature has translated this as 'crosslinking modification rate' or mistranslated it as 'crosslinking', this specification uses 'degree of modification' consistently) and 'cross-linking ratio' (CrR; defined as the ratio of the number of crosslinked agent molecules to the total number of crosslinking agent molecules bound to hyaluronic acid, and while some literature has translated this as 'crosslinking ratio' or 'effective crosslinking rate', this specification uses 'crosslinking ratio' consistently).

[0027] In the case of cross-linked hydrogels cross-linked with alkylenediamine, it is disclosed that they generally exhibit a degree of cross-linking of 5 mol% to 20 mol% when manufactured using conventional manufacturing methods. For example, in the examples of Korean Registered Patent No. 10-1400907 and Korean Registered Patent No. 10-0674177, although there are differences depending on the molecular weight of hyaluronic acid, the molar ratio of cross-linking agent relative to hyaluronic acid, reaction conditions such as initial concentration and pH, washing and swelling conditions, and the method used in the step of removing residual cross-linking agent (e.g., ethanol precipitation method and use of a dialysis membrane), it is disclosed that when manufactured within the range of hyaluronic acid content and molar percentage of cross-linking agent relative to hyaluronic acid commonly used in the industry, the compositions after the step of removing residual cross-linking agent and unreacted substances exhibit a degree of cross-linking of 5 mol% or more, mainly 6 mol% to 14 mol%.

[0028] Hyaluronidase, a degradation enzyme, degrades hyaluronic acid by recognizing the carboxyl groups (-COOH groups) of the acid. Generally, when the degree of crosslinking is less than 5 mol%, the proportion of unreacted carboxyl groups not involved in crosslinking increases. This leads to the problem that hyaluronic acid is easily degraded by hyaluronidase, making it difficult to achieve the desired level of biocompatibility. Furthermore, in the case of hydrogels with a low degree of crosslinking, viscoelasticity decreases after post-sterilization or heat treatment, making it difficult to obtain a clinically effective storage modulus (G') and thus making it difficult to expect clinical efficacy. On the other hand, when the degree of crosslinking exceeds 35 mol%, the swelling properties of the hydrogel decrease, and problems may arise where it is difficult to exhibit viscoelasticity sufficient for joint protection. In addition, there is a concern that biocompatibility may be reduced as the content of residual crosslinking agents increases after in vivo degradation, raising the likelihood of side effects such as inflammatory reactions. In conventional manufacturing processes, dialysis membranes are used to remove residual crosslinking agents; however, fine grinding to facilitate dialysis can lead to a decrease in storage modulus. In hydrogels of typical crosslinking degrees, residual crosslinking agents are sometimes removed by precipitating hydrogel particles using ethanol precipitation instead of dialysis to eliminate crosslinking promoters soluble in organic solvents such as HOBt. However, it is known that this process results in a decrease in storage modulus (G'). Therefore, if residual crosslinking agents can be sufficiently removed to maintain the storage modulus (G') without the need for dialysis or ethanol precipitation, it would not only enable various formulations such as adjusting dosing intervals but also allow for a reduction in the initial amount of hyaluronic acid input, thereby contributing to cost reduction through shortened process steps and reduced quality control costs.

[0029]

[0030] Accordingly, in order to solve the above technical problem, the present invention provides a method for manufacturing a composition with a low degree of crosslinking that can remove residual crosslinking agent to a detection limit of less than 2 ppm without using dialysis or ethanol precipitation processes, and a composition that enables the application thereof. Through this, it was confirmed that a clinically effective storage modulus (G') can be maintained without a decrease in storage modulus (G').

[0031]

[0032] The present invention will be described in more detail below.

[0033] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The entire contents of all publications cited as references in this specification are incorporated by reference into this specification.

[0034]

[0035] According to one aspect of the present invention, as a composition for treating joint diseases,

[0036] Comprising a cross-linked hyaluronic acid hydrogel obtained by a cross-linking reaction with hyaluronic acid or a pharmaceutically acceptable salt thereof and an alkylenediamine cross-linking agent represented by the following Formula 1 or Formula 2;

[0037] The storage modulus (G', 2.5 Hz, 25℃) is 250-850 Pa;

[0038] The degree of cross-linking (CrD; number of cross-linking agents on both sides / number of hyaluronic acid monomers) of the cross-linked hyaluronic acid is less than 5 mol%;

[0039] A composition for treating joint disease, administered once as an injection to a patient over a period of three months or more, and a method for manufacturing the same are provided:

[0040] [Chemical Formula 1]

[0041] [HA] x -C(O)-NH-R1-NH-C(O)-[HA] y

[0042] [Chemical Formula 2]

[0043] [HA] z -C(O)-NH-R1-NH2

[0044]

[0045] In the above chemical formulas 1 and 2:

[0046] HA is hyaluronic acid excluding one carboxyl group or a pharmaceutically acceptable salt thereof;

[0047] R1 is unsubstituted or substituted C3-C 10 It is an alkylene group;

[0048] x, y, and z are each independently integers from 100,000 to 5,000,000.

[0049]

[0050] In this invention, hyaluronic acid is a biopolymer in which disaccharide repeating units of β-N-acetyl-D-glucosamine and β-D-glucuronic acid are linearly linked, and may be hyaluronic acid and a pharmaceutically acceptable salt thereof. The molecular weight of the hyaluronic acid or the pharmaceutically acceptable salt thereof is 100,000 to 5,000,000 Daltons (Da), and the intrinsic viscosity is 1.0 to 4.0 m 3 It may be / kg, but is not limited thereto.

[0051] According to one embodiment of the present invention, the pharmaceutically acceptable salt of the hyaluronic acid may be an inorganic salt such as a sodium salt, potassium salt, calcium salt, magnesium salt, zinc salt, or cobalt salt, or an organic salt such as a tetrabutylammonium salt, but is not limited thereto. According to one embodiment of the present invention, the pharmaceutically acceptable salt of the hyaluronic acid may be a sodium salt. According to one embodiment of the present invention, the hyaluronic acid or the pharmaceutically acceptable salt thereof may be isolated from microorganisms, synthesized, or purchased commercially available, but is not limited thereto.

[0052] According to one embodiment of the present invention, R1 may be an unsubstituted C4-C6 alkylene group substituted with a hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy. According to one embodiment of the present invention, R1 may be an unsubstituted C4-C6 alkylene group. According to one embodiment of the present invention, the alkylenediamine crosslinking agent may be hexamethylenediamine (HMDA). According to one embodiment of the present invention, the alkylenediamine crosslinking agent may be hexamethylenediamine dihydrochloride (1,6-hexamethylenediamine dihydrochloride) applied to the reaction.

[0053]

[0054] In this invention, the crosslinking reaction refers to a reaction in which an alkylenediamine crosslinker is attached to the carboxyl group of hyaluronic acid or a pharmaceutically acceptable salt thereof, and one or both of the alkylenediamine crosslinkers may be crosslinked with hyaluronic acid or a pharmaceutically acceptable salt thereof, and each hyaluronic acid or crosslinker unit may be irregularly continuous.

[0055] According to one embodiment of the present invention, the storage modulus (G', unit Pascal, Pa) of the composition may be 250 Pa to 850 Pa (250-850 Pa), preferably 342 Pa to 721 Pa (342-721 Pa), under analysis conditions of 2.5 Hz and 25°C. In this case, the composition may be in the form of a composition for treating joint disease consisting only of cross-linked hyaluronic acid, a mixture of unmodified hyaluronic acid and cross-linked hyaluronic acid, or a mixed form including a pharmaceutically acceptable carrier, filled into a syringe or the like, and is a finished pharmaceutical product immediately before administration to a patient after heat treatment or sterilization treatment, and the storage modulus (G') refers to the storage modulus at the time of release of the finished pharmaceutical product or the expected storage modulus (G') estimated at clinical application to a patient.

[0056] According to one embodiment of the present invention, the degree of crosslinking (CrD) of the crosslinked hyaluronic acid in the composition may be greater than 0 and less than 5 mol%, preferably greater than 0 and less than or equal to 1 mol%. In this invention, 'degree of crosslinking (CrD)' refers to the mole (mol%) ratio of hyaluronic acid that is crosslinked on both sides relative to hyaluronic acid units within the crosslinker, and is defined by the following formula (Kenne et al., Carbohydrate Polymers 2013;91:410-418):

[0057]

[0058]

[0059] According to one embodiment of the present invention, the composition may be a composition in which a cross-linked hyaluronic acid hydrogel is diluted to an appropriate concentration using the same phosphate buffer solution (pH 6.0-8.0) used for washing the hydrogel, and then mixed with unmodified hyaluronic acid. According to one embodiment of the present invention, the composition may be a composition characterized by mixing the cross-linked hyaluronic acid hydrogel and the unmodified hyaluronic acid at a weight ratio (w / w) of 70:30 to 99:1 by adjusting the cross-linked hyaluronic acid hydrogel and the unmodified hyaluronic acid to a reference content. Preferably, the composition may be characterized by a reference content of the cross-linked hyaluronic acid hydrogel and the unmodified hyaluronic acid being 2 w / w% and a weight ratio of the cross-linked hyaluronic acid hydrogel to the unmodified hyaluronic acid being 90:10.

[0060]

[0061] According to one embodiment of the present invention, the joint disease may be osteoarthritis, rheumatoid arthritis, or psoriatic arthritis, but is not limited thereto, and the injectable preparation containing the composition may be used in conjunction with physical therapy with non-steroidal anti-inflammatory drugs, immunosuppressants, anti-rheumatic agents, biological agents, etc. In one embodiment of the present invention, intra-articular administration of the composition reduced the release of MMP-3 and VEGF 165 a in the synovial fluid of Beagle dogs, and these substances are suggested as important therapeutic targets not only 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). In the above, joint disease may occur in the knee, spine, shoulder, arm, leg, fingertips, etc., but is 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 improvement of symptoms, healing, or inhibition of progression, and preferably may be improvement of joint pain or improvement of joint function, but is not limited thereto.

[0062]

[0063] According to another aspect of the present invention, a kit comprising a syringe filled with the composition may be provided, preferably a kit comprising a pre-filled syringe filled to a clinical single-dose effective volume of 2 mL to 6 mL, taking into account the specificity of treatment methods for joint diseases such as intra-articular injection, the risk of infection during injection, and the limited volume of the human joint cavity. According to the literature, the average volume of human synovial fluid is reported to be 6.7 ± 2.3 mL (Heilmann H et al., Z Orthop Unfall. 1996;134(2):144-8). In addition, it has been reported that in a clinical trial involving 253 patients, tolerable safety was demonstrated up to 26 weeks when 6 mL of a cross-linked hyaluronic acid preparation was administered twice as a single injection over a period of 6 months (Chevalier X et al., Ann Rheum Dis. 2010;69(1):113-9). Furthermore, the maximum dose of currently approved intra-articular injections is known to be 6 mL (Synvisc One®, Genzyme), which supports the validity of the maximum clinical single-dose effective volume (6 mL) presented above. When cross-linked hyaluronic acid is applied to the human body, particularly via intra-articular injection, high extrusion force may occur during injection due to the limited volume of the joint cavity and the high storage modulus (G') of cross-linked hyaluronic acid. Therefore, the design and filling volume of the above-mentioned pre-filled syringe must take into account not only the effective volume considering clinical efficacy, such as the initial storage modulus (G'0) of cross-linked hyaluronic acid and the dilution ratio in synovial fluid after injection, but also the easy protrusion pressure during injection and the change in storage modulus (G') during heat treatment or sterilization steps. Through this, it needs to be specially designed to satisfy the convenience of the operator and patient during injection, as well as the formulation characteristics.

[0064]

[0065] According to another aspect of the present invention, a method for manufacturing the composition or a syringe kit filled with the composition,

[0066] i) dissolve hyaluronic acid or a pharmaceutically acceptable salt thereof in a solvent;

[0067] ii) Mixing an alkaline aqueous solution, prepared by dissolving an alkylenediamine crosslinking agent, a peptide bond promoter, and a carboxyl group activator in the above solution and filtering it;

[0068] iii) Prepare cross-linked hyaluronic acid by performing a cross-linking reaction at constant temperature conditions (30℃ to 50℃) for at least 10 hours;

[0069] iv) The above-mentioned cross-linked hyaluronic acid is crushed, washed, and swollen to prepare a hydrogel;

[0070] v) A mixed hydrogel composition is prepared by homogenizing the above hydrogel particles, either as is or by mixing;

[0071] vi) A manufacturing method is provided comprising the step of filling the above hydrogel composition into a storage container or syringe and heat-treating or sterilizing it.

[0072]

[0073] According to one embodiment of the present invention, the solvent may be water, i.e., distilled water or water for injection. According to one embodiment of the present invention, the concentration of hyaluronic acid or a pharmaceutically acceptable salt thereof in the solution of step (i) may be 1 w / w% to 20 w / w%, preferably 5 w / w% to 10 w / w%. According to one embodiment of the present invention, in step (ii), an alkylenediamine crosslinking agent may be mixed with 3.5 mol% to 80 mol%, preferably 10 mol% to 30 mol% of the hyaluronic acid monomer. The alkaline aqueous solution may be, but is not limited to, an aqueous lithium hydroxide solution, an aqueous sodium hydroxide solution, or an aqueous hydroxide solution. Preferably, it is sodium hydroxide.

[0074]

[0075] According to one embodiment of the present invention, in order to compensate for the disadvantages of conventional high-viscosity stirrers in which a uniform mixture of the crosslinking agent and hyaluronic acid is not achieved in step (iii) and an overreaction occurs, a constant temperature stirring bath and a revolution-rotation planetary centrifugal mixer may be used. When the crosslinking reaction is overreacted, not only is it difficult to control uniform quality, but depending on the conditions, film-shaped foreign substances that are difficult to completely remove in subsequent manufacturing processes may be generated, which can lead to a serious deterioration in quality that makes it impossible to ship the finished injectable pharmaceutical product. The revolution-rotation planetary centrifugal mixer suppresses the overreaction of the crosslinking reaction by providing a strong homogeneous mixing force through centrifugal force generated by clockwise revolution at low temperatures and rotation that is automatically set to rotate at a certain ratio (0.3-0.6) of the revolution speed in a counterclockwise direction. According to one embodiment of the present invention, when the rotational speed of the orbiting electron centrifugal stirrer is repeated at 300 rpm and the mixture is uniformly mixed for a total of 60 minutes or more, the over-reaction of the crosslinking reaction can be suppressed. According to one embodiment of the present invention, in step (iii), the crosslinking reaction can be performed for 10 hours or more under constant temperature conditions of 30°C to 50°C.

[0076] According to one embodiment of the present invention, a peptide bonding promoter may be additionally mixed in step (ii). According to one embodiment of the present invention, the peptide bonding promoter may be selected from the group consisting of 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 peptide bond promoter may be used in an amount of 10 to 100 mol% of hyaluronic acid repeating units.

[0077] According to one embodiment of the present invention, a carboxyl group activator may be additionally mixed in step (ii). According to one embodiment of the present invention, the carboxyl group activator comprises 1-alkyl-3-(3-dimethylaminopropyl)carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC); and 1-alkyl-3-(3-(trimethylammonio)propyl)carbodiimides such as 1-ethyl-3-(3-(trimethylammonio)propyl)carbodiimide. Alternatively, it may be a carbodiimide such as 1-cycloalkyl-3-(2-morpholinoethyl)carbodiimide, such as 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, but is not limited thereto. According to one embodiment of the present invention, the carboxyl group activator may be used in an amount of 10 to 100 mol% of a hyaluronic acid repeating unit.

[0078]

[0079] According to one embodiment of the present invention, the washing solution refluxed to remove unreacted residual crosslinking agent in the crosslinked hyaluronic acid in the washing machine of step (iv) may be a phosphate buffer solution, but is not limited thereto. According to one embodiment of the present invention, the phosphate buffer solution may be prepared using sodium chloride (NaCl), sodium phosphate dibasic (Na2HPO4), and sodium phosphate monobasic dihydrate (NaH2PO4·2H2O), and filtered through a 0.22 μm filter. According to one embodiment of the present invention, the pH of the phosphate buffer solution may be 6.0 to 8.0. According to one embodiment of the present invention, the time required for the washing and swelling reaction in step (iv) may be 9 hours or more, preferably 13 hours or more. According to one embodiment of the present invention, in order to remove unreacted residual crosslinking agent in step (iv), the washing solution may be replaced several times, and the mesh used during washing may have a pore size that allows for easy inflow and outflow of the buffer solution while preventing the pulverized crosslinked hyaluronic acid from escaping.

[0080]

[0081] According to one embodiment of the present invention, after washing and swelling are completed in step (vi), the washing solution is removed and the hydrogel is dehydrated, and then the composition is homogenized into particles. The homogenization of the composition into particles may be performed using a mesh having a pore size greater than 100 μm and less than or equal to 500 μm, preferably greater than or equal to 180 μm and less than or equal to 500 μm. As the particle size of the composition becomes smaller, the storage modulus (G)' decreases, which may affect efficacy, and as it becomes larger, the protrusion pressure during injection increases (Shizomo AAM et al., J. Appl. Polym. Sci., 2013;128: 2180-2185). In the case of excessive protrusion pressure, the probability of injection detachment during the procedure may increase, and since the operator experiences significant discomfort, considering the use of syringes and needles generally used in injectables, a maximum particle size value exceeding 500 μm may be avoided.

[0082]

[0083] According to one embodiment of the present invention, in step (vi), the composition may be filled into a syringe such that the clinical single injection effective volume is 2 mL to 6 mL, preferably 3 mL to 5 mL. According to one embodiment of the present invention, when a mixture having a weight ratio of cross-linked hyaluronic acid hydrogel to unmodified hyaluronic acid of 90:10 is filled, the clinical single injection effective volume that was effective in a clinical trial for knee osteoarthritis was 3 mL. According to one embodiment of the present invention, when only cross-linked hyaluronic acid hydrogel is filled for knee osteoarthritis, the clinical single injection effective volume was 5 mL. In the case of pre-filled syringes for intra-articular injection, in accordance with aseptic manufacturing principles, the effective volume required for a single injection must be filled into the pre-filled syringe at once and sterilized by autoclave to prevent infection and contamination during injection, and a disposable sterile needle must be enclosed and sterilized. Additionally, the initial storage modulus (G') at the time of filling must be carefully planned, taking into account the decrease in storage modulus (G') caused by autoclave sterilization.

[0084]

[0085] In order to improve process efficiency and yield, the method for manufacturing a composition for treating joint diseases according to the present invention may include some of the processes or other manufacturing processes.

[0086] The cross-linked hyaluronic acid-based composition for treating joint diseases, crosslinked with an alkylenediamine crosslinking agent provided according to the present invention, exhibits superior biosustainability compared to unmodified hyaluronic acid and superior biocompatibility compared to other crosslinking agents. The present invention introduces a novel manufacturing method capable of maintaining a storage modulus (G') above a certain level even with a low degree of crosslinking, thereby maintaining high biosustainability, biocompatibility, and a clinically significant storage modulus despite the low degree of crosslinking. This reduces the burden on patients caused by frequent administration and enables flexible administration intervals, while simultaneously providing a composition for treating joint diseases that can demonstrate a significant improvement in clinical symptoms for more than 3 months in patients with joint diseases such as osteoarthritis, as well as a syringe kit filled with this composition and a method for manufacturing the same. Since the invention supports treatment methods using the same, it has industrial applicability in the pharmaceutical industry and the like.

[0087] FIG. 1 is a crosslinked hyaluronic acid crosslinked with hexamethylenediamine measured to calculate the degree of crosslinking according to one embodiment of the present invention. 1 This is a schematic diagram illustrating the results of H NMR analysis.

[0088] Figure 2 is a graph showing the degree of improvement in the knee joint tissue evaluation score (Mankin score) on day 28 after administering Examples 22 to 27 and Comparative Example 5 to an SD rat chemical (MIA)-induced arthritis model in Experimental Example 7 of the present invention.

[0089] Figure 3 is a graph illustrating the reduction effect of MMP-3 and VEGF165a inflammatory factors in knee joint synovial fluid after administering Examples 28 to 30 and Comparative Example 5 to a Beagle dog osteoarthritis model in Experimental Example 8 of the present invention. (#P>0.05 vs. Solvent Control Group)

[0090] Figure 4 is a graph showing the degree of improvement in weight-bearing pain (measured by 100 mm VAS), WOMAC score, and the proportion of patients taking rescue medication compared to baseline over 24 weeks after administering Examples 31 to 32 and a placebo once into the joint cavity of a patient with knee osteoarthritis in Experimental Example 9 of the present invention.

[0091] Figure 5 is a graph showing the degree of improvement in joint line tenderness at 12 weeks after the first administration and the amount of improvement in weight-bearing pain at 12 weeks after re-administration in knee osteoarthritis patients administered Example 33 and Comparative Example via intra-articular injection in Experimental Example 10 of the present invention.

[0092] The present invention will be explained in more detail below through examples. However, the following examples are provided merely to aid in understanding the invention and do not limit the scope of the invention.

[0093]

[0094] Analysis method

[0095] (1) Crosslinking analysis: 2,4,6-trinitrobenzenesulfonic acid (TNBS) test

[0096] The amount of crosslinking agent bound to both sides can theoretically be calculated as the difference if the total crosslinking agent content and the residual crosslinking agent content are known and the amount of crosslinking agent bound to only one side during crosslinking can be determined. The amount of alkylenediamine, a crosslinking agent bound to only one side, that is, the amount of unbound primary amine terminals of alkylenediamine that are not bound to hyaluronic acid, can be quantified by measuring absorbance after reacting with 2,4,6-trinitrobenzenesulfonic acid (TNBS, TNBSA).

[0097] Number of crosslinkers bound to both sides = (Total number of crosslinkers - Number of crosslinkers bound to one side - Number of residual crosslinkers)

[0098] Crosslinking (CrD) = Number of crosslinking agents bound to both sides / Number of hyaluronic acid monomers

[0099]

[0100] Approximately 1 mL of the sample was mixed with 4 mL of hyaluronidase (50 U / ml) solution and reacted at 37°C with stirring at 8 g for 8 hours, after which 35 mL of 0.1 M sodium bicarbonate buffer solution (pH 8.0) was added to dilute to 1:8. The TNBS reaction test was performed according to the manufacturer's instructions provided by TNBS (Thermo Scientific, USA; Cat. No. 28997). 0.5 mL of the diluted sample or standard was mixed with 0.25 mL of 0.01 w / v% TNBS solution and reacted by stirring at 37°C for 2 hours; then, the reaction was terminated by adding 0.25 mL of 10% sodium dodecyl sulfate (SDS; Sigma Aldrich, USA; Cat. No. 4509) solution and 0.125 mL of 1 N hydrochloric acid (HCl). Absorbance was measured at 335 nm using an ultraviolet spectrophotometer (Infinite M200; Tecan GmbH, Austria). The TNBS solution was prepared to 5 w / v% using 0.1 M sodium bicarbonate buffer and diluted to 0.01 w / v% for use. The standard curve was measured by dissolving HMDA·2HCl in a 0.1 M sodium bicarbonate solution and setting at least 5 concentrations in the range of 4-24 μg / ml.

[0101]

[0102] (2) Crosslinking analysis: Nuclear magnetic resonance spectroscopy (NMR)

[0103] For a novel manufacturing method of HMDA crosslinked hyaluronic acid hydrogels that does not include a drying-hydration step, crosslinkers having a low degree of crosslinking are used for more accurate crosslinking degree analysis. 1The results were analyzed using nuclear magnetic resonance spectroscopy (NMR) spectra. The amount of hyaluronic acid monomers can be determined from the integral of the N-acetyl (-OCH3) peak on the hyaluronic acid monomers, while the amounts of crosslinking agents crosslinked on only one side and those crosslinked on both sides can be determined from the integral of the non-overlapping alkylene (-CH2) peaks. Additionally, the total amount of crosslinked agent was calculated relative to the integral of the alkylene (-CH2) peaks, and the degree of crosslinking (CrD) was derived from this ratio. For example, representative of HMDA-crosslinked hyaluronic acid 1The H-NMR spectrum is as shown in Fig. 1. The chemical formula of HMDA cross-linked on only one side and HMDA cross-linked on both sides is shown in the upper left corner of Fig. 1, and the carbon regions of HMDA cross-linked on only one side are labeled a, b, c, d, e, and f, while the carbon regions of HMDA cross-linked on both sides are labeled 1, 2, and 3. In the spectrum, the relative amount of hyaluronic acid monomers is calculated by dividing the integral of the N-OCH3 group peak appearing at 1.8 ppm by 3, the amount of HMDA cross-linked on one side is calculated by dividing the integral of -CH2 e near 1.45 ppm by 2, and the amount of cross-linking agent on both sides is calculated from the integral of -CH2 2 near 1.35 ppm, provided that since the peak near 1.35 ppm is an overlap between the -CH2 b peak of HMDA cross-linked on one side and the -CH2 2 peak of HMDA cross-linked on both sides, the result is calculated by subtracting the signal value of -CH2 e (which has the same signal value as -CH2 b) from the total peak integral and dividing by 2. Similarly, the integral value due to the 3rd hydrogen of the cross-linking agent on both sides is calculated by subtracting the integral values ​​due to d and c from the integral value due to the resonance line at 1.1-1.2 ppm. By averaging the integral values ​​obtained from hydrogens 2 and 3 and dividing by 4—which corresponds to the number of crosslinker hydrogens bound to only one side—a value proportional to the relative amount of the crosslinker bound to both sides can be obtained. The resonance line near 3 ppm was not used because it overlapped with the resonance line caused by hyaluronic acid.

[0104] Crosslinking (CrD) = Number of crosslinking agents bound to both sides / Number of hyaluronic acid monomers

[0105]

[0106] As an acid digestion pretreatment method for NMR analysis, the trifluoroacetic acid (TFA)-heating method was applied to break down the monomer bonds of the polymeric cross-linked hyaluronic acid composition and increase solubility. At this time, the TFA treatment time was optimized to between 30 minutes and 1 hour to preserve the bond between the crosslinking agent and hyaluronic acid while cleaving only the bonds between the repeating units of hyaluronic acid. 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 heated in a water bath at 90°C for 30 minutes. The solution was diluted with approximately three times the volume of distilled water to reduce the TFA concentration to 1.5 M or less, then sufficiently frozen in a -70°C ultra-low temperature freezer for more than 2 hours, followed by freeze-drying to remove the solvent. The freeze-dried sample was dissolved in at least 700 μL of D2O solvent, filled into an NMR tube, and analyzed at 25°C under conditions of 300 scans (at least 50), a spin rate of 20, and a relaxation time (delay) of 2 seconds. 1 It was analyzed using H NMR (AVANCE III 400 MHz NMR, BRUKER Corp., USA; Magnet System Ascend 400'54).

[0107]

[0108] (3) Storage modulus analysis

[0109] 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 instrument temperature was set to 25°C, and calibration was performed by mounting a 40 mm diameter 2° aluminum cone plate geometry. A sufficient amount of sample was loaded into the center between the upper and lower geometries of the instrument plate, ensuring that it did not adhere to the sides or top of the geometry. The geometry was lowered to the set gap, and after verifying that the sample was filled beneath the geometry, any remaining sample outside the geometry was removed. The geometry was subjected to frequency oscillation at a strain of 1% Shear strain was periodically applied to the sample at 0.1–10 Hz, and among the obtained modulus values, the value at a frequency of 2.53 Hz was defined as the viscoelastic value. The damping factor (tanδ) is a value indicating whether the material's properties are closer to a solid or a liquid, and was calculated as G" / G'. The closer the value of tanδ is to 1, the closer it is to a liquid state with low elasticity and high viscosity; the closer it is to 0, the closer it is to a solid state with high elasticity and low viscosity.

[0110]

[0111] (4) Analysis of residual crosslinking agent

[0112] To detect residual HMDA in the samples, approximately 8 g of the sample was accurately weighed, hyaluronidase solution (6,800 units / ml phosphate buffered saline, PBS; Hyaluronidase from bovine testis, type IS) was added to make 10 mL, and the mixture was decomposed at 40°C for 3 hours. Approximately 160 mg of HMDA standard was accurately weighed, dissolved in water, and diluted to 100 mL. An appropriate amount was taken after diluting 1:100, 2 mL of hyaluronidase was added, and PBS was added to make 10 mL to prepare the standard solution for the calibration curve. To 10 mL of each 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, followed by 0.5 mL of ethylchloroformate. The mixture was then reacted by shaking at approximately 1,000 rpm for 3 minutes. The internal standard solution was prepared by diluting a 1,3-diaminopropane 4 v / v% solution to 1:80. After the reaction, 2.5 mL of toluene was added and the mixture was mixed by shaking at approximately 1,000 rpm, followed by centrifugation to collect the supernatant. The amount of HMDA in the supernatant was measured by gas chromatography (GC) to determine the ratio of the peak area of ​​HMDA to the peak area of ​​the internal standard. A calibration curve was constructed from the standard solution peak area ratio, and the residual HMDA was quantified from the calibration curve.

[0113] The analysis conditions for gas chromatography are as follows.

[0114]

[0115]

[0116] (5) Protrusion pressure measurement test

[0117] The protrusion pressure of the sample was analyzed using a Universal Testing Machine (EZ-S-500N, Shimadzu Corp., Japan). A syringe containing the sample was placed under the load cell of the device, and a 21G needle was attached. The distance was adjusted so that the load cell was just before touching the tip of the syringe, and the applied force was measured by pressing the tip of the syringe at a speed of 5 mm / min. At this time, a load cell with an allowable value higher than the pressure range to be measured was selected, and a flat plunger was attached to the tip of the syringe to ensure that a constant force was applied to the syringe grip. At the initial point of pressure measurement, the pressure generated when the sample flows into the needle is lower than the pressure required to inject the syringe, and at the final point of measurement, when the injection of the fluid is almost complete, force may continue to be applied even though there is no sample. Therefore, the average of the protrusion pressure values ​​measured during the injection of the middle portion of the syringe filling fluid, excluding the initial and final measurement values, was defined as the protrusion pressure.

[0118]

[0119] Example 1: Preparation of the HMDA-crosslinked hyaluronic acid hydrogel of the present invention

[0120] 60 g of sodium hyaluronate having 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 (1,6-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, EDC·HCl) as a carboxyl group activator were completely dissolved in a 1 w / w% sodium hydroxide solution and mixed with the sodium hyaluronate solution. The mixture was placed in a constant temperature stirring bath and stirred three times for a total of at least 60 minutes at an orbital speed of 500 rpm using an orbital centrifugal stirrer (OST-CM-18000-C; Ostar Corp., South Korea) to homogeneously mix the sodium hyaluronate solution and the crosslinking reaction solution. After mixing was complete, the constant temperature stirring bath was placed in a constant temperature chamber, and the crosslinking reaction was carried out at 40°C for at least 10 hours. After the reaction was completed, the HMDA crosslinked hyaluronic acid hydrogel was ground, and the ground composition was washed and swollen in a washing machine (OST-FWM-040; Ostar Corp., South Korea) for at least 9 hours. A phosphate buffer solution (pH 6.0-8.0) was prepared by dissolving 1,060 g of sodium chloride (NaCl), 137.5 g of anhydrous sodium hydrogen phosphate (Na2HPO4), and 45 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) in 125 L of water for injection, followed by filtration through a 0.22 μm filter. Once the residual crosslinking agent (HMDA) was removed to the standard detection limit of 2 ppm or less, the wash solution was removed, and the hydrogel was dehydrated at 3 rpm for 30 minutes. Subsequently, the hydrogel was milled using a milling machine (OST-PM-60; Ostar Corp.) equipped with a 300 μm mesh.It was homogenized through ( , South Korea) and heat-treated or high-pressure steam sterilized at 122℃.

[0121]

[0122] Example 2: Preparation of a 3 mL pre-filled syringe filled with HMDA-crosslinked hyaluronic acid hydrogel

[0123] Example 2 was prepared by diluting the HMDA cross-linked hyaluronic acid hydrogel prepared in Example 1 with a solution having the same composition as the buffer solution used for washing to a concentration of 20 mg / mL based on the active ingredient sodium hyaluronate, then filling 3 mL of this solution into a 3 mL syringe and sterilizing it by autoclaving.

[0124]

[0125] Examples 3 to 4: Preparation of a 5 mL pre-filled syringe filled with HMDA-crosslinked hyaluronic acid hydrogel

[0126] Examples 3 and 4 were prepared by diluting the HMDA cross-linked hyaluronic acid hydrogel prepared in Example 1 with a solution having the same composition as the buffer solution used for washing to a concentration of 20 mg / mL based on the active ingredient sodium hyaluronate, then filling 5 mL of this solution into a 5 mL syringe and sterilizing it by autoclaving.

[0127]

[0128] Examples 5 to 10: Preparation of HMDA-crosslinked hyaluronic acid hydrogels for comparison of crosslinking reaction times

[0129] Examples 5 to 10 were prepared in the same manner as described in Example 1, but the crosslinking reaction was carried out for 0.66, 2, 4, 10, 22, and 48 hours, respectively, and the storage modulus was measured.

[0130]

[0131] Examples 11 to 15: Preparation of HMDA-crosslinked hyaluronic acid hydrogel for comparison of washing process times

[0132] Examples 11 to 15 were prepared in the same manner as described in Example 1, except for washing process times of 1, 5, 7, 20, and 48 hours.

[0133]

[0134] Examples 16 to 19: Preparation of a 5 mL pre-filled syringe filled with HMDA-crosslinked hyaluronic acid hydrogel for comparison of particle homogenization network pore sizes

[0135] Examples 16 to 19 were prepared in the same manner as described in Example 3, except that mesh pore sizes of 400 μm, 300 μm, 180 μm, and 100 μm were applied during particle homogenization.

[0136]

[0137] Examples 20 to 21: Preparation of a Hexamethylenediamine (HMDA)-Crossed Cross-linked Hyaluronic Acid Hydrogel Pre-filled Syringe for Animal Experiments for Confirmation of Spur Pressure

[0138] Examples 20 and 21 were prepared by diluting the HMDA cross-linked hyaluronic acid hydrogel prepared in Example 1 with a solution having the same composition as the buffer solution used for washing to a concentration of 20 mg / mL based on the active ingredient sodium hyaluronate, then filling 3 mL and 5 mL of the diluted solution into 3 mL and 5 mL syringes, respectively, and sterilizing them by autoclaving.

[0139]

[0140] Examples 22 to 30: Preparation of a pre-filled syringe mixed with cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid for animal efficacy evaluation tests

[0141] Examples 22 to 30 prepared a composition for treating joint diseases by diluting the prepared HMDA cross-linked hyaluronic acid hydrogel of Example 1 with a solution having the same composition as the buffer solution used for washing to a concentration of 20 mg / mL based on the active ingredient sodium hyaluronate, and then mixing this with 20 mg / mL of unmodified hyaluronic acid in a certain ratio as shown in Tables 7 and 8, filling it into a 3 mL pre-filled syringe, and then sterilizing it by autoclaving.

[0142]

[0143] Examples 31 to 33: Preparation of HMDA-crosslinked hyaluronic acid hydrogel pre-filled syringes for clinical trials

[0144] Examples 31 to 33 were prepared by diluting the HMDA cross-linked hyaluronic acid hydrogel prepared in Example 1 with a solution having the same composition as the buffer solution used for washing, so that the concentration was 20 mg / mL based on the active ingredient sodium hyaluronate, and then filling it into a clinical pre-filled syringe specially designed to have low protrusion pressure and ease of use during injection so that the effective single injection volume was 3 mL (Example 31) or 5 mL (Examples 32 and 33), and then sterilizing it by autoclaving.

[0145]

[0146] Examples 34 to 35: Preparation of a pre-filled syringe with a mixed composition of HMDA-crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid for clinical trials

[0147] Examples 34 and 35 prepared a composition for treating joint diseases by diluting the HMDA cross-linked hyaluronic acid hydrogel prepared in Example 1 with a solution having the same composition as the buffer solution used for washing to obtain 20 mg / mL based on the active ingredient sodium hyaluronate, and then mixing this with 20 mg / mL of unmodified hyaluronic acid in weight ratios of 90:10 and 20:80. The composition was then filled into a specially designed clinical pre-filled syringe with low protrusion pressure and ease of use during injection, so that the effective single injection volume for clinical use was 3 mL, and then sterilized by autoclaving.

[0148]

[0149] Comparative Examples 1 to 4: Preparation of HMDA-crosslinked hyaluronic acid hydrogel by a comparative conventional manufacturing method

[0150] For 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, and for Comparative Example 4, twice the weight of the above was completely dissolved in distilled water. To facilitate the cross-linking reaction with the hyaluronate carboxyl groups, the cross-linking agent hexamethylenediamine dihydrochloride was added; to control the degree of cross-linking, it was added at 10-20 mol% of sodium hyaluronate repeating units (20 mol% is 1.39 g dissolved in 5 g of distilled water), 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 6.0-6.5. 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 mixture. The mixture was stirred at 30 rpm for 30 minutes at 45°C in a high-viscosity stirrer, and then left undisturbed at 45°C for more than 12 hours to perform the crosslinking reaction. The crosslinked hydrogel was first ground and then passed through a sieve with 180 μm pores to homogenize the particles. 80% ethanol filtered through a 0.22 μm filter was added to obtain the hydrogel powder as a precipitate, and a 1.3% NaCl solution was added at 100 times the volume and stirred for 1 hour. 80% ethanol was added again to obtain a precipitate, and the obtained hydrogel precipitate was placed in 100% ethanol for 10 minutes, then dried under reduced pressure at 40°C for 12 hours to confirm that residual EDC, HOBt, and HMDA were removed to a detection limit of 2 ppm or less, after which the dried material was recovered. This dried material was swollen with water for injection at a concentration of 20 mg / ml, and the hydrated cross-linked hyaluronic acid hydrogel was autoclaved at 121°C for 15 minutes.

[0151]

[0152] Comparative Examples 5 to 6: Comparative BDDE cross-linked hyaluronic acid hydrogel pre-filled syringe

[0153] Comparative Examples 5 and 6 are commercially available BDDE cross-linked sodium hyaluronate gel injections, specifically Synovian Injection from LG Chem (Republic of Korea). ® Injection; 3 g per 1 pre-filled syringe (3.0 mL); also available as Hyruan ONE ® Hyalone shot Injection (3.03 g per 3.0 mL pre-filled syringe) from Yifan Pharmaceutical, China and Shin Poong Pharm. Co., Ltd., Republic of Korea was used.

[0154]

[0155] Experimental Example 1: Measurement of the degree of crosslinking (CrD) of HMDA-crosslinked hyaluronic acid according to the present invention

[0156] The degree of crosslinking of Example 2 and Comparative Example 1 was measured by the method described in analysis methods (1) and (2) above, and the results are shown in Table 1 below. As a result of the experiment, in the case of Comparative Example 1, which was prepared by a conventional method of removing residual crosslinking agent through an ethanol precipitation method following the drying-hydration step according to the present invention, the degree of crosslinking could be analyzed by both TNBS analysis and NMR analysis. However, in the case of the crosslinked hyaluronic acid composition according to the present invention, such as Example 2, which has a low degree of crosslinking of about 0 to 2 mol%, it was confirmed that accurate measurement was impossible by TNBS analysis, so the degree of crosslinking was measured by NMR analysis.

[0157] [Table 1]

[0158]

[0159]

[0160] Experimental Example 2: Measurement of Physical Properties of the HMDA Crosslinked Hyaluronic Acid Hydrogel of the Present Invention

[0161] When examining the knee joint, a representative joint, the average storage modulus (G'; elasticity) and loss modulus (G''; viscosity) of joint fluid in healthy adults aged 21-27 have been reported to be 117±13 Pa and 45±8 Pa, respectively (both 2.5 Hz) (Stitik et al., Future Rheumatol. 2008;3(3):215-222). In contrast, the storage modulus (G', 2.5 Hz) of joint fluid in normal elderly individuals aged 52-78 years was reported to be reduced to 19±3 Pa, and in particular, the storage modulus of joint fluid in adult osteoarthritis patients aged 21-45 years or patients immediately before total knee arthroplasty at a moderate or higher stage was reported to be significantly reduced to 8.5±0.5 Pa and 1.9±0.5 Pa (both 2.5 Hz), respectively (Stitik et al., Future Rheumatol. 2008;3(3):215-222; Mazzucco D et al., J Orthop Res 2002;20(6):1157-1163). Therefore, a hyaluronic acid-based composition that helps lubricate the joint when administered via intra-articular injection and restores the reduced storage modulus of the patient's joint fluid to the level of a young adult is expected to be utilized for the treatment of joint diseases, exhibiting therapeutic effects such as improvement of joint pain and function. Accordingly, the target final storage modulus in the joint fluid after injection of the composition of the present invention was set to 117 Pa, which is the storage modulus of the joint fluid of a young adult, and the reduced storage modulus of the joint fluid before injection was estimated to be a minimum value of 1.9 Pa. Meanwhile, considering the average volume of human bone-joint synovial fluid (6.7±2.3 mL) and the expected volume of the therapeutic composition (2-6 mL), and taking into account the dilution factor due to the synovial fluid, the lower limit of the target storage modulus of the therapeutic composition was calculated as 232.1 Pa by the following formula.

[0162] Lower limit of target storage modulus of a composition for treating joint diseases

[0163] = [117 (Target) X 2 (Expected Minimum Dilution Factor)] - 1.9 (Estimated Initial Synovial Fluid Hypoelasticity)

[0164] = 232.1 (Pa)

[0165] In addition, to meet the biocompatibility required in clinical practice as a therapeutic composition, the amount of residual crosslinking agent must be controlled to 2 ppm or less.

[0166] 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 by the method described in analysis methods (2) to (4) above, and the results are shown in Table 2 below. As a result of the experiment, with conventional manufacturing methods including the ethanol precipitation method, if a composition is prepared such that the residual crosslinking agent is 2 ppm or less, a degree of crosslinking of 5 mol% or more is required at the same hyaluronic acid concentration (20 mg / ml) to produce a therapeutic composition with a target storage modulus of 232.1 Pa or higher. In contrast, it was confirmed that the composition according to the present invention can reach the target storage modulus even with a lower degree of crosslinking, particularly a low degree of crosslinking of 1 mol%, and a composition with a high storage modulus could be obtained without increasing the hyaluronic acid concentration (Comparative Example 4 vs. Examples 3 to 4).

[0167] [Table 2]

[0168]

[0169]

[0170] Experimental Example 3: Change in Storage Modulus According to Crosslinking Reaction Time

[0171] The storage modulus of Examples 5 to 10 above was measured by the method described in the analysis method (3) above, and the results are shown in Table 3 below. As a result of the experiment, the storage modulus increased as the crosslinking reaction time increased, but showed a tendency to saturate after 10 hours.

[0172] [Table 3]

[0173]

[0174]

[0175] Experimental Example 4: Change in Residual Crosslinking Agent (HMDA) According to Washing Time

[0176] The residual HMDA of Examples 11 to 15 above was measured by the method described in the analysis method (4) above, and the results are shown in Table 4 below. As a result of the experiment, it was confirmed that a washing process time of at least 20 hours with a phosphate buffer solution (pH 6.0-8.0) is required to control the residual HMDA to 2 ppm or less. To estimate a more accurate time, it was estimated that a washing process of at least 9 hours is required when fitting a linear regression curve for log concentration using the entire data, or 13.12 hours when fitting with data around 7-20 hours, or 8.82 hours.

[0177] [Table 4]

[0178]

[0179]

[0180] Experimental Example 5: Change in Storage Modulus According to Pore Size of Particle Homogenization Network

[0181] The storage modulus of Examples 16 to 19 above was measured by the method described in the analysis method (3) above, and the results are shown in Table 5 below. As a result of the experiment, a rapid decrease in storage modulus was confirmed when the mesh pore size was 180 μm or less when the cross-linked hyaluronic acid hydrogel particles were homogenized.

[0182] [Table 5]

[0183]

[0184]

[0185] Experimental Example 6: Comparison of protrusion pressure upon injection of the HMDA cross-linked hyaluronic acid hydrogel of the present invention

[0186] The injection pressure of the above Examples 20, 21, and Comparative Example 3 was measured by the method described in the above analysis method (5), and the results are shown in Table 6 below. As a result of the experiment, although the injection pressure increased with the filling volume, it showed a significantly lower injection pressure at the same hyaluronic acid concentration (20 mg / ml) compared to a commercially available BDDE cross-linked hyaluronic acid product (Comparative Example 3).

[0187] [Table 6]

[0188]

[0189]

[0190] Experimental Example 7: Evaluation of therapeutic efficacy for joint disease in a rodent arthritis model

[0191] The physical properties of Examples 22 to 27 and Comparative Example 5 were measured using the method described in analysis methods (2) to (4) above and are shown in Table 7. The results of evaluating the arthritis therapeutic efficacy of Examples 22 to 27 and Comparative Example 5 using a rat monosodium iodoacetate (MIA)-induced arthritis model, which is commonly used as an arthritis model, are shown in Table 7 and Figure 2.

[0192] After thoroughly removing the hair around the right knee of rats (Sprague-Dawley Rat; Daehan-Bio, South Korea; 7-8 weeks old), osteoarthritis was induced by administering 50 μL of a solution of MIA (I2512, Sigma-Aldrich, USA), dissolved in 0.9% physiological saline at a concentration of 50 mg / mL and filtered through a 0.22 μm filter, into the joint cavity of the right knee using a Hamilton syringe equipped with a 21-30G needle (Udo M et al., Osteoarthritis and cartilage 2016 24:1284-1291). Seven days after the MIA injection, 50 μL of the solvent (physiological saline) and the compositions of Examples 22 to 27 and Comparative Example 5 were injected into the joint cavity of selected animals in which stable arthritis induction had occurred. Joint cavity edema, body weight, and blood inflammatory markers were evaluated for 28 days. On day 28 after MIA injection (day 21 after sample administration), a necropsy was performed to collect knee joint tissue samples. The tissues were stained to assess the condition of the tissues and chondrocytes, and the joint tissue evaluation score (Mankin's score) was calculated. The Mankin score is the most basic histopathological observation for evaluating osteoarthritis. It is determined based on surface damage to articular cartilage, staining properties, numerical changes in chondrocytes, and the presence or absence of clone formation induced by osteoarthritis; a higher score indicates a higher degree of osteoarthritis induction. Statistical analysis between groups was performed using SPSS ver 2 (IBM, USA). Normality was tested using the Shapiro-Wilk's test; if normality was observed, group comparisons were conducted via ANOVA, while if normality was not observed, the Kruskal-Wallis test / Man-Whitney test was used for analysis.

[0193] As a result of the test, when HMDA cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid were mixed at various weight ratios ranging from 90:10 to 20:80, as the proportion of cross-linked hyaluronic acid decreased, the storage modulus (G'), i.e., elasticity, decreased and tanδ'' / G') increased, whereas the loss modulus (G''), i.e., viscosity, increased until it reached its highest value at a ratio of 50:50 between the cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid, and then decreased again.

[0194] The efficacy of the rat arthritis models of Examples 22 to 27 and Comparative Example 5 was evaluated using the Mankin score, a joint area evaluation score that assesses the morphology and structure of tissues and chondrocytes. As a result of the test, tissue damage was significantly increased in the solvent control group, in which arthritis was induced by MIA, compared to the sham control group, in which arthritis was not induced; furthermore, when a mixture of cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid was administered intra-articularly, a tendency was observed to reduce tissue damage as the storage modulus increased (Example 22, 90:10 mixture of cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid). In addition, even if the storage modulus was low, a higher proportion of unmodified hyaluronic acid (e.g., 70% or more) showed a tendency to reduce tissue damage, and compared to the solvent control group, the group administered in Example 27—that is, the group administered with a composition ratio of cross-linked hyaluronic acid hydrogel to unmodified hyaluronic acid of 20:80—showed a significant effect of reducing tissue damage even with a low storage modulus.

[0195] [Table 7]

[0196]

[0197]

[0198] Experimental Example 8: Evaluation of therapeutic efficacy for joint disease in a non-rodent osteoarthritis model

[0199] In human joint diseases, since the load caused by body weight has a significant impact on the exacerbation of symptoms such as pain, medium-sized non-rodent models may be more useful for evaluating therapeutic efficacy than rodent models. The results of evaluating the therapeutic efficacy of Examples 28 to 30 and Comparative Example 5 for osteoarthritis using a surgery-induced Beagle dog osteoarthritis model are shown in Table 8 and Figure 3.

[0200] Beagle dogs (Covance, USA; 12 months old) were anesthetized with 50 mg / kg zoletil 50 (Virbac, France) and 2.5 mg / kg xylazine (BayerAg, Germany). After shaving both knee areas and disinfecting with povidone and 70% ethanol, the skin was incised. The surrounding tissues were blunt dissected to expose the articular surface of the distal right femur, and the cartilage of the Beagle dogs was damaged through medial meniscectomy and anterior cruciate ligament transection. The sham control group underwent only wound closure using 4-0 nylon without meniscectomy or anterior cruciate ligament transection. The antibiotic cephardine (30 mg / kg) and the analgesic tramadol (3 mg / kg) were administered for 7 days after the induction of osteoarthritis. One week after inducing cartilage damage, osteoarthritis was induced by performing artificial exercise for three weeks (30 minutes / session, once a day), and when the average gait evaluation score was 2.5-3.0 points or higher, samples were administered by random distribution to each group. After anesthetizing the animals, a single dose of 1 mL each of the solvent (physiological saline) and the compositions of Examples 28 to 30 and Comparative Example 5 were administered into the joint cavity at a dose of 20 mg / mL per knee joint using a C-arm (Arcadis Varic, Siemens Co.). Gait pain evaluation was performed once a week for a total of 12 weeks, and at the 12th week, a necropsy was performed to confirm the tissue evaluation score (Mankin's score) of the joint area. The content of MMP-3 and VEGF165a in the joint fluid was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. Statistical analysis was performed in accordance with Experimental Example 6.MMP-3 is a degrading enzyme produced by synovial and chondrocytes in osteoarthritis or rheumatoid arthritis and plays an important role in the destruction of cartilage matrix such as collagen and connective tissue (Pulik L et al., Reumatologia 2023; 61(3):191-201). VEGF 165a is also known to act as an inflammatory factor in addition to angiogenesis in osteoarthritis or rheumatoid arthritis, contributing to abnormal cell growth or the intensification of joint inflammatory responses (Kim J et al, Exp. Mol. Med. 2020; 52: 843-853).

[0201] The efficacy of Examples 28 to 30 and Comparative Example 5 in the Beagle osteoarthritis model was evaluated by analyzing the effects on gait score evaluation, histopathological evaluation, and the secretion of inflammatory factors in the joint synovial fluid. As a result of the test, in the non-rodent osteoarthritis model, the higher the storage modulus, the better the symptom improvement effect (Table 8). In particular, when Example 30, in which HMDA cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid were mixed in a weight ratio of 90:10, was administered, the target gait improvement score (2 points) was reached most quickly (28 days) compared to the group administered other compositions, and was maintained at 2 points until week 12. In addition, at week 9, a significant improvement in the gait evaluation score was shown compared to the solvent control group. In the histopathological evaluation at week 12, the articular cartilage structure score improved in all composition administration groups compared to the solvent control group; however, in the mixed composition of HMDA-crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid, a tendency was observed where the degree of improvement in the articular cartilage structure score increased with higher storage modulus. In particular, when Example 30, a mixture of HMDA-crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid at a weight ratio of 90:10, was administered, the target gait improvement score (2 points) was reached most rapidly (28 days) compared to other composition administration groups and was maintained at 2 points until week 12. Furthermore, the release of MMP-3 and VEGF-165 a in the joint fluid was statistically significantly inhibited compared to the solvent control group, in which the secretion of inflammatory factors increased due to osteoarthritis induction, indicating potential efficacy in treating joint diseases (Fig. 3).

[0202] [Table 8]

[0203]

[0204]

[0205] These results in the medium-sized Beagle dog arthritis model subjected to weight bearing show a different trend from the correlation between storage modulus and efficacy observed as a U-shape in the small animal rat arthritis model of Experimental Example 7. However, given the small sample size and the commercially available BDDE cross-linked hyaluronic acid injection used as a control, which has proven efficacy in clinical trials, it was considered that there is a high probability of showing efficacy when the weight ratio of cross-linked hyaluronic acid hydrogel to unmodified hyaluronic acid is composed of 70:30 or higher and exhibits a high storage modulus.

[0206]

[0207] Experimental Example 9: Evaluation of clinical efficacy of the HMDA-crosslinked hyaluronic acid hydrogel of the present invention in patients with knee osteoarthritis (Phase 1 / 2 clinical trial)

[0208] The clinical efficacy (efficacy) of the above Examples 31 to 32 was evaluated by conducting a Phase 1 / 2 clinical trial on 20 patients with knee osteoarthritis, and the results are shown in Fig. 4.

[0209]

[0210] Clinical trial design

[0211] The efficacy of administering the investigational drug—specifically, the test drug (Examples 31 to 32), which is the composition for treating joint disease according to the present invention—or an equal dose of placebo once over a period of 6 months into adult patients with mild to moderate knee osteoarthritis was evaluated in a randomized, double-blind, placebo-controlled, stepwise weighting clinical trial. The test drug and placebo used were as follows:

[0212] - Test drug 1: Subject substance (Example 31, 20 mg / ml, 3 mL of intra-articular injection composition)

[0213] - Placebo 1: 3 mL of injectable normal saline solution not containing the subject substance

[0214] - Test drug 2: Subject substance (Example 32, 20 mg / ml, 5 mL of intra-articular injection composition)

[0215] - Placebo 2: 5 mL of injectable saline solution not containing the subject substance

[0216]

[0217] Subjects meeting the following selection and exclusion criteria were randomly assigned to Cohorts A and B, administered two different doses of Test Drug 1 and Test Drug 2, and followed up for 6 months. Efficacy was evaluated using the following evaluation methods at the time of administration and at 6, 12, and 24 weeks after administration. Within each cohort, a total of 10 subjects were randomly assigned, consisting of 8 subjects in the test drug group and 2 subjects in the placebo group. After 1 subject from the test drug group and 1 subject from the placebo group dropped out of each cohort, efficacy was evaluated on 8 subjects (7 test drug subjects and 1 placebo subject) in Cohorts A and B, respectively. As in Experimental Example 9 above, a rescue drug (acetaminophen 500 mg) was allowed up to a maximum of 4 g per day.

[0218]

[0219] Key Selection and Exclusion Criteria

[0220] Patients who met all selection criteria, including the following major selection criteria 1-4, were selected.

[0221] (Main Selection Criteria)

[0222] 1. Adult men and women aged 40 or older

[0223] 2. Patients with osteoarthritis classified as KL Grade 1–3 within 6 months of the screening date or at the time of the screening visit

[0224] 3. Patients diagnosed with unilateral or bilateral knee arthritis according to the clinical diagnostic criteria of the American College of Rheumatology (ACR) at the time of the screening visit who have knee pain and satisfy three or more of the following conditions

[0225] ① Age > 50

[0226] ② Morning rigidity <30 minutes

[0227] ③ Friction sound when moving

[0228] ④ Bony tenderness

[0229] ⑤ Bony enlargement

[0230] ⑥ No palpable warmth of synovium

[0231] 4. Weight Bearing Pain (WBP) - Those with a 100 mm VAS measurement of 40 mm or more

[0232] (Major exclusion criteria)

[0233] Patients who met any of the exclusion criteria, including the following major exclusion criteria 1-10, were excluded.

[0234] 1. BMI ≥35 kg / m 2

[0235] 2. Individuals with severe hip osteoarthritis or osteoarthritis of other joints at the time of screening that interferes with the evaluation of knee osteoarthritis.

[0236] 3. Cases involving a disease that may affect the evaluation of efficacy and safety

[0237] 4. Persons with joint infections or skin diseases unsuitable for administration of injectables

[0238] 5. Those with complete loss of the patello-femoral joint space

[0239] 6. Those who have received the following treatments within 14 days of the screening visit date (provided that those who have completed a 14-day wash-out period are eligible for registration)

[0240] - Glucosamine, anti-inflammatory analgesics including chondroitin sulfate and NSAIDs, herbal preparations, or physical therapy, etc.

[0241] 7. Patients who have used systemic steroids at the injection site within the last 3 months

[0242] 8. Persons who received intra-articular hyaluronic acid injections at the injection site within the last 6 months / Persons who received intra-articular injections such as intra-articular corticosteroids

[0243] 9. Persons who have undergone surgical procedures within 6 months or are scheduled to undergo surgery within 10 months

[0244] 10. Those with moderate or severe joint effusion detected by the Patella tap test during the screening visit

[0245]

[0246] Validity evaluation method

[0247] The primary efficacy endpoints for evaluating efficacy were the improvement in weight-bearing pain (WBP) relative to baseline, 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 of which were evaluated using a 100 mm visual analogue scale (VAS). In the VAS, patients subjectively indicated the degree of sensation they felt in response to a question regarding the severity of knee osteoarthritis pain by marking their level on a 100 mm straight line from 0 mm (no pain) to 100 mm (unbearable pain). The investigator verified the subjects' markings and recorded them numerically on the evaluation sheet. The improvement effect was quantified and calculated based on the difference between the length indicated by the patient before administration (pre-administration baseline) and the length indicated by the patient at each evaluation after administration. WOMAC evaluation was performed using a questionnaire based on established evaluation methods in the industry, in which patients self-evaluated 5 items regarding pain level, 2 items regarding stiffness level, and 17 items regarding difficulty with daily activities over the past 48 hours. Each item was graded using a 5-point Likert scale (0: none, 1: slight, 2: moderate, 3: severe, 4: very severe), and the scores of the three items (maximum 96 points) 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 was evaluated by assessing the proportion of patients who took acetaminophen, a rescue medication, during the clinical trial period.

[0248] As a result of the test, when the composition for treating joint disease of the present invention, namely Test Drug 1 (Example 31) and Test Drug 2 (Example 32), or a placebo was administered once into the knee joint cavity, a tendency for improvement in WOMAC and weight-bearing pain compared to the placebo was observed in patients who received 5 mL of Test Drug (Example 32) (Fig. 5). On the other hand, in the case of 3 mL of Test Drug (Example 31), no improvement in symptoms such as weight-bearing pain measured by VAS, stiffness measured by WOMAC, or function was observed compared to the placebo. However, while the placebo did not reduce the intake of the rescue drug acetaminophen, the proportion of patients taking the rescue drug in Test Group 1 was reduced to a degree similar to that of the Test Drug 2 group; therefore, caution is required in interpretation as the effect of the placebo may be due to the use of analgesics. For subsequent clinical trials, a 5 mL dose was selected.

[0249]

[0250] Experimental Example 10: Evaluation of clinical efficacy of the HMDA cross-linked hyaluronic acid hydrogel of the present invention in patients with knee osteoarthritis (Phase 3 clinical trial)

[0251] The clinical efficacy of the above Example 33 and Comparative Example 5 was evaluated by conducting a therapeutic confirmatory Phase 3 clinical trial on 223 patients with knee osteoarthritis using the method described below, and the results are shown in Table 9 and Figure 5.

[0252]

[0253] Clinical trial design

[0254] The efficacy of administering the investigational drug—specifically, the test drug (Example 33), which is the composition for treating joint disease according to the present invention, or the active control drug (Comparative Example 5)—into the knee joint cavity once at 6-month intervals followed by repeated re-administration was evaluated in a multi-center, randomized, double-blind, active-controlled, non-inferiority clinical trial in adult patients with mild to moderate knee osteoarthritis by following up for 3 months after re-administration. The test drug and placebo used were as follows:

[0255] - Test drug: Subject substance (Example 33, 20 mg / ml, 5 mL of intra-articular injection composition)

[0256] - Active reference: Comparative Example 5 (commercially available BDDE-crosslinked hyaluronic acid crosslinked single-dose formulation) 3 mL of intra-articular injection composition

[0257]

[0258] The test drug or active control drug was administered into the knee joint cavity of the subjects a total of two times at 24-week intervals. Subjects meeting the following selection and exclusion criteria were randomized into two groups to receive the test drug or active control drug, and efficacy was evaluated according to the evaluation method described below at each evaluation point at weeks 2, 6, 12, 24 (re-administration), and 36 after the first administration. As in Experimental Example 9 above, a rescue drug (acetaminophen 500 mg) was allowed up to a maximum of 4 g per day.

[0259]

[0260] Key Selection and Exclusion Criteria

[0261] The selection and exclusion criteria described in Experimental Example 9 above were generally applied.

[0262]

[0263] Validity evaluation method

[0264] For the efficacy evaluation, the primary efficacy endpoint was the improvement in weight-bearing pain (WBP) relative to baseline, a pain indicator, using a 100 mm visual analogue scale (VAS). The 100 mm VAS was evaluated in the same manner as described in Experimental Example 9 above. As a secondary efficacy endpoint, the improvement in joint-line tenderness on pressure was evaluated. Physical evaluation of joint-line tenderness was performed at each visit using a 4-point scale [0=none, 1=mild, 2=moderate, 3=severe]. A difference value lower than 0 indicates a higher efficacy.

[0265]

[0266] The primary efficacy endpoint was the change in weight-bearing pain (WBP - 100 mm-VAS) from baseline at 12 weeks. To test for non-inferiority regarding the change in weight-bearing pain (WBP) from baseline at 12 weeks for the test group compared to the active control group, an analysis of covariance was performed with baseline WBP values ​​as covariates. When the least squares mean (LS Mean) and standard error (SE) of each group and the difference in least squares mean between groups (LS Mean Difference, the least squares mean of the test group minus the least squares mean of the active control group) were calculated, it was defined that if the lower limit of the two-sided 95% confidence interval (CI) was greater than the pre-set non-inferiority margin of -10 mm, the treatment effect of the test group was judged to be non-inferior to the treatment effect of the active control group. In this case, the baseline was defined as the reference value before administration.

[0267]

[0268] As a result of the test, when the test drug (Example 33), which is a composition for treating joint diseases according to the present invention, was administered once at 6-month intervals into the knee joint cavity and the improvement in weight-bearing pain relative to baseline was measured at week 12, weight-bearing pain in patients with mild to moderate knee osteoarthritis was statistically significantly improved relative to baseline (improvement in weight-bearing pain relative to baseline: 23.71 mm). The pain improvement effect was confirmed by demonstrating non-inferiority in the improvement of weight-bearing pain in patients with mild to moderate knee osteoarthritis compared to an active control group (Comparative Example 5) whose efficacy has already been proven and is currently on the market (difference in corrected improvement in weight-bearing pain between groups: -2.26; refer to the judgment in Table 9 below). In addition, it was confirmed that the test group significantly reduced joint line tenderness compared to the active control group (Comparative Example 5), confirming a significant improvement effect on joint line tenderness in knee osteoarthritis (difference between groups -0.17, P<0.001; Fig. 5).

[0269] In addition, after 6 months (24 weeks), the test drug (Example 33) or the active control drug (Comparative Example 5) was re-administered, and the amount of improvement in weight-bearing pain compared to week 24 was checked again at the 12-week mark. As a result, a trend of superior improvement in weight-bearing pain was confirmed in the test drug compared to the active control drug, confirming that it showed a superior pain improvement effect upon re-administration (difference between groups in corrected weight-bearing pain improvement -4.75; Fig. 5).

[0270]

[0271] [Table 9]

[0272]

[0273]

[0274] Experimental Example 11: Evaluation of clinical efficacy of the HMDA cross-linked hyaluronic acid hydrogel and unmodified hyaluronic acid mixed composition of the present invention in patients with knee osteoarthritis (Phase 1 / 2 clinical trial)

[0275] The clinical efficacy of the above Examples 34 to 35 and Comparative Example 5 was evaluated by conducting a Phase 1 / 2 clinical trial on 40 patients with knee osteoarthritis, and the results are shown in Table 10.

[0276]

[0277] Clinical trial design

[0278] A randomized, double-blind, placebo-active-controlled clinical trial was conducted on adult patients with mild to moderate knee osteoarthritis by administering a single intra-articular administration of the investigational drug—namely, the investigational drug (Examples 34 to 35), the active control drug (Comparative Example 5), or a placebo, which is the composition for treating joint diseases according to the present invention—into the knee joint cavity, followed by 6 months of follow-up. The investigational drug, placebo, and active control drug used were as follows:

[0279] - Test agent 1: 3 mL of a subject substance (Example 34; a composition for intra-articular injection in which the standard 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).

[0280] - Test agent 2: 3 mL of a subject substance (Example 35; a composition for intra-articular injection in which the standard content of HMDA-crosslinked hyaluronic acid hydrogel and unmodified hyaluronic acid is 2 w / w% (20 mg / ml) and the weight ratio is 20:80).

[0281] - Placebo: 3 mL of injectable normal saline solution not containing the test substance

[0282] - Active reference: Comparative Example 5 (commercially available BDDE-crosslinked hyaluronic acid crosslinked single-dose formulation) 3 mL of intra-articular injection composition

[0283]

[0284] Subjects meeting the following selection and exclusion criteria were randomized into two groups, and efficacy was evaluated according to the evaluation method described below at each evaluation time of weeks 2, 6, 12, 16, 24, and 36 after intra-articular administration of test drug 1, test drug 2, placebo, or active control drug. As in Experimental Example 9 above, a rescue drug (acetaminophen 500 mg) was allowed up to a maximum of 4 g per day.

[0285]

[0286] Key Selection and Exclusion Criteria

[0287] The selection and exclusion criteria described in Experimental Example 9 above were generally applied.

[0288]

[0289] Validity evaluation method

[0290] The primary efficacy endpoints for evaluating efficacy were weight-bearing pain measured by a 100 mm VAS and WOMAC, and the 100 mm VAS and WOMAC were evaluated using the same evaluation method as described in Experimental Example 9 above. A difference value between groups lower than 0 indicates a higher effect. Analysis of covariance was performed to analyze the difference between groups.

[0291]

[0292] As a result of the test, when Test Drug 1 (Example 34), a composition for treating joint diseases according to the present invention, was administered once into the knee joint cavity, both weight-bearing pain and WOMAC scores in patients with mild to moderate knee osteoarthritis were significantly improved compared to the active control group (Comparative Example 5), which was a commercially available BDDE cross-linked hyaluronic acid hydrogel injection (Tables 10, 11, and 12). In particular, the composition of the present invention, in which the weight ratio of HMDA cross-linked hyaluronic acid hydrogel to unmodified hyaluronic acid is 90:10, showed an effect of significantly improving weight-bearing pain compared to the active control group and placebo at week 6, despite a lower rescue drug dosage compared to other groups, confirming that the initial pain improvement effect was outstandingly superior (change from baseline -35.10; Tables 10 and 12). In addition, the significantly lower dosage of rescue medication at weeks 12 and 16 supports the excellent pain-improving effect of this composition (Table 12). In contrast, for Test Drug 2 (Example 35; weight ratio 20:80), although lower than Test Drug 1, a superior improvement trend was observed in weight-bearing pain and WOMAC compared to the active control group (Tables 10 and 11), and a tendency to reduce WOMAC and rescue medication dosages compared to the placebo was also observed (Tables 11 and 12). It was considered that caution is required in interpreting the effects of each group, especially the placebo, taking into account the rescue medication dosage.

[0293] [Table 10]

[0294]

[0295]

[0296] [Table 11]

[0297]

[0298]

[0299] [Table 12]

[0300]

[0301]

[0302] Experimental Example 12: Comparison of Storage Modulus and Improvement in Weight-bearing Pain of the HMDA-Cross-linked Hyaluronic Acid Hydrogel-containing Investigational Drug of the Present Invention

[0303] Table 13 compares the storage modulus (G') of the investigational medicinal products containing the compositions of Examples 31 to 35 with the weight-bearing pain (100 mm VAS-WBP) values ​​used as an efficacy evaluation scale in clinical trials targeting patients with knee osteoarthritis in Experimental Examples 9 to 11. The storage modulus range of the investigational medicinal product batches that were clinically used and confirmed to be effective was observed to be 219-721 Pa. Among these, the storage modulus range that showed superior pain improvement effects in clinical trials compared to commercially available products—that is, the storage modulus range of the HMDA cross-linked hyaluronic acid hydrogel of the present invention that guarantees efficacy—was estimated to be 342-721 Pa (2.5 Hz, 25℃).

[0304] [Table 13]

[0305]

[0306]

[0307] Reference Example 1: Clinical Safety Evaluation of the HMDA-Crossed Hyaluronic Acid Hydrogel of the Present Invention in Patients with Knee Osteoarthritis

[0308] Examples 32 to 34 above involved the administration of the investigational drug 213 times to 123 adult patients with knee osteoarthritis in two clinical trials of Experimental Examples 9 to 10, and no adverse drug reactions, serious adverse drug reactions, or deaths related to the investigational drug were reported, and safety evaluation results showed no concerning risks regarding safety and tolerability.

[0309]

[0310] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

1. Comprising a cross-linked hyaluronic acid hydrogel obtained by a cross-linking reaction with hyaluronic acid or a pharmaceutically acceptable salt thereof and an alkylenediamine cross-linking agent represented by the following Formula 1 or Formula 2; The storage modulus (G', 2.5 Hz, 25℃) is 250-850 Pa; The degree of cross-linking (CrD; number of cross-linking agents on both sides / number of hyaluronic acid monomers) of the cross-linked hyaluronic acid is less than 5 mol%; A composition for treating joint disease administered once as an injection to a patient over a period of 3 months or more: [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 the above chemical formulas 1 and 2, HA is hyaluronic acid excluding one carboxyl group or a pharmaceutically acceptable salt thereof; R1 is unsubstituted or substituted C3-C 10 It is an alkylene group; x, y, and z are each independently integers from 100,000 to 5,000,000.

2. A composition according to claim 1, characterized in that the pharmaceutically acceptable salt of the hyaluronic acid is a sodium salt, potassium salt, calcium salt, magnesium salt, zinc salt, cobalt salt, or tetrabutylammonium salt.

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

4. A composition according to claim 3, characterized in that the alkylenediamine crosslinking agent is hexamethylenediamine.

5. A composition according to claim 1, characterized in that the storage modulus (G', 2.5 Hz, 25℃) of the composition is 342-721 Pa.

6. A composition according to claim 1, characterized in that the degree of crosslinking (CrD) of the crosslinked hyaluronic acid is 1 mol% or less.

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

1.

8. A composition according to claim 7, characterized in that the standard 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. A composition characterized in that, in any one of claims 1 to 8, the joint disease is osteoarthritis.

10. A composition according to any one of claims 1 to 9, characterized in that the treatment is the improvement, healing, or inhibition of progression of symptoms.

11. A composition according to claim 10, characterized in that the treatment is the improvement of joint pain or the improvement of joint function.

12. A kit comprising a pre-filled syringe filled with a composition according to any one of claims 1 to 11 to have a clinical single injection effective volume of 2 mL to 6 mL.

13. A method for manufacturing a composition according to any one of claims 1 to 11 or a kit according to claim 12, wherein i) dissolve hyaluronic acid or a pharmaceutically acceptable salt thereof in a solvent; ii) Mixing an alkaline aqueous solution, prepared by dissolving an alkylenediamine crosslinking agent, a peptide bond promoter, and a carboxyl group activator in the above solution and filtering it; iii) Prepare cross-linked hyaluronic acid by performing a cross-linking reaction at constant temperature conditions (30℃ to 50℃) for at least 10 hours; iv) The above-mentioned cross-linked hyaluronic acid is crushed, washed, and swollen to prepare a hydrogel; v) A mixed hydrogel composition is prepared by homogenizing the above hydrogel particles, either as is or by mixing; vi) A manufacturing method comprising the step of filling the above hydrogel composition into a storage container or syringe and heat-treating or sterilizing it.

14. A method of preparation according to claim 13, characterized in that the concentration of the alkylenediamine crosslinking agent is 10 mol% to 30 mol% of hyaluronic acid or a pharmaceutically acceptable salt unit thereof.

15. A manufacturing method according to claim 13, characterized by using a thermostatic stirring bath and an orbital electron centrifugal stirrer to suppress the over-reaction of the crosslinking reaction in step (ii) above.

16. A manufacturing method according to claim 13, characterized in that the rotational speed in the above-mentioned orbital centrifugal stirrer is 300 rpm or more and is performed for a total of 60 minutes or more.

17. A method of preparation according to claim 13, wherein in step (ii) above, the peptide bond promoter is selected from the group consisting of N-hydroxysuccinimide, 1-hydroxybenzotriazole, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, 1-hydroxy-7-azabenzotriazole, sulfo-N-hydroxysulfosuccinimide, and mixtures thereof.

18. A method of preparation according to claim 13, wherein in step (ii) above, the carboxyl group activator is selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-(trimethylammonio)propyl)carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, and mixtures thereof.

19. A manufacturing method according to claim 13, characterized in that, in step (iv) above, washing and swelling are performed with a phosphate buffer solution (pH 6.0-8.0) for 9 hours or more.

20. A manufacturing method according to claim 13, characterized in that, in step (v), particle homogenization is performed using a mesh having a pore size of greater than 180 μm and less than or equal to 500 μm.

21. A method of manufacturing according to claim 13, wherein the joint disease is knee osteoarthritis, the composition consists solely of cross-linked hyaluronic acid hydrogel, and is filled into a pre-filled syringe such that the effective volume for a single clinical injection is 5 mL.

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