Sol-gel transformation of 6-arm PEG hydrate gel over time

Abstract

The present invention relates to a 6-arm PEG hydrated gel composition whose physical properties change from sol to gel within 30 minutes over time. The present invention includes two types of PEG derivatives as main components of the hydrated gel, and when each PEG derivative is dissolved in a buffer solution and the two solutions are mixed, the physical properties can be changed to a gel state within 30 minutes.

Description

[Technical Field]

[0001] The present invention relates to a PEG hydrated gel whose physical properties change from sol to gel over time. [Background technology]

[0002] Osteoarthritis is a joint disease caused by the wear of articular cartilage, which surrounds the articular surface of bones, resulting in bone exposure, pain due to inflammation of the synovial membrane that surrounds and connects the bones, and structural deformation and degeneration of the joint. It primarily affects weight-bearing joints, causing pain, activity limitations, and body shape changes. It is caused by genetic predisposition, trauma to major joints, repeated occupational use of joints, and obesity. It is particularly common among older adults and is gradually increasing in incidence due to the global aging population. To date, the goal of osteoarthritis treatment has been to alleviate symptoms, reduce pain, and slow disease progression, thereby maintaining joint function and quality of life, rather than repairing structural damage. One treatment for osteoarthritis is intra-articular injection therapy, typically steroid injections and hyaluronic acid injections. Steroid injections provide 80-90% temporary pain relief, but frequent injections can worsen joint damage, so it is recommended to administer injections at intervals of 4-6 months. Hyaluronic acid injections have been tried as a method of externally replenishing hyaluronic acid that is lacking due to the inflammatory response of osteoarthritis, and are now being performed under the name of viscosupplement.

[0003] The history of hyaluronic acid injection therapy (viscosupplementation) began in the 1970s as an animal medicine for racehorses, and (商標) , Hylartil-Vet (商標) In 1987, Seikagaku Co., Ltd. in Japan developed and marketed Artz for the treatment of arthritis patients. (商標) and Hyalgan of Italy's FIDIA (商標)Through continuous research and development using hyaluronic acid, Synvisc was developed by Balazs et al. in the 1990s. (商標) was developed, and with the sale of single-injection hyaluronic acid products using a variety of low molecular weights, they have since been widely used in Japan as a replacement therapy to delay surgery for arthritis patients, and improved products have been developed and sold around the world, including in Korea and Europe (Non-Patent Document 1).

[0004] Hyaluronic acid is a natural component of the extracellular matrix and a linear polymeric polysaccharide composed of alternating bonds of β-DN-acetylglucosamine and β-D-glucosamine. When injected into a joint, it acts as a lubricant and shock absorber, alleviating pain from osteoarthritis and improving knee function. The molecular weight of hyaluronic acid in the synovial fluid of healthy young people is 6,000,000 Da, and its dynamic analysis values ​​(at 2.5 Hz) show a viscosity of 45 Pa and an elasticity of approximately 117 Pa (Non-Patent Document 2). Currently, commercially available hyaluronic acid injection products are divided into products made from linear hyaluronic acid itself and products made from crosslinked hyaluronic acid in the form of a hyaluronic acid gel. Hyalgan, a sodium hyaluronate aqueous solution product, is also available. (商標) ), ARTZ (商標) ), Euflexxa (商標) ) and Orthovisc (商標) ) has a molecular weight of about 500,000 to 3,600,000 Da, and is said to provide pain relief for about three months when administered three times in 2 ml doses, and for about six months when administered five times in 2 ml doses. A commercially available product in the form of cross-linked hyaluronic acid is Synvisc. (商標) , Synvisc-one (商標) ), Durolane (商標) ), Gel-One (商標) ) and MONOVISC (商標) ) are products that increase the molecular weight of hyaluronic acid through cross-linking or protect the areas where hyaluronic acid is decomposed by enzymes, thereby increasing the durability of the effect.

[0005] Patent Document 1 discloses a method for forming a crosslinked hyaluronic acid gel by reacting hyaluronic acid (HA) with divinyl sulfone (DVS) in an alkaline aqueous solution, and for controlling the swelling rate and degree of crosslinking of the gel depending on various conditions during the reaction (e.g., polymer / DVS ratio, molecular weight and concentration of hyaluronic acid). The crosslinking reaction is carried out at a pH of 9 and approximately 20°C, with a hyaluronic acid molecular weight of 50,000-8,000,000 Da, a concentration of 1-8%, and an HA / DVS weight ratio of 15:1-1:5. This low reaction temperature reduces the effect on the properties of the crosslinked gel, as HA is rapidly decomposed and its molecular weight is reduced by the high temperature and alkaline solution. Synvisc (Synvisc), an injectable crosslinked hyaluronic acid gel product according to this invention, is a hyaluronic acid gel with a molecular weight of 50,000-8,000,000 Da, a concentration of 1-8%, and an HA / DVS weight ratio of 15:1-1:5. (商標) , Synvisc-one (商標) ) is commercially available.

[0006] Patent Document 2 also discloses a method for producing a biocompatible polysaccharide gel composition, in which hyaluronic acid is reacted with 1,4-butanediol diglycidyl ether (BDDE), a polyfunctional cross-linking agent containing an epoxy functional group, to produce a viscoelastic hyaluronic acid hydrogel. After primary cross-linking to form an ether with 0.2% cross-linking agent and 10% hyaluronic acid at pH 9, acetic acid is added to adjust the pH to 2-6, and a secondary reaction to form an ester is carried out to form a gel. Using the technology of this invention, a high molecular weight (9,000,000 Da) cross-linked hyaluronic acid product, Durolane, is produced. (商標) ) is commercially available.

[0007] Patent Document 3 discloses a method for producing hyaluronic acid hydrogels by synthesizing a photoreactive hyaluronic acid derivative with cinnamic acid and then forming a cyclobutane ring with UV light. The dynamic viscoelasticity measured with a rheometer under 10 Hz conditions shows a storage modulus (G'; elasticity) of 50 to 1,500 Pa and a loss modulus (G'; viscosity) of 10 to 300 Pa, demonstrating superior viscoelasticity compared to existing products. The commercially available GEL-ONE (GEL-ONE) product utilizes the technology of this invention. (商標) ) is a product that has been aminated at the carboxyl group of hyaluronic acid, which is the recognition site for hyaluronic acid-degrading enzymes, and has a lower rate of decomposition by hyaluronic acid compared to other commercially available products, resulting in increased in vivo sustainability.

[0008] Thus, hyaluronic acid has a short half-life (in vivo persistence) of only a few hours after application in the body, and attempts have been made to increase its half-life and persistence by increasing the dosage of the composition, the concentration of hyaluronic acid, and the molecular weight. However, the increased viscosity makes it difficult to inject the composition into the tissue when injecting hyaluronic acid into the joint using a syringe, which causes pain and strain on the patient and the practitioner. Furthermore, although the in vivo persistence of hyaluronic acid crosslinked products has increased compared to uncrosslinked hyaluronic acid, it still has low biodurability, being decomposed within six months.

[0009] To solve these problems, the present invention provides a hydrated gel composition containing a 6-arm PEG derivative, which converts from a sol to a gel within 30 minutes. The hydrated gel composition can be injected in a sol state with low viscosity, reducing injection force, and converts to a gel state within 30 minutes after administration. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 4,582,865 [Patent Document 2] U.S. Patent No. 5,827,937 [Patent Document 3] U.S. Patent No. 6,031,017 [Non-patent literature]

[0011] [Non-Patent Document 1] Advancing Viscosupplementation.2007.Dr Ting Choon Meng [Non-patent document 2] Disorders of the knee 2nd ed.JB Lippincott;1982 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention aims to provide a PEG hydrated gel composition whose physical properties change from sol to gel within 30 minutes. [Means for solving the problem]

[0013] The present invention provides a method for preparing a polymerizable composition comprising: a first solution comprising a first polyethylene glycol derivative having an electrophilic reactive group; and

[0014] a second solution comprising a second polyethylene glycol derivative having a nucleophilic reactive group;

[0015] the first polyethylene glycol derivative comprises 6-arm polyethylene glycol-N-hydroxysuccinimide (6-arm PEG-NHS) and / or 8-arm polyethylene glycol-N-hydroxysuccinimide (8-arm PEG-NHS);

[0016] the second polyethylene glycol derivative comprises a 6-arm polyethylene glycol-thiol (6-arm PEG-SH) and / or an 8-arm polyethylene glycol-thiol (8-arm PEG-SH);

[0017] The first and second solutions form a hydrated gel through a sol-gel reaction when mixed together to provide an injection.

[0018] The present invention also provides a first solution set in which a first polyethylene glycol derivative powder having an electrophilic reactive group and a buffer solution having a pH of 4 to 9 are separately stored; and

[0019] a second solution set in which a second polyethylene glycol derivative powder having a nucleophilic reactive group and a buffer solution of pH 4 to 9 are separately stored,

[0020] the first polyethylene glycol derivative comprises 6-arm polyethylene glycol-N-hydroxysuccinimide (6-arm PEG-NHS) and / or 8-arm polyethylene glycol-N-hydroxysuccinimide (8-arm PEG-NHS);

[0021] the second polyethylene glycol derivative comprises a 6-arm polyethylene glycol-thiol (6-arm PEG-SH) and / or an 8-arm polyethylene glycol-thiol (8-arm PEG-SH);

[0022] The injection kit is provided, wherein the first solution and the second solution form a hydrated gel through a sol-gel reaction when mixed.

[0023] The present invention provides a PEG hydrated gel whose physical properties change from sol to gel within 30 minutes.

[0024] In the present invention, two types of PEG derivatives are used as the main components of the hydrated gel. After dissolving each PEG derivative in a buffer solution, the two solutions are mixed, and the physical properties can be converted from a sol state to a gel state within 30 minutes. The PEG hydrated gel can be easily injected into the body in a liquid (sol) state. After injection, the viscosity and elasticity of the composition increase, converting the physical properties to a gel form. Therefore, it can be used as a viscosupplement for the pain relief of osteoarthritis. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows the gelation time of hydrated gels as a function of pH.

[0026] [Figure 2] FIG. 2 shows the gelation time of hydrated gels as a function of the concentration of the 6-arm PEG derivative.

[0027] [Figure 3] Figure 3 (a, b, c, d) shows the results of viscoelasticity measurements of the hydrated gel.

[0028] [Figure 4] FIG. 4 shows the results of viscoelasticity measurements of the hydrated gel.

[0029] [Figure 5] FIG. 5 shows the results of measuring the joint pain-reducing effect of the hydrated gel. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides a method for preparing a polymerizable composition comprising: a first solution comprising a first polyethylene glycol derivative having an electrophilic reactive group; and

[0031] a second solution comprising a second polyethylene glycol derivative having a nucleophilic reactive group;

[0032] the first polyethylene glycol derivative comprises 6-arm polyethylene glycol-N-hydroxysuccinimide (6-arm PEG-NHS) and / or 8-arm polyethylene glycol-N-hydroxysuccinimide (8-arm PEG-NHS);

[0033] the second polyethylene glycol derivative comprises a 6-arm polyethylene glycol-thiol (6-arm PEG-SH) and / or an 8-arm polyethylene glycol-thiol (8-arm PEG-SH);

[0034] The first and second solutions relate to an injection that forms a hydrated gel through a sol-gel reaction when mixed.

[0035] The present invention will now be described in more detail.

[0036] The present invention uses two types of PEG derivatives and can form a hydrated gel through a crosslinking reaction between the two types of PEG derivatives.

[0037] Specifically, the injectable preparation according to the present invention comprises a first solution and a second solution, the first solution containing a first polyethylene glycol derivative and the second solution containing a second polyethylene glycol derivative, wherein the first polyethylene glycol derivative comprises 6-arm polyethylene glycol-N-hydroxysuccinimide (6-arm PEG-NHS) and / or 8-arm polyethylene glycol-N-hydroxysuccinimide (8-arm PEG-NHS), and the second polyethylene glycol derivative comprises 6-arm polyethylene glycol-thiol (6-arm PEG-SH) and / or 8-arm polyethylene glycol-thiol (8-arm PEG-SH).

[0038] Hereinafter, 6-arm PEG-NHS can be referred to as the first 6-arm PEG derivative, 8-arm PEG-NHS can be referred to as the first 8-arm PEG derivative, 6-arm PEG-SH can be referred to as the second 6-arm PEG derivative, and 8-arm PEG-SH can be referred to as the second 8-arm PEG derivative. Hereinafter, the term "6-arm PEG derivative" can be understood to refer to both the 6-arm PEG-NHS and 6-arm PEG-SH, and the term "8-arm PEG derivative" can be understood to refer to both the 8-arm PEG-NHS and 8-arm PEG-SH.

[0039] In the present invention, a "6-arm PEG derivative" or an "8-arm PEG derivative" refers to a derivative having 6 or 8 linear PEG groups, respectively, from the core, and may have a structure in which the groups are linked to each other from the core.

[0040] In the present invention, the first solution can contain 6-arm polyethylene glycol-N-hydroxysuccinimide (6-arm PEG-NHS) and / or 8-arm polyethylene glycol-N-hydroxysuccinimide (8-arm PEG-NHS) having an electrophilic reactive group and a first solvent.

[0041] In the present invention, "6-arm polyethylene glycol-N-hydroxysuccinimide (6-arm PEG-NHS)" may have a structure in which each terminal of the 6-arm PEG is substituted with N-hydroxysuccinimidyl (NHS). Furthermore, "8-arm polyethylene glycol-N-hydroxysuccinimide (8-arm PEG-NHS)" may have a structure in which each terminal of the 8-arm PEG is substituted with N-hydroxysuccinimidyl (NHS). The NHS can react with a thiol group.

[0042] In one embodiment, the 6-arm PEG-NHS and / or 8-arm PEG-NHS may be a compound represented by the following Chemical Formula 1:

[0043] [ka]

[0044] [ka]

[0045] If the core has a 6-arm structure, [ka] If the core has an 8-arm structure, [ka] may be.

[0046] Specifically, the first polyethylene glycol derivative may be 6-arm PEG-NHS, wherein L may be -(C=0)-, and R is [ka] The Core may be [ka] Furthermore, n may be an integer of 20 to 400, m1 may be 0, m2 may be an integer of 1 to 3, p may be an integer of 1, and q may be an integer of 6.

[0047] In one embodiment, the 6-arm PEG-NHS has the following formula: 7 The compound may be represented by the formula: [ka]

[0048] The chemical formula 7 and n may be an integer between 20 and 400.

[0049] In the present invention, the second solution may contain 6-arm polyethylene glycol-thiol (6-arm PEG-SH) and / or 8-arm polyethylene glycol-thiol (8-arm PEG-SH) having a nucleophilic reactive group and a second solvent.

[0050] In the present invention, "6-arm polyethylene glycol-thiol (6-arm PEG-SH)" may have a structure in which each terminal of a 6-arm PEG is substituted with a thiol. Also, "8-arm polyethylene glycol-thiol (8-arm PEG-SH)" may have a structure in which each terminal of an 8-arm PEG is substituted with a thiol. The thiol can react with NHS.

[0051] In one embodiment, the 6-arm PEG-SH and / or 8-arm PEG-SH has the following formula: 8 The compound may be represented by the formula:

[0052] [ka]

[0053] [ka]

[0054] If the core has a 6-arm structure, [ka] If the core has an 8-arm structure, [ka] may be.

[0055] Specifically, the second polyethylene glycol derivative may be 6-arm PEG-SH, wherein R may be a thiol group, and Core may be: [ka] Furthermore, n may be an integer of 20 to 400, m1 may be 0, m2 may be an integer of 1 to 3, p may be 0, and q may be an integer of 6.

[0056] In one embodiment, the 6-arm PEG-SH has the formula: 13 The compound may be represented by the formula: [ka]

[0057] The chemical formula 13 and n may be an integer between 20 and 400.

[0058] In the present invention, the molecular weight of the first polyethylene glycol derivative may be 5,000 to 100,000 Da, and the molecular weight of the second polyethylene glycol derivative may be 5,000 to 100,000 Da. The structural and physical properties of the hydrated gel to be produced can be controlled by adjusting the molecular weight. The larger the molecular weight, the coarser the structure of the hydrated gel, and the smaller the molecular weight, the finer the structure.

[0059] In the present invention, the first solvent and the second solvent may each be a buffer solution. That is, the first polyethylene glycol derivative and the second polyethylene glycol derivative may each be contained in a buffer solution. The buffer solution may be a phosphate buffer solution or physiological saline. The first solvent and the second solvent may be the same solvent.

[0060] In one specific example, the pH of the first solution may be 4-9, and the pH of the second solution may be 4-9.

[0061] In one specific example, the concentration of the first polyethylene glycol derivative in the first solution may be 1 to 10% (w / v), and the concentration of the second polyethylene glycol derivative in the second solution may be 1 to 10% (w / v). As the concentration of the first polyethylene glycol derivative increases and the pH of the solution becomes more basic during the reaction, the gelation time required for the formation of a hydrated gel may become shorter. That is, when the concentration is less than 1%, the physical properties become similar to a sol, and when the concentration exceeds 10%, the physical properties become such that a hard gel is formed and breaks down, making it unsuitable for use with materials requiring viscoelasticity.

[0062] In the present invention, the first and second solutions can be mixed immediately before injection, and can form a hydrated gel within 30 minutes through a sol-gel reaction upon mixing. That is, the mixture of the first and second solutions can form a hydrated gel after injection.

[0063] In one specific example, the first polyethylene glycol derivative having an electrophilic reactive group and the second polyethylene glycol derivative having a nucleophilic reactive group can be mixed in a weight ratio of 1:0.5 to 1:2 or 1:0.7 to 1:1.1.

[0064] A hydrogel is defined as a structure of natural or synthetic derivatives that swells but does not dissolve in aqueous solution. It also has many advantages applicable to the biomedical field. Because it can absorb and encapsulate aqueous solution, it is similar to living tissue and is permeable to low-molecular-weight substances such as oxygen, nutrients, and metabolic substances. Furthermore, the soft surface structure of swollen hydrogels reduces frictional irritation to surrounding cells and tissues in vivo. The present invention provides an injectable arthritis solution that lasts for a long time in vivo and is highly biocompatible by incorporating two types of PEG derivatives. The injectable solution of the present invention provides a long-lasting analgesic effect and articular cartilage protection after a single administration into the joint (cartilage cavity). Furthermore, two types of biocompatible polymer PEG derivatives are reacted with each other in a neutral or basic aqueous solution, and the physical properties can be converted from sol to gel within 30 minutes of reaction through cross-linking and covalent bonding between them, forming a hydrogel.

[0065] That is, when the first solution and the second solution are mixed, the first polyethylene glycol derivative having an electrophilic reactive group and the second polyethylene glycol derivative having a nucleophilic reactive group can form a covalent bond. Specifically, the thiol group (SH) of the second polyethylene glycol derivative having a thiol group and the NHS of the first polyethylene glycol derivative having an NHS group form a covalent bond to form a hydrated gel (Reaction Scheme 1).

[0066] <Reaction Scheme 1> [ka]

[0067] In the present invention, the pH of the first solution and the pH of the second solution may be different from each other. Solutions with the same pH may gel quickly, which may cause clogging of the syringe needle during injection. Therefore, the gelation time can be adjusted by using a buffer solution with a different pH condition.

[0068] In the present invention, when 6-arm PEG-NHS is used as the first polyethylene glycol derivative, 6-arm PEG-SH can be used as the second polyethylene glycol derivative, and when 8-arm PEG-NHS is used as the first polyethylene glycol derivative, 8-arm PEG-SH can be used as the second polyethylene glycol derivative.

[0069] In one embodiment, the structural and physical properties of the hydrated gel can be adjusted by the molecular weight, concentration, and reaction conditions of the first polyethylene glycol derivative and / or the second polyethylene glycol derivative.

[0070] The injection according to the present invention can be administered into a joint (cartilage cavity), and can form a hydrated gel after injection.

[0071] In one specific example, the viscosity (loss modulus G') of the injection, specifically the mixture of the first solution and the second solution, within 300 seconds upon injection (infusion) may be 1 Pa or less.

[0072] In one specific example, the elasticity (storage modulus G) of the injection, specifically the mixture of the first solution and the second solution, after 2,000 seconds of injection (infusion) may be 1,000 Pa or more.

[0073] The elasticity and viscosity (G', G'; Pa) values ​​can show low viscosity (1 Pa or less) close to that of a sol, and high viscosity (1,000 Pa or more) in a gel state where covalent bonds are formed.

[0074] The injectable solution of the present invention can reduce the drawback of existing products, which have a high viscosity and a high injection force when injected into a joint, causing pain. In addition, by adjusting the crosslinking time, the injectable solution can be easily injected from a syringe with a low viscosity upon injection. After injection, the two types of PEG derivatives react to gradually form covalent bonds, forming a hydrated gel with excellent viscoelasticity.

[0075] The present invention also provides a storage kit for the injection, i.e., an injection kit.

[0076] The injection kit according to the present invention comprises a first solution set in which a first polyethylene glycol derivative powder having an electrophilic reactive group and a buffer solution having a pH of 4 to 9 are separately stored; and

[0077] A second solution set may be included in which a second polyethylene glycol derivative powder having a nucleophilic reactive group and a buffer solution of pH 4 to 9 are stored separately.

[0078] Here, the first polyethylene glycol derivative includes 6-arm polyethylene glycol-N-hydroxysuccinimide (6-arm PEG-NHS) and / or 8-arm polyethylene glycol-N-hydroxysuccinimide (8-arm PEG-NHS), the second polyethylene glycol derivative includes 6-arm polyethylene glycol-thiol (6-arm PEG-SH) and / or 8-arm polyethylene glycol-N-thiol (8-arm PEG-SH), and the first and second solutions can form a hydrated gel through a sol-gel reaction when mixed.

[0079] The kit includes dissolving a first polyethylene glycol derivative powder in a buffer solution of a first solution set to prepare a first solution immediately before use;

[0080] A second polyethylene glycol derivative powder is dissolved in a buffer solution of a second solution set to prepare a second solution, and then

[0081] The first and second solutions can be mixed and used.

[0082] The present invention will be described in detail below through examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. The examples are provided so that the disclosure of the present invention will be complete and will fully convey the scope of the invention to those skilled in the art to which the present invention pertains, and the present invention is defined only by the scope of the claims.

[0083] (Best Mode for Carrying Out the Invention) [Example]

[0084] Preparation Example 1. Synthesis of 6-arm PEG-succinimidyl glutarate (6-arm PEG-SG) <Reaction Scheme 2> [ka]

[0085] 6-arm PEG-succinimidyl glutarate (6-arm PEG-SG) was synthesized according to Reaction Scheme 2.

[0086] The compound of formula 5 was dissolved in methylene chloride at room temperature and triethylamine was added to prepare a reaction solution. Glutaric anhydride was added to the reaction solution and stirred at room temperature for 20-24 hours. The reaction solution was washed with 14% ammonium chloride aqueous solution, and after the layers were separated, the lower organic solution layer was collected. The aqueous layer was extracted with methylene chloride. The combined organic solution layers were dehydrated using magnesium sulfate, and the solvent was concentrated and then precipitated by adding diethyl ether. The precipitate was filtered and dried under vacuum at room temperature for 24 hours to obtain the compound of formula 6.

[0087] The compound of formula 6 was dissolved in methylene chloride and N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) were added to prepare a reaction solution. The reaction solution was stirred at room temperature for 15-20 hours. After the reaction, the by-product dicyclohexyl urea (DCU) was filtered using a glass filter. The filtered solution was concentrated and then precipitated in diethyl ether. The precipitate was filtered, dissolved in ethyl acetate at 55±5°C, and recrystallized at 0-5°C for 15-17 hours. The recrystallized material was filtered, washed three times with diethyl ether, and dried under vacuum at room temperature for 24 hours to obtain the compound of formula 3 (n=151) with a weight-average molecular weight of 40,000.

[0088] Preparation Example 2. Synthesis of 6-arm PEG-SH <Reaction Scheme 3> [ka]

[0089] 6-arm PEG-SH was synthesized according to Reaction Scheme 3.

[0090] The compound of formula 7 was dissolved in methylene chloride at room temperature and triethylamine was added to prepare a reaction solution. P-toluenesulfonyl chloride was added to the reaction solution and stirred at room temperature for 20-24 hours. The reaction solution was washed with 14% ammonium chloride solution, and after the aqueous layer was separated, the lower organic solution layer was collected. The aqueous layer was extracted with methylene chloride. The combined organic solution layers were dehydrated using magnesium sulfate, and the solvent was concentrated. The solution was then precipitated by adding it to diethyl ether. The precipitate was filtered and dried under vacuum at room temperature for 24 hours to obtain the compound of formula 8.

[0091] The compound of formula 8 was dissolved in water and then thiourea was added to prepare a reaction solution. The reaction solution was stirred at reflux temperature for 15-20 hours. Then, 1N NaOH solution was added and stirred at reflux temperature for 2 hours. The reaction solution was extracted twice with methylene chloride. The combined organic layers were dehydrated using magnesium sulfate, and the solvent was concentrated. The mixture was then precipitated in diethyl ether. The precipitate was filtered and dried at room temperature under vacuum for 24 hours to obtain the compound of formula 4 (n=151) with an average molecular weight of 40,000.

[0092] Examples 1 to 5 The 6-arm PEG-SG prepared in Preparation Example 1 and the 6-arm PEG-SH prepared in Preparation Example 2 were added to PBS buffer solution to prepare Solutions 1 and 2, respectively, and hydrated gels were prepared according to the method shown in Table 1 below depending on the pH of the buffer solution.

[0093] [Table 1]

[0094] Experimental Example 1: Measurement of gelation time depending on the pH of solution 1 1 ml of each of Solutions 1 and 2 from Examples 1 to 5 was placed in a glass test tube, vortexed for 10 seconds, and the time it took for the mixture to gel at room temperature was measured. The gelation time was measured by turning the glass test tube upside down and measuring the time it took for the mixture to stop flowing, settle to the bottom of the test tube, and solidify without moving. The test was repeated three times, and the average and standard deviation were plotted.

[0095] FIG. 1 shows the gelation time of hydrated gels as a function of pH.

[0096] 1, Examples 1 to 5 show a tendency for the gelation time to decrease depending on the pH of Solution 1, and it can be seen that, in particular, Examples 3 to 5 show that the gelation time is significantly shortened as the pH of Solution 1 increases. The gelation times of Examples 1 to 3 were measured to be approximately 15 minutes, 12 minutes for Example 4, and within 10 minutes for Example 5.

[0097] Examples 6 and 7 The 6-arm PEG-SG prepared in Preparation Example 1 and the 6-arm PEG-SH prepared in Preparation Example 2 were added to PBS buffer to prepare solutions 1 and 2, respectively, and hydrated gels were prepared according to the method shown in Table 2 below.

[0098] [Table 2]

[0099] Experimental Example 2. Measurement of gelation time depending on the concentration of 6-arm PEG derivative The gelation times of Solutions 1 and 2 of Examples 3, 6 and 7 were measured in the same manner as in Experimental Example 1.

[0100] FIG. 2 shows the gelation time of hydrated gels as a function of the concentration of the 6-arm PEG derivative.

[0101] As shown in FIG. 2, it can be seen that the gelation time is shortened as the concentration of the 6-arm PEG derivative increases.

[0102] Experimental Example 3. Physical Property Experiment - Rheological Test Viscosity refers to viscosity, and elasticity refers to the force that tends to return to its original state, i.e., the force that tends to return a spring to its original state when pulled. A material that possesses both of these physical properties is called viscoelasticity. When a force is applied to an object, a flow phenomenon that possesses both elasticity and viscosity occurs, and generally, polymer solutions possess both viscosity and elasticity (restoring force), i.e., viscoelasticity.

[0103] Hydrated gels were prepared using 6-arm PEG derivatives as described in the Examples, and their viscoelasticity was measured using a rheometer. The analysis was performed using a DHR1 (Discovery Hybrid Rheometer, TA Instruments Ltd., USA) at 37°C with a 40 mm plate, under conditions of 0.5 mm gap, 1% strain, and 2.5 Hz frequency oscillation.

[0104] 3 and 4 show the results of viscoelasticity measurements of the hydrated gel.

[0105] In Figure 3, G' is the storage modulus or elastic modulus, which indicates elasticity. The harder the sample is and the stronger its resistance to deformation, the higher the value. G" is a value indicating viscosity. Tan delta is G" / G', and the higher the value, the greater the viscosity is. Complex viscosity is a value that reflects both viscosity and elasticity, and indicates the degree of deformation due to an external force.

[0106] In Figure 3, (a) and (b) show the results of rheometer analysis of the hydrated gel prepared with the composition of Example 7, and (c) and (d) show the results of rheometer analysis of the control group, 1% hyaluronic acid (HA; high viscosity 3.3, 3,500,000-4,200,000 Da; Bioland).

[0107] As shown in Figure 3, the G' elasticity value of the control group (1% hyaluronic acid) was approximately 33-35 Pa, the G" viscosity value was 13-16 Pa, the Tan delta value was 0.4, and the complex viscosity was 2.3-2.5 Pa·s. In other words, 1% hyaluronic acid exhibited constant viscosity and elasticity that did not change over time.

[0108] On the other hand, in the case of the hydrated gel of Example 7, the G' elasticity value was 0.32 Pa and the G" viscosity value was 0.09 Pa (viscosity of water at 10°C, 0.001 Pa) in the first 6 seconds, showing low viscosity. However, as time passed, at 1500 seconds (25 minutes), G' exceeded 10,000 Pa, G" exceeded 1,000 Pa, the Tan delta value was 0.03, and the complex viscosity was 2,000 Pa·s, showing a significant increase in viscosity and elasticity.

[0109] FIG. 4 shows the results of a comparison of the complex viscosity of the hydrated gel of Example 7 and the control group, 1% HA.

[0110] As shown in FIG. 4, compared to the constant value of 1% HA, the hydrated gel of Example 7 exhibited lower viscoelasticity than the control group within about 300 seconds of the initial analysis, but after about 300 seconds, it was confirmed to exhibit higher viscoelasticity.

[0111] Experimental Example 4: Confirmation of joint pain relief effect Using a rat MIA arthritis model, which is commonly used as an arthritis model, the arthritis pain-reducing effects of the injection of Example 7 and the positive control group were confirmed. First, the hair around the knee of the right hind leg of each rat was removed, and then 50 μl (60 mg / ml) of the osteoarthritis inducer MIA (Monosodium iodoacetate, Sigma Chemical Co. Ltd., Cat No. I9148) was administered into the knee joint cavity using a Hamilton syringe (Corinne Guingamp et al., Mono-iodoacetate-induced experimental osteoarthritis, Arthritis & Rheumatism, 1997, 40(9), 1670-1679; Kai Gong et al., Journal of the Formosan Medical Association, 2011, 110(3), 145-152).

[0112] One week after the MIA injection, 50 μl of each of the injection preparation of Example 7 and 1% (10 mg / ml) sodium hyaluronate as a positive control was injected into the knee joint cavity.

[0113] The effect of reducing joint pain was evaluated by measuring the weight (g) of each left and right hind paw using an incapacitance tester (Stoelting Co., Wood Dale, IL) before osteoarthritis induction (week 0) and on weeks 1, 2, 3, and 4 after osteoarthritis induction, and calculating the change in weight of the right hind paw (Change in Hind Paw Weight Distribution; HPWD, %) using the following mathematical formula 1. Paw weight was measured three times per animal.

[0114] The weight change rate on the right hind leg is a percentage calculated from the additional weight placed on the left hind leg due to pain in the right knee caused by arthritis in the right leg, and a measurement value of approximately 50% can be said to be in a normal state without arthritis.

[0115] (Number 1) Right hind leg weight (%) = [right hind leg weight / (right hind leg weight + left hind leg weight)] x 100

[0116] The ratio of the weight of the right hind paw to the weight of both hind paws was calculated and expressed as mean (%)±standard error.

[0117] The measurement results are shown in Table 3 and FIG.

[0118] [Table 3]

[0119] In Table 3, each value is expressed as mean ± SD, and parameter comparisons were performed using one-way ANOVA or Student's t-test ((N=7), *P<0.05, **P<0.01: vs. vehicle control group (G2)).

[0120] As shown in Table 3 and Figure 5, the weight change rate of the right hind paw was maintained at a low level of 26-33% from week 1 to week 4 in the vehicle control group (G2, a group not treated with hydrated gel after arthritis induction), while the injection of Example 7 (G3) showed a significant drug effect, with the weight change rate of the right hind paw recovering to 38% at week 3 and 40% at week 4 after administration of the test substance. The weight rate of the right hind paw in the positive control group (G4) also increased to 34% at week 3 and 36% at week 4, demonstrating that the injection of the present invention had a similar or better pain-reducing effect than the positive control group. Industrial Applicability

[0121] In the present invention, two types of PEG derivatives are used as the main components of the hydrated gel. After dissolving each PEG derivative in a buffer solution and mixing the two solutions, the PEG hydrated gel can be converted from a sol state to a gel state within 30 minutes. The PEG hydrated gel can be easily injected into the body in a liquid (sol) state. After injection, the viscosity and elasticity of the composition increase, converting it to a gel state. Therefore, it can be used as a viscosupplement for the pain relief of osteoarthritis.

Claims

1. a first solution comprising a first polyethylene glycol derivative having an electrophilic reactive group; and a second solution comprising a second polyethylene glycol derivative having a nucleophilic reactive group; the first polyethylene glycol derivative comprises 6-arm polyethylene glycol-N-hydroxysuccinimide (6-arm PEG-NHS) and / or 8-arm polyethylene glycol-N-hydroxysuccinimide (8-arm PEG-NHS); the second polyethylene glycol derivative comprises a 6-arm polyethylene glycol-thiol (6-arm PEG-SH) and / or an 8-arm polyethylene glycol-thiol (8-arm PEG-SH); The first solution and the second solution form a hydrated gel through a sol-gel reaction when mixed together, The viscosity (loss modulus, G") of the mixture of the first and second solutions within 300 seconds of injection (infusion) is 1 Pa or less; The elasticity (storage modulus, G') of the mixture of the first and second solutions is 1,000 Pa or more after 2,000 seconds of injection; It is administered into the joint cavity and has an effect of suppressing pain induced by arthritis or protecting articular cartilage. Injectable.

2. The injection according to claim 1, wherein the 6-arm polyethylene glycol-N-hydroxysuccinimide and / or the 8-arm polyethylene glycol-N-hydroxysuccinimide is a compound represented by the following chemical formula 1: 【Chemistry 1】 【Chemistry 2】

3. The injection according to claim 1, wherein the 6-arm polyethylene glycol-N-hydroxysuccinimide is a compound represented by the following chemical formula 3: 【Transformation 3】 In Formula 3, n is an integer of 20 to 400.

4. The injection according to claim 1, wherein the 6-arm polyethylene glycol-thiol and / or the 8-arm polyethylene glycol-thiol is a compound represented by the following chemical formula 4: 【Chemistry 4】 【Transformation 5】

5. The injection according to claim 1, wherein the 6-arm polyethylene glycol-thiol is a compound represented by the following chemical formula 6: 【Transformation 6】 In Formula 6, n is an integer of 20 to 400.

6. the molecular weight of the first polyethylene glycol derivative in the first solution is 5,000 to 100,000 Da; 2. The injection according to claim 1, wherein the molecular weight of the second polyethylene glycol derivative in the second solution is 5,000 to 100,000 Da.

7. The solvents in the first solution and the second solution are each a buffer solution; the pH of the first solution is 4 to 9; 2. The injection according to claim 1, wherein the second solution has a pH of 4 to 9.

8. the concentration of the first polyethylene glycol derivative in the first solution is 1 to 10% (w / v); 2. The injection according to claim 1, wherein the concentration of the second polyethylene glycol derivative in the second solution is 1 to 10% (w / v).

9. mixing the first and second solutions immediately prior to injection; The injection according to claim 1 , wherein the mixture of the first solution and the second solution forms a hydrated gel after injection.

10. 2. The injection according to claim 1, wherein the weight ratio of the first polyethylene glycol derivative to the second polyethylene glycol derivative is 1:0.5 to 1:

2.

11. The injection according to claim 9, wherein the first polyethylene glycol derivative and the second polyethylene glycol derivative form a covalent bond when the first solution and the second solution are mixed.

12. a first solution set in which a first polyethylene glycol derivative powder having an electrophilic reactive group and a buffer solution having a pH of 4 to 9 are separately stored; and a second solution set in which a second polyethylene glycol derivative powder having a nucleophilic reactive group and a buffer solution having a pH of 4 to 9 are separately stored, the first polyethylene glycol derivative comprises 6-arm polyethylene glycol-N-hydroxysuccinimide (6-arm PEG-NHS) and / or 8-arm polyethylene glycol-N-hydroxysuccinimide (8-arm PEG-NHS); the second polyethylene glycol derivative comprises a 6-arm polyethylene glycol-thiol (6-arm PEG-SH) and / or an 8-arm polyethylene glycol-thiol (8-arm PEG-SH); The first solution and the second solution form a hydrated gel through a sol-gel reaction when mixed together, The viscosity (loss modulus, G") of the mixture of the first and second solutions within 300 seconds of injection (infusion) is 1 Pa or less; The elasticity (storage modulus, G') of the mixture of the first and second solutions is 1,000 Pa or more after 2,000 seconds of injection; It is administered into the joint cavity and has an effect of suppressing pain induced by arthritis or protecting articular cartilage. Injection kit.

13. Immediately before use, a first solution is prepared by dissolving the first polyethylene glycol derivative powder in the buffer solution of the first solution set; A second polyethylene glycol derivative powder is dissolved in a buffer solution of a second solution set to prepare a second solution, and then 13. The injection kit according to claim 12, wherein the first solution and the second solution are mixed.

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