WO2013036072A1

WO2013036072A1 - Injectable therapeutic agent for arthritis

Info

Publication number: WO2013036072A1
Application number: PCT/KR2012/007225
Authority: WO
Inventors: 조일환; 황의진; 서무석; 박영수; 김봉주; 김형준
Original assignee: 신풍제약 주식회사
Priority date: 2011-09-08
Filing date: 2012-09-07
Publication date: 2013-03-14
Also published as: TW201322988A; KR20130028012A

Abstract

The present invention relates to an injectable solution composition to be injected into a joint, for treating arthritis or ameliorating the symptoms of arthritis, including a hyaluronic acid hydrogel crosslinked with alkylene diamine, wherein the composition exhibits superior biocompatibility and long-lasting in-vivo sustainability, and thus exhibits the effects of long-lasting joint pain alleviation, cartilage protection, and synovial inflammation inhibition through a single injection, and therefore, can be effectively used as an injectable therapeutic agent for arthritis.

Description

Arthritis Injection Therapy

The present invention relates to an injection solution composition which can be used in arthritis injection therapy, which has long-term analgesic effect, cartilage protection effect and synovial inflammation inhibitory effect in a joint by a single injection, and a kit comprising the same.

Osteoarthritis (OA), which is caused by joint pain and degenerative arthrosis, is a common disease among older age groups, and is increasing with the aging trend of the world. The cause is not clear, but pain, swelling, and joint motion restriction occur as a result of secondary inflammatory reactions that occur in the supernatural bone, synovial membrane, and bone tissue. Until now, the purpose of treating osteoarthritis injection therapy is to maintain joint function and quality of life by reducing pain and delaying disease progression, rather than focusing on repairing structural damage of joints. As one of the treatments for subjects with osteoarthritis, hyaluronic acid-injection therapy is administered in which intra-articular injection of hyaluronic acid is performed.

Hyaluronic acid is a biopolymer material in which repeating units consisting of N-acetyl-D-glucosamine and D-glucuronic acid are linearly connected, and are present in many of the eye's vitreous fluids and joint synovial fluids. It acts as a shock absorber to relieve joint pain and improve joint function. For example, Synvisc ^® , Hyalgan ^® and the like are commercially available in the United States. However, these preparations are made of hyaluronic acid itself, and since hyaluronic acid has a short half-life of only a few hours after application in the body, three to five consecutive injections are performed to show efficacy for osteoarthritis for a certain period of time. It was inevitable. However, since the multiple injection method is cumbersome and inefficient for both the patient and the surgeon, a single injection with the same efficacy is required. In the United States and Europe, research on the development of a single injection with cross-linking hyaluronic acid to increase its half-life (persistence) has been conducted. Examples of synthesizing crosslinked hyaluronic derivatives using a compound having two functional groups as a crosslinking agent, such as divinyl sulfone, bisepoxide, bishalide, formaldehyde, etc., have been reported in several documents. Are marketed as Viscosupplement.

For example, the United States, and Patent No. 4,582,865 discloses discloses a hyaluronic derivative crosslinked using divinyl sulfone (DVS) as the crosslinking agent, the counter hydrogel composite form is sold under the trade name of Synvisc-one ^®, U.S. Patent No. 5,827,937 No. discloses a method for preparing a hyaluronic derivative crosslinked product using a multifunctional epoxy compound as a crosslinking agent, of which hyaluronic acid prepared using 1,4-butanediol diglycidyl ether (BDDE) as a crosslinking agent as a multifunctional epoxy compound. Durolane ^® , a hydrogel in the form of a lonic acid crosslink, is commercially available in many countries with European CE certification. All of these products are produced by combining a hydroxyl group of a hyaluronic acid with a crosslinking agent, and compared to uncrosslinked hyaluronic acid, its in vivo persistence is increased, but there is still a problem of low biopersistence, such as degradation within 6 months. Accordingly, the present applicant is aware that the recognition site of the hyaluronic acid degrading enzyme is a carboxyl group of hyaluronic acid, and thus the hyaluronic acid-ADH having high bio-durability using adipic acid dihydrazide (ADH) which binds to the carboxyl group of hyaluronic acid as a crosslinking agent Although a method for preparing a crosslinked product was developed and filed with Korean Patent Application No. 10-2008-0074260, there was a problem of low biocompatibility. On the other hand, recently PMA approved product GEL-ONE ^® in the United States FDA is expected by taking contrast to commercially available products of the above carboxyl group and the method (U.S. Patent No. 6,031,017) that a crosslinking agent is bonded to be superior in vivo persistence.

On the other hand, hyaluronic acid crosslinked products are manufactured by using a chemical that can be recognized as a foreign body in the body as a crosslinking agent.Therefore, since there is a problem of biocompatibility such that an immune reaction occurs due to the remaining crosslinking agent when it is decomposed after being administered to a living body, There is a need for crosslinks with better biocompatibility.

As described above, an injection solution composition which can be used for an arthritis injection therapy which shows long-lasting in vivo and high biocompatibility, and has long-lasting analgesic effect, cartilage protection effect and synovial inflammation inhibitory effect in a joint by a single injection, It is requested.

[Preceding technical literature]

(Patent Document 1) U.S. Patent No. 4,582,865 (published April 15, 1986)

(Patent Document 2) U.S. Patent No. 5,827,937 (published October 27, 1998)

(Patent Document 3) Korean Patent Application No. 10-2008-0074260 (published February 5, 2009)

(Patent Document 4) US Patent No. 6,031,017 (published February 29, 2000)

The present invention is to solve the problems of the prior art as described above, injection composition comprising a cross-linked hyaluronic acid hydrogel excellent in in vivo persistence and biocompatibility, so that it can be used as a one-time arthritis injection therapy and its It is an object to provide a manufacturing method.

In order to achieve the above object, the present invention provides an injection solution composition for intraarticular administration for the treatment or symptomatic improvement of arthritis diseases, comprising an alkylenediamine crosslinked hydrogel of hyaluronic acid of formula 1 as an active ingredient;

[Equation 1]

[HA] _m -C (O) -NH-R1-NH-C (O)-[HA] _n

Wherein HA represents hyaluronic acid or a salt thereof except one carboxyl group, and R 1 represents a C ₃ -C ₁₀ alkylene group unsubstituted or substituted with hydroxy, C ₁ -C ₆ alkyl, or C ₁ -C ₆ alkoxy M and n are independently integers of 10,000 to 4,000,000.

Preferably, the hyaluronic acid used in the composition of the present invention has a molecular weight of 20,000 Daltons to 4,000,000 Daltons.

Preferably, the alkylenediamine in the composition of the present invention is hexamethylenediamine.

Preferably, the crosslinking rate of the crosslinked hydrogel in the composition of the present invention is 5 to 35%.

Preferably, the crosslinked hydrogel in the composition of the present invention is included in 0.4 to 3.0 (w / w)% based on the total weight of the injection solution.

Preferably, the composition of the present invention has a long-lasting analgesic effect at the time of one injection for at least two weeks after administration of the injection solution.

Preferably the composition of the present invention further comprises unmodified hyaluronic acid.

When the composition of the present invention further comprises unmodified hyaluronic acid, preferably the alkylenediamine crosslinked hydrogel: unmodified hyaluronic acid weight ratio of hyaluronic acid is 5: 5 to 9.5: 0.5, more preferably 7: 3 to 9.5: 0.5.

Preferably the composition of the present invention exhibits an analgesic effect to reduce joint pain caused by arthritis disease, cartilage degeneration inhibitory effect caused by arthritis disease, synovial inflammation inhibitory effect caused by arthritis disease, treatment of arthritis disease or It is useful as symptomatic injection therapy.

Preferably, the composition of the present invention is usefully used as a single injection therapy for treating or improving symptoms of osteoarthritis.

The remainder of the composition consists of pharmaceutically suitable carriers such as water, saline, and the like.

In addition, the term "hyaluronic acid" is used herein to mean both hyaluronic acid itself as well as salts and derivatives thereof. Accordingly, the term "aqueous solution of hyaluronic acid" as used below is a concept including all of aqueous solutions of hyaluronic acid, aqueous solutions of hyaluronic acid salts, and mixtures thereof.

The preparation of the alkylenediamine crosslinked hydrogel of hyaluronic acid used in the injectable liquid composition according to the present invention will be specifically described below.

Alkylenediamine crosslinked hydrogel of hyaluronic acid used in the present invention is prepared by reacting hyaluronic acid with an alkylene diamine compound in the presence of a carboxyl group activator and a peptide bond promoter.

Hyaluronic acid (HA) having a molecular weight of 10,000 Daltons to 4,000,000 Daltons, more preferably 20,000 Daltons to 4,000,000, is used. In vivo degradation rate of the crosslinked hydrogel prepared by adjusting the molecular weight or concentration of HA can be controlled. Usually, the higher the initial concentration of HA, the higher the crosslinking rate of the crosslinked hydrogel to be produced, and thus lower degradation rate in vivo. Preferably HA is used at a concentration of 1.0 to 3.5 (w / w)%, more preferably 3.0 (w / w)%. If it is less than 1.0 (w / w)%, it is difficult to obtain a desired degree of biodegradation, and if it exceeds 3.5 (w / w)%, the prepared hydrogel is easily dried and broken.

The alkylenediamine compound is a C ₃ -C ₁₀ alkylenediamine compound unsubstituted or substituted with hydroxy, C ₁ -C ₆ alkyl, or C ₁ -C ₆ alkoxy, preferably C ₃ -C ₇ alkylenediamine , More preferably C ₄ -C ₆ alkylenediamine, most preferably C ₆ alkylenediamine (hexamethylenediamine), 3.5 to 80 mol%, preferably 10 to 30 mol of hyaluronic acid repeat units %, Most preferably 10 to 25 mol%.

As a carboxyl activator, 1-alkyl-3- (3-dimethylaminopropyl) such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) ) Carbodiimides; 1-alkyl-3- (3- (such as 1-ethyl-3- (3- (trimethylammonio) propyl) carbodiimide (ETC, 1-ethyl-3- (3- (trimethylammonio) propyl) carbodiimide) Trimethylammonio) propyl) carbodiimides; 1-cycloalkyl-3- (2-morpholinoethyl) such as 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (CMC, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide) Carbodiimide of water-soluble carbodiimide, such as carbodiimide, is used, More preferably, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is used, and per mole of hyaluronic acid It is preferable to use it 1 to 5 times by molar ratio.

Examples of the peptide bond promoters include 1-hydroxybenzotriazole (HOBt) and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). , 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine), 1-hydroxy-7-azabenzotriazole (HOAt, 1-hydroxy-7-azabenzotriazole), sulfo-N -Hydroxysulfosuccinimide (Sulfo-NHS, Sulfo-N-hydroxysulfosuccinimide), O- (1H-benzotriazole-1-y) -N, N, N ', N'-tetramethyluronium tetrafluoro Borate (TBTU, O- (1H-benzotriazole-1-yl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate) is used, preferably 1-hydroxybenzotriazole (HOBt), hyaluronic acid It is preferable to use 1 to 5 times in molar ratio per 1 mol of lonic acid.

1 shows a hyaluronic acid-hexamethylenediamine crosslinked product prepared using hexamethylenediamine (HMDA) as the alkylene diamine compound. As can be seen in Figure 1, in the presence of a carboxyl group activator and a peptide bond promoter, it can be seen that the carboxyl group of hyaluronic acid and the amine group of the alkylene diamine compound are crosslinked through an amide bond. Crosslinking may occur between molecules and may occur within molecules. The carboxyl group of hyaluronic acid is an important group involved in the recognition of hyaluronic acid by hyaluronidase, a hyaluronic acid degrading enzyme, and the alkylenediamine crosslinked hydrogel of hyaluronic acid of the present invention is used by the carboxyl group of hyaluronic acid for crosslinking. When introduced into a living body, the degradation of hyaluronic acid by a degrading enzyme is minimized, thereby increasing the biological sustainability.

The crosslinking reaction is usually carried out in water. The hyaluronic acid, the alkylenediamine compound, the carboxyl activator and the peptide bond promoter may be dissolved in water and mixed, respectively.The solution in which the hyaluronic acid and the alkylenediamine compound is dissolved is mixed with the solution of the carboxyl group activator and the peptide bond promoter. Also good. In particular, in the case of EDC used as a carboxyl group activator, the transformation may occur at weak acidity into N -ethyl- N- (3-dimethylaminopropyl) urea (EDU), so that the EDC is dissolved in water and crosslinked with hyaluronic acid and alkylene diamine mixture. As the time required for the addition to a long time, the physical properties of the prepared crosslinked hydrogel may be deteriorated, such as the elastic modulus of the composition is lowered after sterilization. Preferably the time required from dissolution to input is within 10 to 30 minutes, and most preferably within 10 to 20 minutes. Moreover, reaction temperature is maintained at 30-50 degreeC, Preferably it is 43-45 degreeC. The hydrogelation reaction proceeds efficiently at this temperature, and usually hydrogel is produced within 30 minutes. It is also possible to prepare a hydrogel having good viscoelasticity by standing at this temperature for at least 9 hours. At this time, it is preferable to leave it without stirring. As the additional reaction time increases, physical properties such as viscoelasticity are increased, and there is almost no change in physical properties such as viscoelasticity of the prepared hydrogel at 9 hours or more. In addition, the crosslinking rate of the prepared crosslinked hydrogel varies according to pH at the time of manufacture, and physical properties may be changed. The pH can be adjusted by adding NaOH aqueous solution to the reaction solution, preferably 5.5 to 6.5, more preferably 6.0 to 6.3. In particular, by adjusting the pH to 5.5 to 6.5, even a relatively small amount of crosslinking agent (e.g., hexamethylenediamine) and reaction aid (e.g., carboxyl group activator such as EDC and peptide bond promoter such as HOBt) It is possible to prepare an alkylenediamine crosslinked hydrogel of hyaluronic acid having physical properties and no change in physicochemical properties such as viscoelasticity even after sterilization. Hydrogels prepared in an acidic environment with a pH of less than 5.5 exhibit a relatively low modulus of elasticity (G ′) after preparation, and may further degrade elasticity during sterilization. The prepared hydrogel is passed through a sieve or homogenizer is used to form a uniform particle. In particular, it is preferable to homogenize using a sieve, and a hydrogel having a narrow particle distribution and a low protrusion pressure can be obtained. After the end of the crosslinking reaction, unreacted materials may be removed by dialysis in PBS using a dialysis membrane for 24 hours to 3 days, or below a detection limit (2 ppm) using an aqueous ethanol solution and / or buffer. However, accelerators such as HOBt tend to be more soluble in organic solvents, and thus are not easily purified by dialysis using dialysis membranes and PBS, but can be easily removed by using ethanol aqueous solutions and / or buffers. Can be. At this time, the buffer is preferably a PBS solution or sodium chloride (NaCl) solution, more preferably using a sodium chloride (NaCl) solution. The higher the concentration of NaCl used, the easier the purification, preferably 0.5 to 1.5%, most preferably 1 to 1.3%. In addition, hydrogel particles are produced by precipitation when an aqueous ethanol solution, such as an 80% aqueous ethanol solution, is added, and unreacted substances are easily removed in the process. The precipitated hydrogel may be dried and powdered with nitrogen gas, and the powdered hydrogel may be rehydrated with a physiologically suitable aqueous solution. In this case, PBS or saline is preferable as a physiologically suitable aqueous solution, and may be used by rehydrating a physiologically suitable aqueous solution at a ratio of 20 to 30 (volume ratio) with respect to the hyaluronic acid-alkylenediamine crosslinked hydrogel powder. have.

The hyaluronic acid-alkylenediamine crosslinked hydrogel of the present invention has a crosslinking rate of 5 to 35%, and preferably has a crosslinking rate of 10 to 20%. If the crosslinking rate is less than 5%, the ratio of unreacted COOH groups not involved in crosslinking in hyaluronic acid is increased, so that it is easily decomposed by hyaluronidase, a degrading enzyme, and thus it is difficult to obtain a desired degree of biopersistence. . If the crosslinking rate exceeds 35%, it is difficult to show the degree of viscoelasticity useful for hydrogel swelling and joint protection, and the biocompatibility is low such that the residual crosslinking agent content increases after biodegradation, which may cause an inflammatory reaction. . In addition, when the crosslinking rate of the hydrogel is low, physical properties such as viscoelasticity may be degraded after sterilization. The crosslinking rate of the hyaluronic acid-alkylenediamine crosslinked product of the present invention means the percentage of hyaluronic acid content participating in the crosslinking with respect to the total hyaluronic acid content, and controls the ratio of the alkylenediamine used as the crosslinking agent to the total hyaluronic acid. Or, the crosslinking rate can be adjusted by adjusting the pH at the time of manufacture. Determination of the crosslinking rate is known in the art, such as NMR analysis and TNBS assay (Habeeb, AFSA, Determination of free amino groups in proteins by trinitrobenzenesulfonic acid.Anal. Biochem., 1966. 14: pp. 328-33 Can be easily implemented. The hydrogel of the hyaluronic acid-alkylenediamine crosslinked product of the present invention has a significantly lower decomposition rate than the HA-DVS crosslinked hydrogel using divinyl sulfone (DVS) as a crosslinking agent, and adipic acid dihydrazide ( Compared with the HA-ADH crosslinked hydrogel using ADH) as a crosslinking agent, physical properties such as good swelling property are exhibited.

The composition of the present invention preferably comprises the alkylenediamine crosslinked hydrogel of hyaluronic acid in an amount of 0.4 to 3.0 (w / w)%, more preferably 1.0 to 2.0 (w / w)%, based on the total weight of the composition. It includes. If the hydrogel contains less than 0.4 (w / w)%, the composition prepared does not have the desired in vivo persistence and protrusion pressure, and if it contains more than 3.0 (w / w)%, the composition will protrude The pressure may be too high, making injection into the tissue difficult.

Meanwhile, the composition of the present invention may further include unmodified (ie, uncrosslinked) hyaluronic acid in addition to the hyaluronic acid-alkylenediamine crosslinked hydrogel. Unmodified HA typically uses the same hyaluronic acid used to prepare the hyaluronic acid-alkylenediamine crosslinked hydrogel. Unmodified HA acts as a lubricant to lower the pressure over the composition, thereby filling the syringe and injecting the composition into the tissue at a low pressure when injected into the tissue. When the composition of the present invention further comprises unmodified hyaluronic acid, preferably the alkylenediamine crosslinked hydrogel: unmodified hyaluronic acid weight ratio of hyaluronic acid is 5: 5 to 9.5: 0.5, more preferably 7: 3 to 9.5: 0.5. In addition, preferably the unmodified hyaluronic acid is included in 0.1 to 2.0 (w / w)% with respect to the total weight of the composition, more preferably included in 0.15 to 1.0 (w / w)%. When included in less than 0.1 (w / w)%, the extrusion pressure does not decrease to a desired degree, and when included in excess of 2.0 (w / w)%, the physical property is deteriorated due to deterioration of physical properties such as excessively low elastic modulus after sterilization. Persistence can be reduced.

Joint including the alkylenediamine crosslinked hydrogel of hyaluronic acid of the present invention, more preferably the alkylenediamine crosslinked hydrogel of hyaluronic acid and unmodified hyaluronic acid in a weight ratio of 5: 5 to 9.5: 0.5 Injectable injectable compositions are useful as single injection therapy for excellent joint treatment or symptom improvement because they have excellent physical properties necessary for biocompatibility and joint surface protection and joint surface protection and shock absorption necessary for biocompatibility and joint protection and can be sustained in vivo. Can be used.

1 is a schematic diagram of the synthesis process of the HA-HMDA crosslinked product of the present invention and the synthesized crosslinked structure.

Figure 2 shows the rat joint pain relief effect of the composition administration group of Examples 1 to 4 of the present invention compared to the negative control group.

Figure 3 shows a comparison of the rat joint pain alleviating effect of Example 1 composition administration group of the present invention, the conventional comparative formulation administration group and the negative control group.

Figure 4 shows a comparison of the area of rat arthroscopic cartilage damage between the administration group of the embodiments 1, 3 and 4 of the present invention and the negative control group.

Figure 5 shows a comparison of the area of severe cartilage damage in the rat joint of the control group and the negative control group of Examples 1, 3 and 4 of the present invention.

Figure 6 shows a comparison of the degree of severe cartilage damage (%) of the rat joint of the composition administration group of the present invention, the conventional comparative formulation administration group and the negative control group.

Figure 7 shows a comparison of the rabbit knee femoral injuries of the composition administration group of the present invention, the conventional comparison formulation administration group and the negative control group.

FIG. 8 illustrates the comparison of rabbit knee tibial damage between the composition-administered group of the present invention, the conventional control group, and the negative control group.

Figure 9 shows the effect of the composition comprising the HA-HMDA cross-linked hydrogel of the present invention in comparison with the normal group, staining the tissue samples of the back portion of each group of mice using an optical microscope after staining with H & E The rods indicated were 10 µm ((A): normal group (B): HA-HMDA hydrogel administration group of Preparation Example 2, E: epidermis, D: dermis, H: subcutaneous).

Figure 10 shows the degradation effect of hyaluronidase enzyme of the composition of Example 2 of the present invention and the control product in association with the elastic modulus (G ') (○: Example 2, ●: control).

11 is a result of observing damage and cartilage thickness and chondrocyte reduction in the femur of the composition and the control agent of Example 2 of the present invention, and the indicated bars represent 220 μm ((A): normal control (B): Osteoarthritis control group (C): Comparative control group (D): Example 2 administration group). The dashed line indicates the area for preparation for histological examination, and the arrow indicates the thickness of the cartilage.

Figure 12 shows a comparison of the total Mankin score in the femur and tibia cartilage of the composition and the control agent of Example 2 of the present invention.

제조예 1: 본 발명의 히알루론산-헥사메틸렌디아민(HA-HMDA) 가교물 하이드로겔의 제조Preparation Example 1 Preparation of Hyaluronic Acid-Hexamethylenediamine (HA-HMDA) Crosslinked Hydrogel of the Present Invention

Hyaluronic acid (HA, manufacturer: Lifecore Co.) having a molecular weight of about 230 kDa was completely dissolved in distilled water at a concentration of 1 (w / w)%, and then hexamethylenediamine (HMDA) was added to crosslink by reaction with a carboxyl group. . HMDA was added at 72 mol% of the repeat units of HA. The carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) were dissolved in distilled water in an amount of 1.4 times the HA repeating unit, and the HA was And HMDA were added to the mixture. The mixed solution was reacted at 37 ° C. for 1 hour for complete crosslinking of the HA-HMDA crosslinked product. The pH of the solution was 5.0-5.5. Thereafter, the prepared HA-HMDA hydrogel was sealed with a pre-washed dialysis membrane (molecular weight cut-off of 7 kDa) and dialyzed in 0.01 M PBS (phosphate buffered saline, pH 7.4) for 24 hours to maintain residual EDC, HOBt and HMDA. Was removed. The degree of crosslinking of the prepared HA-HMDA hydrogel was 8-9%.

제조예 2: 본 발명의 히알루론산-헥사메틸렌디아민(HA-HMDA) 가교물 하이드로겔의 제조Preparation Example 2 Preparation of Hyaluronic Acid-hexamethylenediamine (HA-HMDA) Crosslinked Hydrogel of the Present Invention

Hyaluronic acid (HA, manufacturer: Lifecore Co.) having a molecular weight of about 1,000 kDa was completely dissolved in distilled water at a concentration of 1 (w / w)%, and then hexamethylenediamine (HMDA) was added to crosslink by reaction with a carboxyl group. . HMDA was added at 72 mol% of the repeat units of HA. The carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) were dissolved in distilled water in an amount of 1.4 times the HA repeating unit, and the HA was And HMDA were added to the mixture. The mixed solution was reacted at 37 ° C. for 1 hour for complete crosslinking of the HA-HMDA crosslinked product. The pH of the solution was 5.5-5.9. Thereafter, the prepared HA-HMDA hydrogel was sealed with a pre-washed dialysis membrane (molecular weight cut-off of 7 kDa) and dialyzed in 0.01 M PBS (phosphate buffered saline, pH 7.4) for 24 hours to maintain residual EDC, HOBt and HMDA. Was removed. The degree of crosslinking of the prepared HA-HMDA hydrogel was 11-13%.

제조예 3: 본 발명의 히알루론산-헥사메틸렌디아민 (HA-HMDA) 가교물 하이드로겔의 제조Preparation Example 3 Preparation of Hyaluronic Acid-hexamethylenediamine (HA-HMDA) Crosslinked Hydrogel of the Present Invention

Hyaluronic acid (HA, manufacturer: Lifecore Co.) having a molecular weight of 1,000 kDa was completely dissolved in water at a concentration of 3 (w / w)%, and then hexamethylenediamine (HMDA) was added to crosslink by reaction with a carboxyl group. HMDA was added at 20 mol% of the repeating units of HA. The pH was adjusted to 6.0-6.5 by addition of 0.25 N NaOH aqueous solution. Carboxyl activator 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) were added to distilled water in an amount of 1.0 times the HA repeating unit for 20 minutes. After stirring, the mixture was added to the mixed solution of HA and HMDA aqueous solution. Stir at 45 ° C. for 30 minutes. The mixed solution was allowed to stand at 45 ° C. for 10 hours without agitation so that the HA-HMDA crosslinked material was completely crosslinked. The prepared HA-HMDA hydrogel was first ground and then passed through a 200 um pore size sieve to obtain a homogeneous hydrogel. 80% ethanol was added to obtain hydrogel powder as a precipitate, 1.3% NaCl solution was added to 100-fold volume and stirred for 1 hour. 80% ethanol was added again to obtain a precipitate, and the obtained hydrogel precipitate was put in 100% ethanol for 10 minutes, and then dried under reduced pressure at 40 ° C. for 12 hours to remove residual EDC, HOBt, and HMDA. As a result of LC analysis, it was confirmed that the detection limit was controlled to 2 ppm or less. The degree of crosslinking of the prepared HA-HMDA hydrogel was 10-14%.

실시예 1 내지 4: 본 발명의 히알루론산-헥사메틸렌디아민(HA-HMDA) 가교물 하이드로겔 함유 조성물Examples 1 to 4: Hyaluronic acid-hexamethylenediamine (HA-HMDA) crosslinked hydrogel containing composition of the present invention

Hyaluronic acid-alkylenediamine hydrogel prepared by the method of Preparation Example 3, unmodified hyaluronic acid and physiological saline were mixed in the ratio of the following Table 1, and then sterilized for 15 minutes at 121 ℃ intra-articular injection solution Was prepared.

Table 1

제조비교예 1: 아디프산 디하이드라자이드(ADH)를 가교제로 사용한 히알루론산-아디프산 디하이드라자이드(HA-ADH) 가교물 하이드로겔의 제조Preparation Example 1 Preparation of Hyaluronic Acid-Adipic Acid Dihydrazide (HA-ADH) Crosslinked Hydrogel Using Adipic Acid Dihydrazide (ADH) as Crosslinking Agent

100 mg of hyaluronic acid (HA, Lifecore Co.) having a molecular weight of about 234 kD according to the method of Hahn et al. (Hahn SK et al., Int. J. Biol. Macromol., 2007; 40; pp. 374-380). Was dissolved in 20 ml of water to prepare a 5 mg / ml aqueous solution of HA. A 40-fold excess of adipic acid dihydrazide (ADH) powder (1.736 g) was mixed in the aqueous HA solution in a molar ratio of HA and completely dissolved for 10 minutes. The pH of the obtained HA / ADH mixed aqueous solution was adjusted to 4.8 using 1N hydrochloric acid aqueous solution, and then stirred thoroughly for 30 minutes. To this was added a 4-fold excess of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC, 0.191 g) powder in a molar ratio with good stirring to activate the carboxyl group of HA. 1N HCl aqueous solution was added to the aqueous solution and reacted for 2 hours while maintaining the pH at 4.8. After two hours, the reaction was stopped by adding 1N aqueous sodium hydroxide solution to raise the pH to 7.0. In order to reduce the viscosity and impurities (unbound ADH) of the obtained product, it was placed in a pre-washed dialysis tube (molecular weight cut-off of 7 kDa) and dialyzed in 100 mM NaCl aqueous solution for 60 hours. Thereafter, dialysis was repeated in 25% ethanol and distilled water, respectively. Aqueous dialysis solution was lyophilized for 3 days to obtain a HA-ADH derivative. HA-ADH obtained by the above method was dissolved in 0.01 M PBS (pH 7.4) for 2 hours. Bis [sulfosuccinimidyl] suberate (BS3), a crosslinking agent specific for hydrazide, was also dissolved in PBS and added to HA-ADH solution. At this time, the amount of BS3 added was 20 mol% of HA-ADH hydrazide. After thoroughly mixing the aqueous solution, the reaction was carried out at 37 ° C. for 1 hour to complete the crosslinking reaction to prepare a HA-ADH crosslinked hydrogel.

제조비교예 2: 디비닐설폰(DVS)을 가교제로 사용한 히알루론산-디비닐설폰(HA-DVS) 가교물 하이드로겔의 제조Preparation Example 2 Preparation of Hyaluronic Acid-DivinylSulfone (HA-DVS) Crosslinked Hydrogel Using DivinylSulfone (DVS) as a Crosslinking Agent

68 mg of hyaluronic acid (HA, Lifecore Co.) having a molecular weight of about 230 kDa according to the method of Oh et al. (Oh EJ et al., J. Biomed. Mater. Res A., 2008; 86; pp. 685-693) Was dissolved in 1.68 mL of 0.2 N aqueous sodium hydroxide (NaOH) solution (= pH 13). After complete dissolution, divinylsulfone (DVS) was added to crosslink by reaction with the hydroxyl groups of HA. At this time, the molar ratio of the hydroxyl group of hyaluronic acid and the added DVS was 1: 1. The solution was reacted at 37 ° C. for one hour to prepare HA-DVS hydrogel. The prepared hydrogel was then sealed with a pre-washed dialysis membrane (molecular weight cut-off of 7 kDa) and dialyzed in PBS for 24 hours to diffuse and release the ions (Na ⁺ and OH ⁻ ) through the dialysis membrane and sealed HA The pH of the -DVS crosslink was neutralized.

비교예:Comparative example:

As a control of the injectable composition of the present invention, a commercially available "hyal-forte state" (Shin Poong Pharm., 1% sodium hyaluronate) was used as a triple injection therapy for treating arthritis.

실험예 1: 히알루론산 가교물 하이드로겔의 생체외 분해 및 팽윤성 확인시험Experimental Example 1: In vitro degradation and swelling test of hyaluronic acid crosslinked hydrogel

In order to predict the in vivo persistence of the hyaluronic acid crosslinked hydrogel used in the present invention, the hyaluronic acid degrading enzyme was tested for the hyaluronic acid crosslinked hydrogels of Preparation Example 1 and Comparative Examples 1 and 2. .

The hyaluronic acid crosslinked hydrogels of Preparation Example 1 and Comparative Examples 1 and 2 were packed in the same mass in each vial. 0.2 M PBS (= pH 6.2) with 50 U of hyaluronic acid degrading enzyme (hyaluronidase from Streptomyces hyalurolyticus , Sigma-Aldrich) was added. The mixture was reacted at 37 ° C. for a predetermined 48 hours. Thereafter, the supernatant was completely removed and the mass of the remaining hyaluronic acid crosslinked material was measured. The degree of decomposition of the crosslinked product was calculated as the mass ratio (%) of the remaining crosslinked product and the original crosslinked product, and the decomposition rate (%) was shown in the following table with time. As can be seen in Table 2, the HA-DVS crosslinked product of Comparative Example 2 was completely decomposed within about 25 hours, while the hyaluronic acid-alkylenediamine crosslinked hydrogel according to the present invention was partially removed even after 40 hours. Only decomposition took place. From this, it can be seen that the hyaluronic acid-alkylenediamine crosslinked hydrogel of the present invention has excellent biopersistence superior to commercial hydrogels.

In addition, it can be seen that the hyaluronic acid-alkylenediamine crosslinked hydrogel according to the present invention exhibits a swelling ratio of 2 times higher than that of the HA-ADH crosslinked product of Comparative Example 1, which shows a similar decomposition rate. From this, it can be seen that the hyaluronic acid-alkylenediamine crosslinked hydrogel according to the present invention exhibits physical properties such as high biopersistence and excellent swelling property.

TABLE 2

실험예 2: 본 발명의 히알루론산 가교물 하이드로겔을 포함하는 조성물의 물성 확인Experimental Example 2: Checking the physical properties of the composition comprising a hyaluronic acid crosslinked hydrogel of the present invention

In order to predict the injection force according to the syringe and use needle of the composition comprising the hyaluronic acid crosslinked hydrogel of the present invention, this experiment was performed.

Protruding pressure was measured using the Universal Tester (EZ-S-500N, SHIMADZU Corp, Japan) for the compositions of Examples 1-4 and Comparative Example 1 of the present invention. After the sample was filled in a 3 ml glass syringe, the pressure was measured at a rate of 1 mm / min by attaching an 18, 20, or 22 gauge needle which is commonly used for injection of arthritis injection therapy. The results are shown in Table 3 below.

TABLE 3

As can be seen from the above results, it was confirmed that the injectable composition Example 1 consisting of a single hyaluronic acid-alkyldiamine crosslinked hydrogel showed a significantly higher protrusion pressure than that of Comparative Example 1. This is believed to be due to the high viscoelasticity of the hydrogels used in the composition. On the other hand, Examples 2 to 4 with the mixture of unmodified hyaluronic acid was significantly lower than the pressure of the composition of Example 1, in particular, the higher the proportion of unmodified hyaluronic acid, the lower the pressure of the protrusion, thereby unmodified hyaluronic acid By adjusting the mixing ratio of the lon acid, it was confirmed that the physical properties of the composition can be adjusted to have an appropriate protrusion pressure in the syringe and the needle used in consideration of the operator's convenience and patient safety.

실험예 3: 본 발명의 히알루론산 가교물 하이드로겔을 포함하는 조성물의 물성 확인Experimental Example 3: Checking the physical properties of the composition comprising a hyaluronic acid crosslinked hydrogel of the present invention

Rheological properties are one of the important factors indicative of the properties of crosslinked hydrogels. The modulus of elasticity of the composition of Examples 1-4 was measured using the method of Ghosh et al (Biomacromolecules 2005; 6: pp. 2857-2865) with an AR 2000 controlled stress rheometer (TA Instruments Ltd., USA) instrument and 4-cm, The 2 ° -cone and plate geometry was used to measure 0.1-20 Hz in 1% strain and oscillation mode, and the elastic modulus (G ') values measured at 3 Hz are shown in Table 4 below.

Table 4

As can be seen from the above results, as the mixing ratio of the unmodified hyaluronic acid increases, the elastic modulus tended to decrease. The lower the modulus of elasticity, the lower the protrusion resistance and increase the lubrication effect during the injection into the joint. However, since the in vivo persistence is shortened, the mixing ratio of the unmodified hyaluronic acid should be determined in consideration of complex factors.

According to the results of Experimental Examples 2 and 3, in Example 1, the elastic modulus related to the in vivo persistence was the highest, but the unexposed hyaluronic acid was not added, so the actual protrusion pressure was considerably high. Although the pressure is low, the elastic modulus may be low, and thus the persistence in vivo may be lowered. In Example 2, an appropriate elastic modulus and an appropriate protrusion pressure are shown.

실험예 4: 본 발명의 조성물의 관절 통증 경감효과의 확인Experimental Example 4: Confirmation of the joint pain alleviating effect of the composition of the present invention

The rat MIA arthritis induction model commonly used as the arthritis model was used to confirm the arthritis pain relief efficacy of the compositions of Examples 1-4. After depilation of the rat's right foot knee, osteoarthritis was induced by administering 25 μl of osteoarthritis-inducing substance, MIA (monosodium iodoacetate) into the joint cavity of the right foot knee using a Hamilton syringe (Bove SE et al., Osteoarthritis and cartilage. 11 : 821-830; Combe R et al., Neurosci. Lett. 370 (2-3): 236-240). Seven days after MIA injection, 35 μl of the compositions of Examples 1-4 were injected into the knee joint cavity.

Joint pain reduction effect was measured by the incapacitance tester (Incapacitance tester, Linton instrument Co., UK, Model No. 600R) on the 1st, 7th, 14th and 21st day after osteoarthritis induction (g) ) Is calculated and evaluated by calculating the Change in Hind Paw Weight Distribution (HPWD,%) on the left foot.

% Change in weight on left foot = [weight on left foot / (weight on left foot + weight on right foot)] × 100

The measurement is calculated as the ratio of the left foot to the weight of both feet and expressed as mean (%) ± standard error.

The change in weight on the left foot is calculated by calculating the percentage of the load on the left hind foot due to the pain in the right knee caused by arthritis in the right leg. The 50% measurement is a normal state without arthritis. .

As a result, the weight change rate on the left foot is maintained at 75% or more from 14 days to 21 days in the control group, whereas the groups of the Examples 1, 2, 3, and 4 compositions of the present invention are loaded on the left foot at 14 days. The weight change rate decreased by 14.0%, 13.8%, 10.8%, and 10.8%, respectively, compared to the negative control group, and significant drug effects were observed in all test groups. In addition, on the 21st day of administration, the weight ratio of the left foot of the administration groups of Examples 1, 2, and 4 was reduced to 17.0%, 15.7%, and 11.5%, respectively, compared to the control group, and significant drug effects were confirmed (Table 5 And FIG. 2).

Table 5

(Each value is expressed as mean ± S.D (N = 5). * P <0.05 for negative control, ** P <0.01 for negative control)

실험예 5: 본 발명의 조성물의 관절 통증 경감효과의 확인Experimental Example 5: Confirmation of the joint pain alleviating effect of the composition of the present invention

The osteoarthritis pain relief effect of the composition of Example 1 of the present invention and the control agent of the comparative example was compared using a rat MIA arthritis induction model. All test methods proceed in the same manner as in Experimental Example 4, except that the composition of Example 1 of the present invention is administered only once after 7 days of arthritis induction, and the comparative formulation is 3 over 7 days, 17 days, and 27 days after arthritis induction After administration in turn, over 1 week to 7 weeks after the administration, the weight change rate on the left foot was calculated and shown in Table 6 below.

Table 6

The change in weight on the left foot before the induction of osteoarthritis ranged from 49% to 50% in all groups. The weight change on the left foot increased in all groups 7 days after MIA-induced osteoarthritis. The composition of Example 1 of the present invention showed a change in weight on the lower left foot compared to the negative control group from 7 days to 21 days after administration, and it was confirmed that it showed a significant drug effect. 35 days after the administration, the drug effect was significantly (P <0.01) in both Example 2 and Comparative Example 1 administration group. Significant (P <0.05) drug effects were observed in both the Example 1 composition and the Comparative Example administration group 49 days after administration (Table 6, FIG. 3). As can be seen from the above results, the composition of the present invention showed the effect of alleviating pain for a long period of 7 weeks with only one administration, and showed much better pain alleviating effect than the comparative formulation administered three times in the same period.

실험예Experimental Example 6: 본 발명의 조성물의 관절 내 연골 보호 효과 확인 6: Confirmation of cartilage protection effect in the joints of the composition of the present invention

The rat MIA arthritis induction model commonly used as an arthritis model was used to confirm the arthritis pain relief efficacy of the Examples 1, 3 and 4 compositions. By the method described in Experiment 3, arthritis was induced in rats. 21 days after arthritis induction, the whole blood was collected through the abdominal aorta, and then the right knee was incised to separate the femur and tibia, and the cartilage damage area of the separated tibia was photographed under a microscope (VHX-600, 20x magnification). Measured with an image analyzer. The whole cartilage area (WA) is the whole area of the knee joint where the cartilage is distributed in the tibia, and the white rough area (WRA) is the area where the cartilage is damaged to lose shine and the roughness of the cartilage damage is severe. The hollow area (HA) is the area of severe cartilage damage in which the cartilage is damaged and the spongy bone at the base of the cartilage is exposed.

1) Results of cartilage damage area

Significant decrease (P <0.05) of mild cartilage damage area and severe cartilage damage area of 38.5% and 67.7%, respectively, was observed in the composition-treated group of Example 1 of the present invention (Table 7, FIG. 4). On the other hand, a decrease in the area of severe cartilage damage was observed in the Examples 3 and 4 composition administration group.

2) mild cartilage damage

Mild cartilage damage indicates the ratio of mild cartilage area (WRA) to total cartilage area (WA). That is, the degree of mild cartilage damage (%) = WRA / WA × 100. Mild cartilage damage in Examples 1, 3 and 4 administration group of the present invention was reduced to 40.3%, 15.8%, 12.0% than the negative control group (Table 8, Figure 4).

3) severe cartilage damage

Severe cartilage damage indicates the ratio of severe cartilage area (HA) to total cartilage area (Whole area, WA). That is, the degree of severe cartilage damage (%) = HA / WA × 100. Severe cartilage damage was reduced by 39.1%, 26.5% and 35.2% in the negative control group, respectively, in Examples 1, 3 and 4 of the present invention (Table 9, Figure 5).

TABLE 7

(Each value is expressed as mean ± S.D (N = 5). * P <0.05 for negative control)

Table 8

Table 9

실험예 7: 본 발명의 조성물의 랫드 관절 내 연골 보호 효과 확인Experimental Example 7: Confirmation of cartilage protection effect in the rat joint of the composition of the present invention

The cartilage protection effect of the composition of Example 1 of the present invention and the control agent of the comparative example was compared using a rat MIA arthritis induction model. All test methods proceed in the same manner as in Experiment 5, except that the composition of Example 1 of the present invention is administered only once after 7 days of inducing arthritis, and the control agent of Comparative Example is 7 days, 17 days and 27 days after arthritis induction. Administration was performed three times and the degree of severe cartilage damage was calculated and shown in FIG. 6.

As can be seen in Figure 6, the effect of reducing the area of severe cartilage damage in both the composition administration group and the comparative formulation administration group of Example 1 of the present invention. On the other hand, the administration group of the composition of Example 1 of the present invention, which is a one-time therapy, showed a significant severe cartilage damage area reduction effect of 24% and 19%, respectively, compared to the negative-control group, which was administered as a three-time therapy, and the present invention was carried out. Example 1 The administration group of the composition showed a better cartilage protection effect than the administration of Comparative Example 1 by one injection.

실험예 8: 본 발명 조성물의 토끼 관절 내 연골 보호 효과 확인Experimental Example 8: Confirmation of cartilage protection effect in the rabbit joint of the composition of the present invention

Using osteoarthritis-induced model through rabbit meniscal chondrectomy, the osteoarthritis cartilage protection effect of the composition of Example 1 of the present invention and the control agent of the comparative example was compared.

After dissecting one leg of the rabbit aseptically, the medial cartilaginous plate was excised from the anterior to the medial area, and then injected with a certain amount of painless injection and spontaneously exercised on a treadmill to induce arthritis. Five weeks after the operation, the composition of Example 1 of the present invention and the Comparative Example were administered 0.3 ml each in the knee joint cavity, and the second and third administrations of the Comparative Example were performed at 7 and 9 weeks. Finally, at the 7th week of induction, cartilage damage areas in the femur and tibia were measured and shown in Tables 10, 7, and 11 and 8, respectively.

Table 10

Table 11

(Each value is expressed as mean ± S.D (N = 5). * P <0.05: for negative control)

As can be seen from the results of the above table, the cartilage damage area in the knee joint was reduced in the composition administration group of Example 1 and the comparative formulation administration group of the present invention. That is, in both the composition of Example 1 of the present invention, which is a one-time therapy, and the comparative formulation, which was a three-time therapy, the area of cartilage damage in the femoral region was reduced compared to the negative control. In the tibial area, cartilage damage area was reduced in both the administration group of Example 1 and the administration of Comparative Example of the present invention.

실험예 9: 본 발명 조성물의 생체적합성 확인Experimental Example 9: Confirmation of biocompatibility of the composition of the present invention

The biocompatibility of the hyaluronic acid crosslinked hydrogel composition of Example 1 used in the present invention was evaluated.

According to the method of Fujimura et al. (Fujimura T et al., J. Dermatol. Sci., 2000; 24; pp. 105-111), a 6-week-old female hairless mouse (SKH; Jung-Ang Lab Animal Inc., Korea) and the square (1.5 × 1.5 cm ² ) of the tattoo and the hyaluronic acid cross-linked hydrogel of Example 1 of the present invention was administered subcutaneously in the square tattoo. 0.4 ml of the hyaluronic acid crosslinked hydrogel of the present invention was injected through a 27 gauge syringe needle into the tattooed rectangle of the dorsal subcutaneous layer, respectively. Twelve weeks after hyaluronic acid crosslinked hydrogel administration, histological examination was performed using hematoxyl-iosin (H & E) staining. Skin samples were taken from each mouse and fixed in 10% by volume buffered formaldehyde, dehydrated with ethanol, then stored in paraffin to make samples, which were cut into 4 μm sections and stained with H & E. The result of photographing the stained sample using an optical microscope is shown in FIG. As shown in Figure 9, the mice administered the hydrogels of the present invention did not show any inflammatory response as in the control mice (in the case of H & E, hematozain is stained blue in the inflammatory response, Iosin is stained red as a contrast dye with hematozain). That is, when the HA-HMDA crosslinked hydrogel (FIG. 9B) of the present invention was treated, the epidermis was changed similarly to the epidermis of normal mice, and no hemolysis or inflammatory reaction was observed in the dermis as in the normal mice.

From the above results, the composition containing the hyaluronic acid-alkylene diamine crosslinked hydrogel of the present invention shows very excellent biocompatibility, and is long-lived in vivo, and injects once intra-articularly, thereby effectively preventing pain due to arthritis for two weeks. It can be seen that it can be used as an injection for intra-articular administration to effectively alleviate abnormalities for a long time and protect the cartilage of the joint, and effectively treat arthritis diseases.

실험예 10: 본 발명 조성물 내 히알루론산 가교물 하이드로겔의 생체외 분해 정도와 물성(G')에 따른 지속 효과 확인Experimental Example 10: Confirmation of the sustained effect according to the degree of ex vivo decomposition and physical properties (G ′) of the hyaluronic acid crosslinked hydrogel in the composition of the present invention

The determination of the sensitivity of hyaluronic acid degrading enzyme of the hyaluronic acid cross-linked hydrogel-containing sterile composition of the present invention can be carried out using a known method (WE Hennink, Rheological monitoring of long-term degrading polymer hydrogels. J. Rheol., 1999. 43 (4). ): 933-950). Determination of susceptibility to degrading enzymes correlated with the G 'value, using a BDDE (1,4-butanediol diglycidyl ether) commercially available from Q-Med AB and crosslinking the hydroxyl group of hyaluronic acid (20 mg / ml). ) Was evaluated as a control. The sterile composition and the control of Example 2 of the present invention were each dispensed 800 μl into a 1.5 ml test tube. Hyaluronic Acid Degrading Enzyme (Hase,Bovine Testes, Sigma-Aldrich) was dissolved in PBS (pH 7.4) at 2 mg / ml (2,000 U), and 15 μl were dispensed into each test tube at 37 ° C. for each time period, followed by elastic modulus (G ′) at 3 Hz. The change is shown in FIG. 10. The control exhibited an initial modulus of 620 Pa at 3 Hz and the sterile composition of Example 2 exhibited an initial modulus of 330 Pa at 3 Hz. The change of elastic modulus was confirmed over 25 hours. As shown in FIG. 10, in the case of the control product, the initial modulus of elasticity (G ′) was high but was rapidly degraded by hyaluronic acid degrading enzyme up to 3 hours, indicating a very low modulus of elasticity. It was stable to degradation up to 25 hours. In addition, it was confirmed that the decomposition of the crosslinked hyaluronic acid (HA) was independent of the initial modulus of elasticity G '.

실험예 11: 본 발명 조성물의 대퇴골 연골 보호 효과 및 조직학적 변화 관찰Experimental Example 11 Observation of Femoral Cartilage Protective Effects and Histological Changes

The osteoarthritis-induced rabbit model was used to confirm the osteoarthritis cartilage protection effect of the composition of Example 2 of the present invention and the control agent of the comparative example, and the specific degree of damage was confirmed by histopathological examination confirming the thickness of the cartilage and the reduction of cartilage cells. .

The animals were infused with an intraperitoneal injection of a 25 mg / kg Zoletile mixture (Zoletile 50; Vibac Lab., France), followed by 1.5% isoflurane (Hana Pharm. Co. in 70% N ₂ O and 28.5% O ₂ gas). , Hwasung, Korea), while maintaining general anesthesia with a mixed anesthetic gas, the left knee joint was exposed and the anterior cruciate ligament, according to previous methods (Hanashi et al., 2002; Jiang et al., 2010). Osteoarthritis was induced by cruciate ligament resection and partial medial menisectomy. In the normal control group, the articular capsule was excised to confirm the anterior cruciate ligament and medial meniscus, and then the articular capsule was closed without ablation. Five weeks after the induction of arthritis, the composition of Example 2 of the present invention and the comparative agent were administered 0.3 ml each in the knee joint cavity, and the second and third administrations of the comparative agent of the comparative example were performed at 7 and 9 weeks after surgery. In the normal control group, only 0.3 ml of sterile saline solution was administered in a single intra articular capsule 5 weeks after arthritis induction.

For histopathological observation, knee joints were taken and fixed on 10% neutral formalin buffer. After fixing for 24 hours, descaling process was performed for 5 days with descaling solution [24.4% formic acid, and 0.5N sodium hydroxide]. Thereafter, the surface cartilage was separated, cut vertically, fixed with paraffin, and then sliced into sections having a thickness of 3 to 4 μm, and the samples were observed after staining with Safranin O. In histomorphometry, the previously stained sample sections were analyzed using iSolution FL ver 9.1 (IMT i-solution Inc., Vancouver, Canada). The number of chondrocytes was measured using an image analysis program within the same area of the joint. The results are shown in FIG. As shown in FIG. 11, damage to the entire femur (black stained surface), and reduction of cartilage thickness and cartilage cells were observed in osteoarthritis control. However, the control agent of the comparative example and Example 2 composition treatment group of the present invention showed a marked improvement effect.

실험예 12: 본 발명 조성물의 Mankin score 및 조직형태계측을 이용한 평가Experimental Example 12 Evaluation Using Mankin Score and Tissue Morphometric Measurement of the Invention Composition

The degree of rabbit joint damage of the composition of Example 2 of the present invention and the control agent of the comparative example was evaluated using a Mankin score. The Mankin score is the most basic histopathological observation for evaluating osteoarthritis, and the surface damage of articular cartilage induced during osteoarthritis. Judgment is based on the change in staining, chondrocyte count, and the formation of clones. The higher the score, the higher the incidence of osteoarthritis (Armstrong et al. [1994] and Lovasz et al [2001]). using Safranin O stain). In addition, histologic measurements of the femoral and tibial articular surface cartilage were observed. The results of histomorphometry and Mankin scores of the femur and tibia joint surface cartilage are shown in Table 12 and Table 13, respectively.

Table 12

(Each value is expressed as mean ± SD. ^Ad P <0.01 ^be P <0.05: for normal control, ^cf P <0.01 ^g P <0.05: for osteoarthritis control)

Table 13

As can be seen from the results of the above table, cartilage thickness and chondrocytes were increased in the Example 2 composition-administered group and the Comparative Example-controlled group of the present invention. In the osteoarthritis control group, surface damage, decreased chondrocyte count, clone formation and decreased staining were observed in the articular cartilage of the femur and tibia, indicating a significant (p <0.01) increase in the Mankin score compared to the normal control group. In femoral and tibial articular cartilage, respectively, the control group of Comparative Example and the composition of Example 2 showed a significant (p <0.01) reduction in Mankin score of femur and tibia articular cartilage, respectively, compared to the osteoarthritis control group. That is, in both the composition of Example 2 of the present invention and the comparative example preparation administration group, which is a triple therapy, there was a cartilage protection effect at the femur and tibia area. In addition, an overall decrease in the Mankin score was observed. In the osteoarthritis control group, significant decreases in the thickness of femur and tibia joint cartilage and the number of chondrocytes per unit area were observed (p <0.01) compared to the normal control group. Significant (p <0.01) femoral and tibial articular cartilage thicknesses and an increase in the number of chondrocytes per unit area were observed compared to osteoarthritis controls. The Mankin score of the femoral articular cartilage showed 550.00% change in the osteoarthritis control group compared to the normal control group, and -42.31 and -57.69% change in the comparative control group and the Example 2 composition administration group of the present invention, respectively. Indicated. The tibial Mankin score showed a change of 507.69% in the osteoarthritis control group compared to the normal control group, and -50.63% and -65.82% in the control group of the comparative example and the Example 2 administration group, respectively. The thickness of the femoral articular cartilage was 63.19 and 111.34% in the osteoarthritis control group compared to the normal control group. The thickness of the tibial articular cartilage was -63.06% in the osteoarthritis control group compared to the normal control group, and 80.82 and 135.40% in the comparative control group and Example 2 administration group of the present invention, respectively. The number of chondrocytes per unit area in the femoral articular cartilage was -73.61% in the osteoarthritis control group compared to the normal control group, and 166.67 and 209.06% in the osteoarthritis control group compared to the osteoarthritis control group, respectively. Indicated. The number of chondrocytes per unit area in the tibial articular cartilage was -70.97% in the osteoarthritis control group compared to the normal control group, and 84.40% and 127.27% in the osteoarthritis control group compared to the osteoarthritis control group, respectively. Change.

In addition, the totals of Mankin scores are shown in FIG. 12. The significance in the femur and tibia is ^a p <0.01 and ^b p <0.05 for the normal control group and ^c p <0.01 for the osteoarthritis control group.

Claims

Injectable liquid composition for intraarticular administration for treating or improving symptoms of arthritis diseases, comprising as an active ingredient an alkylenediamine crosslinked hydrogel of hyaluronic acid of formula 1 below:

[Equation 1]

[HA] m -C (O) -NH-R1-NH-C (O)-[HA] n

Wherein HA represents hyaluronic acid or a salt thereof except one carboxyl group, and R 1 represents a C 3 -C 10 alkylene group unsubstituted or substituted with hydroxy, C 1 -C 6 alkyl, or C 1 -C 6 alkoxy M and n are independently integers of 10,000 to 4,000,000.
The composition of claim 1, wherein the hyaluronic acid has a molecular weight of 20,000 Daltons to 4,000,000 Daltons.
The composition of claim 1 wherein said alkylenediamine is hexamethylenediamine.
The composition of claim 1, wherein the crosslinking rate of the crosslinked hydrogel is 5 to 35%.
The composition of claim 1, wherein the crosslinked hydrogel is comprised in an amount of 0.4 to 3.0 (w / w)% based on the total weight of the injection solution.
The composition of claim 1, wherein the analgesic effect lasts for at least two weeks after administration of the injection.
The composition of claim 1, further comprising unmodified hyaluronic acid.
The composition of claim 7, wherein the weight ratio of the alkylenediamine crosslinked hydrogel: unmodified hyaluronic acid of the hyaluronic acid is 5: 5 to 9.5: 0.5.
The composition of claim 8, wherein the alkylenediamine crosslinked hydrogel: unmodified hyaluronic acid weight ratio of hyaluronic acid is 7: 3 to 9.5: 0.5.
An analgesic composition for reducing joint pain caused by arthritis diseases, comprising as an active ingredient an alkylenediamine crosslinked hydrogel of hyaluronic acid according to claim 1.
A composition for inhibiting cartilage degeneration caused by arthritis diseases, comprising as an active ingredient an alkylenediamine crosslinked hydrogel of hyaluronic acid according to claim 1.
A composition for inhibiting synovial inflammation caused by arthritis diseases, comprising an alkylenediamine crosslinked hydrogel of hyaluronic acid of claim 1 as an active ingredient.
The composition according to any one of claims 1 to 10, wherein said arthritis disease is osteoarthritis.
A kit comprising a syringe filled with a composition according to claim 1.
A kit comprising a syringe filled with a composition according to claim 7.