KR102425496B1 - Crosslinked hyaluronic acid having high elasticity, high viscosity and high effective cross-linker ratio, and preparing method thereof - Google Patents

Abstract

The present invention is a crosslinked hyaluronic acid crosslinked with an epoxy-based crosslinking agent, and has an effective crosslinking ratio (CrR) of 0.3 or more, a viscosity of 90 Pa·s or more, and an elasticity of 400 Pa when contained in an amount of 2.0 wt% or more in the composition It provides a cross-linked hyaluronic acid, characterized in that the above. The present invention also comprises the steps of (a) mixing hyaluronic acid or a salt thereof, an epoxy-based crosslinking agent, and an aqueous alkali solution; (b) crosslinking the mixture obtained in step (a) after concentration under reduced pressure; (c) washing and swelling the crosslinked product obtained in step (b); (d) pulverizing and purifying the swollen material obtained in step (c); and (e) swelling and dialysis of the purified product obtained in step (d). The cross-linked hyaluronic acid of the present invention has high viscosity, high elasticity, and high effective cross-linking rate while minimizing the amount of residual cross-linking agent, so it can be usefully used for the treatment of osteoarthritis or filler injection.

Description

Crosslinked hyaluronic acid having high elasticity, high viscosity and high effective cross-linker ratio, and a method for preparing the same

The present invention relates to a cross-linked hyaluronic acid having high elasticity, high viscosity, and high effective cross-linking rate, and a method for preparing the same.

Hyaluronic acid is a biopolymer material in which repeating units composed of N-acetyl-D-glucosamine and D-glucuronic acid are linearly connected, and is present in connective tissue, subcutaneous tissue, nerve tissue, or joint synovial fluid in a living body. It is known to be present in many places, such as the placenta of animals or chickens. In addition, hyaluronic acid is a substance that has been extracted from fermentation of microorganisms such as Streptococcus genus or chicken rice bran, and used as a filler, an osteoarthritis treatment agent, an anti-adhesion agent, and a raw material for ophthalmic medicines or cosmetics.

However, hyaluronic acid is decomposed by enzymes and free radicals and is rapidly discharged from the body, and for this reason, it has a disadvantage that it cannot be continuously injected into the body. In order to extend the decomposition period of hyaluronic acid, many studies have been introduced to synthesize hyaluronic acid cross-linked products using various cross-linking agents.

A typical example of using cross-linked hyaluronic acid is a filler. Looking at the general method for producing a cross-linked hyaluronic acid, if the amount of the cross-linking agent is reduced, the viscosity and elasticity are lowered, and the decomposition time in vivo tends to be shorter. Conversely, when the amount of the crosslinking agent is increased, the viscosity and elasticity are increased, and the decomposition time in vivo is prolonged, but problems of causing an inflammatory reaction may occur. It is known that such an inflammatory reaction is caused by the recognition of the cross-linking agent component as a foreign substance when the hyaluronic acid cross-linker is decomposed in vivo.

Hyaluronic acid fillers currently on sale have a monophasic or biphasic form depending on the manufacturing method. The monophasic filler manufactured by the method described in US Patent No. 8,357,795 is known to be useful for making a delicate shape with high viscosity and soft injection because the whole is made of a homogeneous gel. The biphasic filler manufactured by the method described in US Patent No. 5,827,937 has advantages of maintaining shape and increasing volume due to high elasticity. Accordingly, in recent years, the development of fillers having both monophasic high viscosity (numerical) and biphasic high elasticity (numerical) have been mainly developed. However, in the case of the conventionally developed hyaluronic acid crosslinked product having both monophasic and biphasic characteristics, it is difficult to find a single-step synthesis method. For example, in Korean Patent Publication No. 10-1720426, monophasic and biphasic hyaluronic acid crosslinked products are prepared for the production of hyaluronic acid crosslinked products having monophasic and biphasic characteristics, respectively. It is manufactured by a method of mixing after manufacturing. In Korean Patent Laid-Open Publication No. 10-2017-0090965, a cross-linked hyaluronic acid having monophasic and biphasic characteristics is prepared by using a method of mixing a primary cross-linked product and a secondary cross-linked product. .

The present inventors use a minimum amount of a crosslinking agent to improve the above problems, minimize side effects and improve crosslinking efficiency, so that it has the characteristics of monophasic and biphasic properties in a single synthesis method, complex physical properties hyaluronic acid A cross-linked product manufacturing method was developed. Additionally, a crosslinking agent and a method for producing a crosslinked hyaluronic acid that can minimize residues caused by the crosslinking agent were developed.

US Patent No. 8,357,795 US Patent No. 5,827,937 Republic of Korea Patent Publication No. 10-1720426 Republic of Korea Patent Publication No. 10-2017-0090965

An object of the present invention is to provide a crosslinked hyaluronic acid having high elasticity, high viscosity and high effective crosslinking rate, a method for preparing the same, and a use thereof.

Another object of the present invention is to provide a cross-linked hyaluronic acid having reduced residues derived from a cross-linking agent, a method for preparing the same, and a use thereof.

The present inventors have demonstrated that by increasing the effective crosslinking rate while minimizing the amount of crosslinking agent in the crosslinked hyaluronic acid, it is possible to provide a crosslinked hyaluronic acid that minimizes crosslinking agent-derived residues and inflammatory reactions as well as high elasticity and viscosity. confirmed, and the present invention was completed.

Hereinafter, the composite physical properties of the cross-linked hyaluronic acid of the present invention, its preparation method, and its use are sequentially looked at.

Composite physical properties hyaluronic acid crosslinked product

The present invention is a cross-linked hyaluronic acid cross-linked with an epoxy-based cross-linking agent, having an effective cross-linking ratio (CrR) of 0.3 or more, a viscosity of 90 Pa s or more, and an elasticity of 400 Pa or more when contained in an amount of 1.5 wt % or more in the composition It provides a cross-linked hyaluronic acid, characterized in that.

In the present invention, hyaluronic acid is a linear polymer in which β-D-N-acetylglucosamine and β-D-glucuronic acid are alternately bonded.

In the present invention, the epoxy-based crosslinking agent means a crosslinking agent known in the art having two or more epoxy functional groups. For example, the epoxy-based crosslinking agent is ethylene glycol diglycidyl ether (EGDGE), 1,4-butanediol diglycidyl ether (1,4-butandiol diglycidyl ether: BDDE), 1,6-hexane Diol diglycidyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, EDC (1-ethyl -3-(3-dimethylaminopropyl)carbodiimide), biscarbodiimidie (BCDI), diglycerol polyglycidylether, divinyl sulfone (DVS), or a combination thereof. In one embodiment, the epoxy-based crosslinker is 1,4-butanediol diglycidyl ether (BDDE).

The epoxy-based crosslinking agent crosslinked to the hyaluronic acid crosslinker of the present invention may be 0.01 to 60 mol% based on the hydroxyl group contained in the hyaluronic acid, specifically 1 to 10 mol%, and more specifically 3 to 6 mol%.

In the present invention, the effective cross-linker ratio (CrR) of the cross-linked hyaluronic acid refers to an effective cross-linking ratio in which both of the cross-linking agents cross-linked to the hyaluronic acid molecules in the cross-linked product are cross-linked, and the formula defined (Kenne et al., Carbohydrate Polymers 91 (2013) 410-418, et al.).

CrR = moles of double crosslinked crosslinker / (number of moles of single crosslinked crosslinker + moles of double crosslinked crosslinker)

Since the cross-linked hyaluronic acid of the present invention has an effective cross-linking ratio of 0.3 or more, it is possible to increase the viscosity and elasticity to a desired level while minimizing the amount of the cross-linking agent used in the cross-linking product. In one embodiment, the crosslinked hyaluronic acid of the present invention may have an effective crosslinking ratio of 0.3 or more and 0.8 or less. For example, the effective crosslinking rate may be 0.3 to 0.6.

Conventional hyaluronic acid crosslinked products have a monophasic or biphasic form depending on the manufacturing method. It is known that the monophasic hyaluronic acid crosslinked product is made of a homogeneous gel with high viscosity, soft injection, and useful for making delicate shapes. The biphasic hyaluronic acid crosslinked product has advantages of maintaining shape and increasing volume due to its high elasticity.

The cross-linked hyaluronic acid of the present invention is characterized in that it exhibits complex physical properties having both monophasic and biphasic characteristics. The crosslinked hyaluronic acid of the present invention is characterized in that when the hyaluronic acid content is 1.5 wt% or more, the viscosity is 90 Pa·s or more, and the elasticity is 400 Pa or more. For example, the crosslinked hyaluronic acid of the present invention has a viscosity of 90 Pa·s or more when contained in an aqueous solution in an amount of 1.5 to 2.6 wt%, preferably 2.0 wt% to 2.6 wt%, more preferably 2.4 wt%, and elasticity This is 400 Pa or more.

In one embodiment, the crosslinked hyaluronic acid of the present invention has a viscosity of 90 to 300 Pa. s, specifically 90 to 250 Pa·s, and more specifically 100 to 200 Pa·s.

In one embodiment, the crosslinked hyaluronic acid of the present invention has an elasticity of 400 to 800 Pa when contained in an aqueous solution in an amount of 1.5 to 2.6% by weight, preferably 2.0% to 2.6% by weight, more preferably 2.4% by weight, and , specifically 500 to 700 Pa.

In one embodiment, the crosslinked hyaluronic acid of the present invention has a viscosity (Pa s) and The relative ratio of elasticity (Pa) is 1 to 3 to 1 to 6.

The crosslinked hyaluronic acid exemplified in Examples of the present invention (Examples 1 to 3) is contained in an aqueous solution in an amount of 2.4% by weight to exhibit a viscosity of 90 to 250 Pa·s and an elasticity of 400 to 800 Pa (Experimental Example 1; Table) One). A representative cross-linked hyaluronic acid of the present invention (Example 1) has an effective cross-linking rate (CrR) of 0.34, a viscosity of 108 Pa s, and an elasticity of 551 Pa, and has a viscosity and It was confirmed that all of the elasticity had excellent composite properties (Experimental Example 2; Table 2).

The crosslinked hyaluronic acid of the present invention has the advantage that the rate of change in viscosity and elasticity is minimized during long-term storage. In one embodiment, the crosslinked hyaluronic acid of the present invention has less than 17% change in viscosity and less than 11% change in elasticity when stored for 6 months.

According to an embodiment of the present invention, the cross-linked hyaluronic acid of the present invention showed little decrease in viscosity and elasticity when stored for 6 months, confirming that the composite properties were maintained for a long time (Experimental Example 3; Table 3; Fig. 1 and Figure 2).

The crosslinking agent in the hyaluronic acid crosslinker may increase the amount of residual material. For example, when the cross-linked cross-linking agent in the cross-linked product is BDDE, BDDE is hydrolyzed during the cross-linking reaction to obtain BGPE (1,4-butanediol glycidyl propan-2,3-diolyl ether), or BDPE (1,4-butanediol di-( propan-2,3-diolyl)ether). The cross-linked hyaluronic acid of the present invention not only reduced the amount of the cross-linked cross-linking agent, but also reduced the amount of residual material derived from the cross-linking agent, thereby minimizing the residual material of the cross-linked hyaluronic acid.

In one embodiment, when the crosslinking agent in the hyaluronic acid crosslinker is BDDE, the residual BDDE is 0.01 ppm or less, the residual BGPE is 0.01 ppm or less, and the residual BDPE is 0.5 ppm or less. According to an embodiment of the present invention, residual BDDE and BGPE were not detected in the BDDE cross-linked hyaluronic acid cross-linked product of the present invention, and BDPE was measured as 0.2 ppm, so that the amount of cross-linking agent residual material compared to a comparative example or a commercially available filler composition It was confirmed that very little (Experimental Example 3; Table 3).

Method for producing cross-linked hyaluronic acid with complex physical properties

The present invention provides a method for preparing a crosslinked product of hyaluronic acid with complex physical properties, comprising the steps (a) to (e) below:

(a) mixing hyaluronic acid or a salt thereof, an epoxy-based crosslinking agent, and an aqueous alkali solution;

(b) concentrating the mixture obtained in step (a) under reduced pressure and then crosslinking;

(c) washing and swelling the concentrate obtained in step (b);

(d) pulverizing and purifying the swollen material obtained in step (c); and

(e) swelling and dialysis of the purified product obtained in step (d).

Step (a) of the manufacturing method is a step of mixing hyaluronic acid or a salt thereof, an epoxy-based crosslinking agent, and an aqueous alkali solution.

In the above preparation method, hyaluronic acid is a linear polymer in which β-D-N-acetylglucosamine and β-D-glucuronic acid are alternately bonded.

The molecular weight of the hyaluronic acid used in the preparation method may be 100,000 to 6,000,000 Da. In one embodiment, the molecular weight of the hyaluronic acid may be 500,000 to 3,000,000 Da, such as, but not limited to, 800,000 to 1,200,000 Da.

The salt of hyaluronic acid used in the above preparation method is an inorganic salt such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, and an organic salt such as tetrabutylammonium hyaluronate. may be included. In one embodiment the salt of hyaluronic acid is sodium hyaluronate.

Hyaluronic acid or a salt thereof used in the preparation method may be isolated from a microorganism, synthesized, or purchased, but is not limited thereto. For example, the hyaluronic acid or a salt thereof may be isolated and purified from a microorganism of the genus Streptococcus ( Streptocossus equi , Streptococcus zooepidemicus ).

The epoxy-based crosslinking agent may be a crosslinking agent known in the art including two or more epoxy functional groups. Specifically, the epoxy-based crosslinking agent is ethylene glycol diglycidyl ether (EGDGE), 1,4-butanediol diglycidyl ether (1,4-butandiol diglycidyl ether: BDDE), hexanediol diglyceride Cydyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether (propylene glycol diglycidyl ether), polypropylene glycol diglycidyl ether (polypropylene glycol diglycidyl ether), EDC (1-ethyl-3- (3-dimethylaminopropyl)carbodiimide), BCDI (biscarbodiimidie), diglycerol polyglycidylether, DVS (divinyl sulfone), or a combination thereof. In one embodiment, the epoxy-based crosslinked product is 1,4-butanediol diglycidyl ether (BDDE).

In one embodiment, the epoxy-based crosslinking agent may be reacted in a range of 0.05 to 300 mg/g, specifically 5 to 50 mg/g, more specifically 15 to 30 mg/g, relative to hyaluronic acid or a salt thereof.

The aqueous alkali solution may be an aqueous lithium hydroxide solution, an aqueous sodium hydroxide solution, or an aqueous potassium hydroxide solution, preferably sodium hydroxide.

The concentration of the aqueous alkali solution is preferably 0.5 to 3%.

The amount of sodium hydroxide aqueous solution used in the above step is preferably 6.2 to 11 mL based on 1 g of hyaluronic acid.

The elasticity and viscosity of the crosslinked hyaluronic acid can be adjusted according to the change in the concentration of the aqueous alkali solution, and in the aqueous alkali solution concentration range, sodium hyaluronate is stably mixed without being extracted into the aqueous alkali solution, so that the crosslinking reaction is possible.

The pH of the aqueous alkali solution may be 9 to 14. For example, the pH of the aqueous alkali solution may be 12.5 to 13.5.

Depending on the pH change of the aqueous alkali solution, the type of hyaluronic acid cross-linking and the physical properties of the cross-linked product can be adjusted, and the mixture required for the subsequent vacuum concentration process in the above pH range can be stably provided.

The mixing may be performed by stirring hyaluronic acid or a salt thereof, a crosslinking agent and an aqueous alkali solution in the mixed solution. The mixing process may be performed at 10 to 40 °C, and may be performed for 0.5 to 7 hours. For example, the mixing process may be performed for 2 hours.

A salt may or may not be additionally added to the mixed solution.

Step (b) of the above preparation method is a step of crosslinking after concentrating the mixture obtained in step (a) under reduced pressure.

In step (a), when hyaluronic acid was dissolved in the basic aqueous solution, at least 6.2 mL of the basic aqueous solution was required per 1 g of hyaluronic acid or a salt thereof. In this case, the basic aqueous solution may be 0.5 to 3% concentration, for example, 1% concentration of lithium hydroxide aqueous solution, sodium hydroxide aqueous solution, or potassium hydroxide aqueous solution.

In order to increase the concentration of the mixed reaction solution, it was concentrated under reduced pressure. It was confirmed that the effective crosslinking rate increased when the injection was prepared by concentrating under reduced pressure, and it was confirmed that the elasticity increased to a certain level. However, when the concentration under reduced pressure was performed more than a certain amount, it was found that hydration was not performed in the method for preparing the injection used by the present inventors (Examples 1 to 3 and Comparative Example 1; Table 1).

In the present invention, reduced pressure concentration is a vacuum evaporation process in which the pressure is adjusted in consideration of the boiling point of water, and is a process of continuously removing water evaporated from the crosslinking reaction mixture through the evaporation process.

In the above preparation method, the concentration under reduced pressure is 40 °C or less, specifically 15 to 40 °C. For example, the reduced pressure concentration temperature may be 15 to 30 ℃, or 30 to 40 ℃. The reduced pressure concentration may be performed under 1 to 30 mmHg.

The concentration under reduced pressure is not particularly limited, but may be performed for 1 to 48 hours, specifically 5 to 20 hours.

The hyaluronic acid concentration after concentration under reduced pressure is preferably 10 to 30%, preferably 15 to 25%. The crosslinking reaction of hyaluronic acid by the crosslinking agent proceeds in a liquid phase. After concentration under reduced pressure according to the present invention, the cross-linked hyaluronic acid may exhibit a solid phase or a gel phase.

The step (b) may further include a step of allowing the mixture to stand after concentration under reduced pressure. The stationary process may be performed by allowing the concentrated mixture under reduced pressure to stand at 15 to 40°C. For example, the mixture concentrated under reduced pressure may be left standing at 20 to 35°C. The stationary reaction may be performed for 1 to 48 hours, and specifically may be performed for 12 to 36 hours.

The crosslinking reaction of hyaluronic acid occurs by the concentration and/or stationary process under reduced pressure of step (b), through which the effective crosslinking rate of the crosslinked hyaluronic acid is improved and elasticity is increased.

Step (c) of the preparation method is a step of washing and swelling the cross-linked product obtained in step (b).

In the above preparation method, washing may be performed through purified water. The amount of purified water is preferably 100 mL or more relative to 1 g of hyaluronic acid. For example, the amount of purified water may be 100 mL or more relative to 1 g of hyaluronic acid, and more specifically, 1,000 mL or less, such as 500 ml or less.

Step (d) of the above preparation method is a step of pulverizing the product according to (c) and then purifying it through an organic solvent to obtain a solid.

In the above preparation method, a hyaluronic acid crosslinked gel having a particle size of 500 μm or less may be produced through pulverization.

In the above step, the organic solvent may be acetone, ethanol, isopropyl alcohol, or a mixture thereof. The organic solvent is preferably isopropyl alcohol.

The present inventors confirmed that residual BDDE, BGPE and BDPE can be minimized when the purification process is performed through the organic solvent (Experimental Example 4; Table 4).

Step (e) of the manufacturing method is a step of swelling and dialysis of the solid obtained according to step (d). In the above step, the swelling may be performed using purified water.

The purified water used for swelling is preferably 7 to 25 g, specifically 8 to 20 g, more specifically 9 to 15 g relative to 1 g of hyaluronic acid, and the dialysate that has undergone the subsequent dialysis process is 25 g relative to 1 g of hyaluronic acid. to 33 g is preferred.

Through the steps (c) to (e) of the manufacturing method, it is possible to effectively remove the BDDE residual material. In addition, the pH of the hyaluronic acid crosslinked product was corrected to 7 to 9.

After step (e), the manufacturing method may further include the step of (f) making an injection from a dialysate.

The content of the cross-linked hyaluronic acid in the injection according to step (f) may be 0.1 wt% or more, specifically 0.5 wt% to 10 wt%, and more specifically 0.8 wt% to 10 wt%. In one embodiment, the crosslinked hyaluronic acid content may be 1.5 to 2.6% by weight, preferably 2.0 to 2.6% by weight.

Additionally, non-crosslinked hyaluronic acid or a salt thereof may be further included in the injection.

Use of crosslinked hyaluronic acid with improved viscosity, elasticity and effective crosslinking rate

The present invention provides a composition comprising a crosslinked hyaluronic acid having improved viscosity, elasticity and effective crosslinking rate. The hyaluronic acid crosslinked product and its preparation method are as described above.

The content of the crosslinked hyaluronic acid included in the composition of the present invention may be 0.1 wt% or more, specifically 0.5 wt% to 10 wt%, and more specifically 0.8 wt% to 10 wt%. In one embodiment, the hyaluronic acid crosslinked content is between 1.5 and 2.6% by weight, preferably between 2.0% and 2.6% by weight.

The cross-linked hyaluronic acid according to the present invention is a biocompatible material and can be used to maintain a certain shape without any side effects when introduced into the body, such as wrinkle improving agents, cosmetic aids, joint function improving agents, and anti-adhesion agents. It can be usefully used as a drug delivery vehicle, a cell culture support, and the like. In addition, it can be prepared in various forms according to the use, such as thread, microbead, film, hydrogel, and sponge.

The composition of the present invention has excellent flowability as an injection, is stable even at high temperature sterilization, and has excellent economic efficiency and quality. Therefore, the composition of the present invention can be used as a food, cosmetic, pharmaceutical, medical device use, such as a stabilizer, a moisturizer, a dispersion aid, a viscosity modifier, a drug delivery carrier, a tissue repair filler, and an anti-adhesion film formation after surgery. More specifically, a composition for an injection drug carrier, a composition for a mucosal and transdermal absorption drug carrier, a composition for an oral drug carrier, a composition for tissue repair, a composition for preventing tissue adhesion after surgery, a filling composition for molding, a composition for filler injection, A composition for joint insertion, a composition for patch-type wound treatment dressing, a composition for treating osteoarthritis, a composition for ocular surgery auxiliary injection, a composition for treating dry eye syndrome, a composition for treating wrinkles, a composition for treating gums, a composition for treating tympanic membrane perforation, health food It can be used as a composition or a cosmetic composition.

In one embodiment, the composition is a composition for preventing or treating osteoarthritis.

In one embodiment, the composition is a filler injectable composition.

The composition may be used by adding other conventional additives such as antioxidants, buffers and/or bacteriostats, diluents, dispersants, surfactants, binders, lubricants, and local anesthetics, if necessary. For example, the composition may further include a local anesthetic.

The cross-linked hyaluronic acid of the present invention has high viscosity, high elasticity, and high effective cross-linking rate while minimizing the amount of residual cross-linking agent, so it can be usefully used for the treatment of osteoarthritis or filler injection.

1 is a view showing the elastic stability test of the crosslinked hyaluronic acid prepared by the method of Example 1.
2 is a view showing the viscosity stability test of the crosslinked hyaluronic acid prepared by the method of Example 1.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereby.

Due to the nature of the hyaluronic acid crosslinked product, the viscosity and elasticity values may vary depending on the concentration, crosslinking agent ratio, crosslinking time, crosslinking temperature, and post-treatment method. Therefore, the present inventors performed a comparative experiment based on the self-developed manufacturing method, and compared the physical properties through analysis of filler products currently on sale. However, the present cross-linked hyaluronic acid is not limited to the filler.

Preparation of Examples and Comparative Examples

Example 1. Preparation of hyaluronic acid crosslinked product 1

45 g of sodium hyaluronate having a molecular weight of 1,000,000 Da (obtained from Shiseido) was dissolved in 324 mL of a 1% sodium hydroxide solution. After dissolution, 0.825 mL (4 mol%) of 1,4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred at 25° C. for 2 hours. 328 g of the mixed solution is taken and concentrated under reduced pressure at 30 to 40 ° C. at a pressure of 1 to 30 mmHg, so that the sodium hyaluronate content is about 23%. The concentrated mixture was further allowed to stand at 25 °C for 21 hours.

The cross-linked product was washed and swollen with 12.3 L of purified water, and the resulting gel was pulverized to 500 μm or less, and then injected into 12.3 L of isopropyl alcohol solution to solidify. After filtering the solidified crosslinked product, it was put into a dialysis network, swollen with purified water in a volume corresponding to 9 times the weight of the hyaluronic acid crosslinked product, and dialyzed with 0.9% brine for about 40 hours. The dialysate was prepared for injection after content correction, grinding and sterilization.

For injection preparation, uncrosslinked sodium hyaluronate (5% by weight of crosslinked sodium hyaluronate) was added to the dialysate, 1×PBS aqueous solution and lidocaine hydrochloride (0.3% by weight of 1 mL of syringe) were added to hyaluronic acid The content was made to be 2.4% by weight. The mixture was ground through a grinder. The pulverized material was placed in a prefilled syringe with an internal volume of 1 mL, sealed with a rubber stopper, and autoclaved at 121° C. for 20 minutes.

Example 2. Preparation of hyaluronic acid crosslinked product 2

The hyaluronic acid crosslinked product of Example 2 was prepared in the same manner as in Example 1, except that the sodium hyaluronate content was about 24.5% after concentration under reduced pressure.

Example 3. Preparation of hyaluronic acid crosslinked product 3

The hyaluronic acid crosslinked product of Example 3 was prepared in the same manner as in Example 1 except that the sodium hyaluronate content was about 20% after concentration under reduced pressure.

Example 4. Preparation of cross-linked hyaluronic acid for osteoarthritis injection

25 g of sodium hyaluronate having a molecular weight of 1,000,000 Da (obtained from Shiseido) was dissolved in 180 mL of a 1% sodium hydroxide solution. After dissolution, 0.458 mL (4 mol%) of 1,4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred at 25° C. for 2 hours. Take 166.23 g of the mixture and concentrate under reduced pressure at 30 to 40 ° C. under a pressure of 1 to 30 mmHg, so that the sodium hyaluronate content is about 18.4%. The concentrated mixture was further allowed to stand at 25° C. for 21 hours.

The crosslinked product was washed and swollen with 6.15 L of purified water, and the resulting gel was pulverized to a size of 500 μm or less and then injected into 6.15 L of isopropyl alcohol solution to solidify. After filtering the solidified crosslinked product, it was put into a dialysis network, swollen with purified water in a volume corresponding to 9 times the weight of the hyaluronic acid crosslinked product, and dialyzed with 0.9% brine for about 40 hours. The dialysate was prepared for injection after content correction, grinding and sterilization.

For the preparation of the injection, 1× PBS aqueous solution was added to the dialysate so that the hyaluronic acid content was 2.5% by weight. The mixture was ground through a grinder. The pulverized material was placed in a prefilled syringe with an internal volume of 1 ml, sealed with a rubber stopper, and autoclaved at 121° C. for 20 minutes.

Comparative Example 1. Preparation of crosslinked hyaluronic acid for comparison 1

After concentration under reduced pressure, the hyaluronic acid crosslinked product of Comparative Example 1 was prepared in the same manner as in Example 1, except that the sodium hyaluronate content was about 90%.

Comparative Example 2. Preparation of crosslinked hyaluronic acid for comparison 2

200 g of sodium hyaluronate was dissolved in 1440 g of 1% sodium hydroxide solution. After dissolution, 8.185 mL (8.9 mol%) of 1,4-butanediol diglycidyl ether (BDDE) was added, and the mixture was stirred at 25° C. for 2 hours. It was statically cross-linked at 25 °C for 24 hours. The obtained gel was put into a dialysis net and dialyzed against purified water and brine. The dialysate is prepared for injection after content correction, grinding and sterilization.

Experimental Example 1. Analysis of elasticity and viscosity according to concentration under reduced pressure

The elasticity and viscosity of Examples 1 to 3 and Comparative Example 1 were measured with an Anton Paar Modular Compact Rheometer 302 model. Shear rate 1.27 s ^-1 , gap size 1 mm, was measured under the conditions of 25 ℃, and the elastic modulus was measured under the conditions of Frequency 1 Hz, shear strain 1%, and 25 ℃. The results are shown in Table 1. As a result of the experiment, it was found that the elasticity increased as the concentration under reduced pressure proceeded. Examples 1 to 3 according to the present invention exhibited elasticity of 400 to 800 Pa, viscosity of 100 to 200 Pa·s, high elasticity, and high viscosity. On the other hand, in the case of Comparative Example 1, when the sodium hyaluronate content is about 90% after concentration under reduced pressure, in the injection preparation method performed by the present inventors, a phenomenon is found that the layers are separated due to no hydration, and it is impossible to measure the viscosity and elasticity did.

[Table 1]

Experimental Example 2. Comparison of physical properties with commercially available hyaluronic acid gel

Effective crosslinking rate (CrR) analysis, viscosity and elasticity analysis for many commercially available gels and Comparative Example 2 and Example 1 were performed. Analysis of effective crosslinking rate, viscosity and elasticity was described in [Kenne et al., Carbohydrate Polymers 91 (2013) 410-418], HPLC system was Waters Acquity UPLC I-Class, MS/MS system was Xevo TQ-S micro QQQ was used. Chondroitinase ABC enzyme was used as a degrading enzyme. The results are shown in Table 2.

As a result of the experiment, it was confirmed that the cross-linked hyaluronic acid prepared by the manufacturing method according to the present invention had a high effective cross-linking ratio (CrR) value of 0.34, and had high viscosity and high elasticity. It can be concluded that the manufacturing method according to the present invention provides a new crosslinked hyaluronic acid having a high effective crosslinking rate (CrR), high viscosity and high elasticity.

[Table 2]

(Unit: Pa for elasticity (G'), Pa s for viscosity (η))

Experimental Example 3. Stability test of viscosity and elasticity

The stability test of pharmaceuticals means confirming the stability of a certain quality over time under certain conditions in order to set the storage method and period of use of the drug. In other words, the shelf life of the product is set by setting the appropriate specifications and evaluating the conformance of the specifications by evaluating significant changes based on the established analytical method.

After concentration under reduced pressure by a method similar to Example 1, a stability sample having a sodium hyaluronate content of about 24% was prepared, and viscosity and elastic retention were confirmed in the same manner as in Experimental Example 1. The results are shown in Table 3, and the change rate graph is shown in FIGS. 1 and 2 . As a result of the experiment, it can be confirmed that the viscosity and elasticity are maintained up to 6 months. As a result of the experiment, it can be concluded that the hyaluronic acid crosslinked product prepared by the manufacturing method according to the present invention is stable.

[Table 3]

(Unit: Pa for elasticity (G'), Pa s for viscosity (η))

Experimental Example 4. Comparison of the amount of crosslinking agent-related residues

Residual BDDE measurement

* Preparation of test solution: Precisely weigh 200 mg of this drug, put it in a vial, add 20 mL of purified water using a whole pipette, and mix. Take a portion of this solution and use the filtered solution as the sample solution.

* Preparation of standard solution: Weigh about 10 mg of each standard of BDDE, BGPE, and BDPE, put it in a 10 mL volumetric flask, and label it with methanol. Take exactly 1 mL of this solution, put it in a 100 mL volumetric flask, and dilute with purified water. Take exactly 10 mL of this solution, put it in a 100 mL volumetric flask, and dilute it with purified water. Take exactly 2 mL of this solution, put it in a 100 mL volumetric flask, and use the solution labeled with purified water as the standard solution.

* LC-MS/MS test conditions

1. HPLC test conditions

- System : Waters Acquity UPLC I-Class

- Column: Waters Acquity UPLC BEH C18

- Column temperature: constant temperature around 30 ℃

- Mobile phase: ACN: 0.1 % Acetic acid = 20: 80

- Flow rate: 0.2 mL/min

- Injection volume: 5 μL

- Analysis time: 3 minutes

2. MS/MS test conditions

- System : Xevo TQ-S micro QQQ

- Source : ESI(+)

- Mode : MRM

- Capillary voltage : 3.0 kV

- Desolvation Temp. : 500℃

- Desolvation gas flow : 800 L/hr

- Cone gas flow : 150 L/hr

- Analyzer MS1 : LM Resolution1 15.0, HM Resolution1 : 15.0

- Analyzer MS2: LM Resolution2 13.0, HM Resolution2: 15.0,

Ion Energy: 0.5

Extended parameter

- Source Temp. : 150℃

- Lens controls

- Ion Guide 2 Offset : 0.5 V

- Collision Cell Lenses

- Entrance : 1.0, Exit : 1.0

- Detector Gain : 1.00

Residual BDDE (1,4-butandiol diglycidyl ether), BGPE (1,4-butanediol glycidyl propan-2,3-diolyl ether) and BDPE [1,4- butanediol di-(propan-2,3-diolyl)ether] was confirmed. The results are shown in Table 4.

[Table 4]

Experimental results In Example 1, residual BDDE (1,4-butandiol diglycidyl ether), BGPE (1,4-butanediol glycidyl propan-2,3-diolyl ether) and BDPE [1,4-butanediol di- (propan-2, 3-diolyl)ether] was confirmed to be regulated.

Experimental Example 5. Comparison of physical properties with commercially available hyaluronic acid gel for osteoarthritis

A commercially available hyaluronic acid gel for osteoarthritis (Sinobian) and Example 4 were analyzed for viscosity and elasticity, and enzyme degradation resistance (elasticity retention %) analysis was performed. Hyaluronidase was used as the degrading enzyme, and the enzyme was dissolved in distilled water at a concentration of 3U/μl and treated on a hyaluronic acid gel. The enzyme-treated hyaluronic acid gel was incubated under shaking conditions for 4 hours, and then measured using Anton Paar's Modular Compact Rheometer 302 model, and calculated as the ratio of elasticity before enzymatic treatment to decomposed elasticity. The results are shown in Table 5.

As a result of the experiment, the hyaluronic acid crosslinked product prepared by the manufacturing method according to the present invention had higher viscosity, elasticity, and enzymatic degradation resistance, as in Example 4, compared to commercial products having a similar content.

[Table 5]

(Unit: Pa for elasticity (G'), Pa s for viscosity (η))

Claims (22)

As a cross-linked hyaluronic acid cross-linked with an epoxy-based cross-linking agent,
an effective crosslinking ratio (CrR) of 0.3 or more,
A crosslinked hyaluronic acid, characterized in that when contained in 1.5 wt% or more in the composition, the viscosity is 90 Pa s or more and the elasticity is 400 Pa or more,
The epoxy-based crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE),
Residual 1,4-butanediol diglycidyl ether (BDDE) is 0.01 ppm or less,
Hyaluronic acid with residual BGPE (1,4-butanediol glycidyl propan-2,3-diolyl ether) less than 0.01 ppm and residual BDPE (1,4-butanediol di-(propan-2,3-diolyl) ether) less than 0.5 ppm ronic acid crosslinked product. delete delete The crosslinked hyaluronic acid according to claim 1, wherein the effective crosslinking ratio (CrR) is 0.3 or more and 0.8 or less. The crosslinked hyaluronic acid according to claim 1, which has both monophasic and biphasic properties. The crosslinked hyaluronic acid according to claim 1, wherein the viscosity is 90 or more and 300 Pa·s or less. The crosslinked hyaluronic acid according to claim 1, wherein the elasticity is 400 to 800 Pa. The crosslinked hyaluronic acid according to claim 1, wherein the viscosity change rate is less than 17% when stored for 6 months. The crosslinked hyaluronic acid according to claim 1, wherein the elastic change rate is less than 11% when stored for 6 months. delete delete (a) mixing hyaluronic acid or a salt thereof, an epoxy-based crosslinking agent, and an aqueous alkali solution;
(b) crosslinking the mixture obtained in step (a) after concentration under reduced pressure;
(c) washing and swelling the crosslinked product obtained in step (b);
(d) pulverizing and purifying the swollen material obtained in step (c); and
(e) swelling and dialysis of the purified product obtained in step (d);
A method for producing a cross-linked hyaluronic acid according to any one of claims 1 and 4 to 9, comprising:
The step of crosslinking in step (b) further comprises a step of standing still
A method for producing a crosslinked hyaluronic acid in which the concentration of hyaluronic acid is 10 to 30% after concentration under reduced pressure in step (b). 13. The method of claim 12,
The molecular weight of the hyaluronic acid or its salt in step (a) is 100,000 Da to 6,000,000 Da production method. 13. The method of claim 12,
In step (a), the aqueous alkali solution is a lithium hydroxide aqueous solution, sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, or a mixture thereof. 13. The method of claim 12,
Concentration under reduced pressure in step (b) is a manufacturing method that is carried out at 15 to 40 ℃. 13. The method of claim 12,
Concentration under reduced pressure in step (b) is a manufacturing method that is carried out at 30 to 40 ℃. 13. The method of claim 12,
In step (d), the purification is performed through an organic solvent that is acetone, ethanol, isopropyl alcohol, or a mixture thereof. 18. The method of claim 17,
A manufacturing method wherein the organic solvent is isopropyl alcohol. 13. The method of claim 12,
After step (e), (f) manufacturing method further comprising the step of making an injection from the dialysate. 10. A composition for preventing or treating osteoarthritis, comprising the cross-linked hyaluronic acid according to any one of claims 1 to 9. A filler injection composition comprising the cross-linked hyaluronic acid according to any one of claims 1 and 4 to 9. The composition for injectable filler according to claim 21, further comprising a local anesthetic.

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