KR102275105B1 - High Density Reticulated Cross-linked Hyaluronic Acid and Process for Preparing the Same - Google Patents

Abstract

The present invention provides hyaluronic acid cross-linked with a high-density network structure having excellent viscoelasticity due to a high cross-linking rate and optimized for biocompatibility and durability, and a method for preparing the same.

Description

High Density Reticulated Cross-linked Hyaluronic Acid and Process for Preparing the Same}

The present invention relates to hyaluronic acid cross-linked with a high-density network structure and a method for preparing the same, and more particularly, hyaluronic acid cross-linked with a high-density network structure having excellent viscoelasticity due to a high cross-linking rate and optimized for biocompatibility and durability; It relates to a manufacturing method thereof.

Hyaluronic acid is a substance naturally present in the body, and when transplanted into the body, it has the property of being decomposed within a few days by hyaluronidase in the body. Therefore, it is not suitable for pharmaceuticals and medical device products that need to be implanted in the body for a long period of time to exhibit their functions. Currently, research on sustained-release compositions and tissue repair biomaterials using cross-linked hyaluronic acid is actively underway. is becoming Among them, as a biomaterial for tissue repair, hyaluronic acid crosslinking technology has been developed by advanced countries such as Europe and the United States to apply biphasic and monophasic fillers.

This cross-linked hyaluronic acid gel is generally prepared by two steps, the first step is to water soluble, and the second step is to chemically bond between the hyaluronic acid chains by a cross-linking agent.

However, the gel prepared by the conventional cross-linking method cannot be directly injected into the human body. This may be caused by a high concentration of hyaluronic acid, an inappropriate pH in the living body, and a residual crosslinking agent, and even if it is properly adjusted, there is a problem in that the in vivo durability is lowered.

In order to overcome this problem, Korean Patent Registration No. 10-1239037 discloses a method for preparing a crosslinked hyaluronic acid gel having a low crosslinking rate and exhibiting excellent viscoelasticity.

Republic of Korea Patent No. 10-1239037

The purpose of the present invention is to maximize the viscosity and cohesiveness by crosslinking hyaluronic acid with a high-density network structure in addition to the existing cross-linked hyaluronic acid manufacturing technology to increase in vivo durability and optimize biocompatibility for direct injection into the body. To provide a method for preparing a gel.

Another object of the present invention is to provide a cross-linked hyaluronic acid with a high-density network structure prepared by the above preparation method.

Another object of the present invention is to provide a wrinkle-removing filler comprising hyaluronic acid cross-linked in the high-density network structure.

One embodiment of the present invention relates to a method for producing hyaluronic acid crosslinked in a high-density network structure, the production method of the present invention comprises:

(i) dissolving the polymer hyaluronic acid in an aqueous alkali solution to linearize;

(ii) crosslinking the linearized hyaluronic acid with a crosslinking agent to obtain a hydrogel;

(iii) cutting the hydrogel;

(iv) swelling the cut hydrogel using a buffer of pH 6.8 or less; and

(v) grinding the swollen hydrogel.

In one embodiment of the present invention, in the step (i), the polymer hyaluronic acid is dissolved in an aqueous alkali solution to form a fully homogenized long chain.

The form and molecular weight of the hyaluronic acid used in the present invention are not particularly limited.

In one embodiment of the present invention, the form of hyaluronic acid may be a physiologically acceptable salt of hyaluronic acid. For example, hyaluronic acid metal salts such as sodium salt, potassium salt, or a mixture thereof may be used, but is not limited thereto. Preferably, a sodium salt may be used.

In addition, the molecular weight of hyaluronic acid may be 500,000 to 3 million.

In addition, hyaluronic acid may have an intrinsic viscosity of 2.97 to 4.5 m ³ /kg.

The hyaluronic acid may be used so that the hyaluronic acid concentration in the aqueous alkali solution is 10 w/w% or more, preferably 10 to 15 w/w%.

The reaction temperature may be 20 to 40 °C, preferably 20 to 35 °C, and the reaction time may be 8 to 20 hours, preferably 8 to 15 hours.

The physical properties of the resulting linearized aqueous hyaluronic acid solution may be a fluid sol having a complex viscosity (η*) of 10 to 50 Pa.s at a frequency of 1 Hz.

In one embodiment of the present invention, in step (ii), the linearized hyaluronic acid is cross-linked with a cross-linking agent to obtain a hydrogel.

The crosslinking agent is for crosslinking the linearized hyaluronic acid into a high-density network structure, and a compound having a bifunctional epoxy group capable of forming a covalent bond by reacting with a carboxyl group, a hydroxyl group or an acetamide group that is a reactive group of the hyaluronic acid molecule may be used. . Specific examples include alkylene diepoxide such as 1,3-butadiene diepoxide and 1,4-hexadiene diepoxide, diglycidyl such as ethylene glycol diglycidyl ether and 1,4-butanediol diglycidyl ether. ether, divinyl sulfone, and the like, and these may be used alone or in combination of two or more. Preferably, 1,4-butanediol diglycidyl ether may be used.

The crosslinking agent may be added in an amount of 0.5 w/w% or more, preferably 0.5 to 1.0 w/w% in the aqueous solution. In addition, the crosslinking agent is added in an amount of 10 w/w% or more, preferably 10 to 15 w/w% with respect to the hyaluronic acid unit, so that a hydrogel can be formed with a high crosslinking rate of at least one for 10 hyaluronic acid units. have.

The reaction temperature may be 30 ° C. or higher, preferably 35 ° C. to 50 ° C., and the reaction time is 2 hours or more, and the reaction is continued for 2 to 7 hours to provide a hydrogel of a high-density network structure.

In addition, the solvent to be diluted before adding the crosslinking agent may be water-soluble ethanol, methanol, acetone, purified water, or the like.

In one embodiment of the present invention, in step (iii), the hydrogel formed in a high-density network structure is cut to a predetermined size.

At this time, the size of the hydrogel may be 1mm*1mm to 20mm*20mm, preferably 1mm*1mm to 10mm*10mm.

In one embodiment of the present invention, in step (iv), the cut hydrogel is swollen using a buffer of pH 6.8 or less to remove the residual cross-linking agent and to provide a hydrogel having excellent biocompatibility.

A phosphate buffer may be used as the buffer, and the pH of the phosphate buffer may be 5.0 to 6.8, preferably 6.0 to 6.8, and the concentration may be 1 to 1,000 mM.

In one embodiment of the present invention, in step (v), the swollen hydrogel is pulverized to make it homogeneous and uniform in size so that it can be directly injected into the body.

Grinding may be performed using an extruder, a colloid mill, a homogenizer, a mesh, and the like.

The average particles of the pulverized hydrogel have a size range of 5 to 1,000 μm, preferably 50 to 500 μm.

The manufacturing method according to an embodiment of the present invention may further include, after step (v), (vi) homogenizing by mixing an analgesic agent or collagen-forming material for the purpose of increasing functionality.

As the analgesic agent, lidocaine, tetracaine, bupivacaine, mepivacaine, etc. may be used, and as the collagen-forming material, peptides that cause collagen types I, II, and III to be formed may be used. In addition, a component that suppresses wrinkles by controlling the formation of a snare complex may also be used.

The manufacturing method according to an embodiment of the present invention may further include sterilizing the homogenized hydrogel after step (v) or step (vi).

At this time, sterilization can be performed by filling a syringe with a homogenized hydrogel, and irradiating radiation, steam, and hydrothermal treatment.

The hyaluronic acid hydrogel prepared by the manufacturing method according to the present invention forms a high-density network structure with a high crosslinking rate of at least 10 w/w% with respect to the hyaluronic acid unit, and a shear rate of 0.05% to 100 Pa.s Above, preferably, it has a very excellent viscosity of 1,000 Pa.s or more, and exhibits elasticity and high viscoelasticity of 100 Pa or more, with a storage modulus (G') at a frequency of 1 Hz.

In addition, the hyaluronic acid hydrogel prepared by the manufacturing method according to the present invention is a single-phase gel, and in particular, the cohesive force is very excellent, and the fluidity is low in the absence of artificial stress, and the enzyme resistance and The durability is far superior to conventional products.

Accordingly, one embodiment of the present invention relates to hyaluronic acid cross-linked in a high-density network structure prepared by the above production method.

The hyaluronic acid crosslinked with a high-density network structure according to the present invention has excellent biocompatibility and can maintain a gel structure without being decomposed for a long time in the body, so a wide range of pharmaceuticals and medical devices such as biomaterials for tissue repair, arthritis treatment, ophthalmic preparations, etc. In particular, it can be used as a filler for removing wrinkles and as a treatment for osteoarthritis.

One embodiment of the present invention relates to a wrinkle removal filler comprising hyaluronic acid cross-linked in the high-density network structure.

The hyaluronic acid crosslinked with a high-density network structure according to the present invention maximizes viscosity and cohesiveness by crosslinking hyaluronic acid with a high-density network structure, thereby increasing in vivo durability, excellent biocompatibility, low injection pressure, and wrinkle removal filler and It can be effectively used in the treatment of osteoarthritis, etc.

1 is a staining photograph showing the resolution test results of hyaluronic acid cross-linked into a high-density network structure according to the present invention in an animal model.

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are for illustrative purposes only, and it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1: Preparation of cross-linked hyaluronic acid with a high-density network structure

10 g of high molecular weight hyaluronic acid (intrinsic viscosity: 3.3 m ³ /kg) was dissolved in 100 ml of 1.0% NaOH at 20 ° C. for 8 hours to linearize hyaluronic acid, and then BDDE as a crosslinking agent was added at 10 Mol% to hyaluronic acid, 45 The reaction was carried out at ℃ for 4 hours. After cutting the resulting gel to a size of 2 mm * 2 mm, swelling (swelling) in 50 mM phosphate buffer of pH 6.8 until the pH is the same, using a homogenizer (Ika), a speed of 5,000 rpm / min After grinding with a furnace for 5 minutes, sterilization was performed in a sterilizer to prepare hyaluronic acid crosslinked in a high-density network structure.

Example 2: Preparation of hyaluronic acid cross-linked into a high-density network structure

10 g of high molecular weight hyaluronic acid (intrinsic viscosity: 3.3 m ³ /kg) was dissolved in 100 ml of 1.0% NaOH at 20° C. for 8 hours to linearize hyaluronic acid, and then BDDE as a crosslinking agent was added at 12 Mol% of hyaluronic acid, 45 The reaction was carried out at ℃ for 4 hours. After cutting the resulting gel to a size of 2 mm * 2 mm, swelling in 50 mM phosphate buffer at pH 6.8 until the pH becomes the same, and then using an extruder 3 times until the particle size distribution becomes uniform After the above grinding, sterilization was performed in a hot water sterilizer to prepare hyaluronic acid cross-linked in a high-density network structure.

Example 3: Preparation of hyaluronic acid crosslinked with lidocaine-containing high-density network structure

10 g of high molecular weight hyaluronic acid (intrinsic viscosity: 3.3 m ³ /kg) was dissolved in 100 ml of 1.0% NaOH at 20° C. for 8 hours to linearize hyaluronic acid, and then BDDE as a crosslinking agent was added at 12 Mol% of hyaluronic acid, 45 The reaction was carried out at ℃ for 4 hours. After cutting the resulting gel to a size of 2 mm * 2 mm, swelling in 50 mM phosphate buffer at pH 6.8 until the pH becomes the same, and then using an extruder 3 times until the particle size distribution becomes uniform over crushed. Then, 1.3 g of lidocaine hydrochloride was added to the pulverized gel, and the mixture was mixed in a power mixer for 30 minutes to ensure homogeneity. The mixed sample was sterilized in a hot water sterilizer to prepare hyaluronic acid crosslinked with a high-density network structure containing an analgesic agent.

Example 4: Preparation of hyaluronic acid cross-linked with a high-density network containing peptides

10 g of high molecular weight hyaluronic acid (intrinsic viscosity: 3.3 m ³ /kg) was dissolved in 100 ml of 1.0% NaOH at 20° C. for 8 hours to linearize hyaluronic acid, and then BDDE as a crosslinking agent was added at 12 Mol% of hyaluronic acid, 45 The reaction was carried out at ℃ for 4 hours. After cutting the resulting gel to a size of 2 mm * 2 mm, swelling in 50 mM phosphate buffer at pH 6.8 until the pH becomes the same, and then using an extruder 3 times until the particle size distribution becomes uniform over crushed. Then, 0.434 g of palmitoyl oligo peptide hydrochloride was added to the pulverized gel, and the mixture was mixed in a power mixer for 30 minutes to ensure homogeneity. The mixed sample was sterilized in a hot water sterilizer to prepare hyaluronic acid crosslinked with a high-density network structure containing an analgesic agent.

Comparative Example 1: Preparation of cross-linked hyaluronic acid

10 g of high molecular weight hyaluronic acid (intrinsic viscosity: 3.3 m ³ /kg) was dissolved in 100 ml of 1.5% NaOH at 29 ° C. for 8 hours to linearize hyaluronic acid, and then BDDE as a crosslinking agent was added at 12 Mol% of hyaluronic acid and 40 The reaction was carried out at ℃ for 5 hours. The resulting gel was pulverized at a speed of 5,000 rpm/min for 5 minutes using a homogenizer (Ika), and then sterilized in a hot water sterilizer to prepare cross-linked hyaluronic acid.

Experimental Example 1: Resolution test

When the cross-linked hyaluronic acid (HA) is injected into the body, the cross-linked hyaluronic acid is subjected to a direct decomposition attack by hyaluronidase to be decomposed. By directly injecting hyaluronidase into the sample, it is possible to check the degradation tendency and measure the resistance to the enzyme.

Using the samples prepared in Example 2 and Comparative Example 1 as specimens, a resolution test was performed with the following materials and methods.

solution composition

- Buffer: 4.05 g of sodium chloride, 0.72 g of monohydrogen phosphate, and 0.31 g of dihydrogen phosphate were weighed, respectively, and dissolved in distilled water.

- HADase Stock Solution: 125 mg of hyaluronidase (Sigma Aldrich, derived from Bovine, H1136) was placed in 10 mL of a volume flask and mass-up with a buffer solution.

- HADase 2000 Units/mL: 1 mL of HADase stock solution was taken, diluted with 4 mL of buffer, and filtered before use.

Experimental method

About 0.5 g of the sample was taken in a glass syringe and centrifuged at 3000 rpm for 5 minutes to collect the liquid down and remove air bubbles. After centrifugation, a rubber stopper was inserted, and the front part of the syringe was separated. 200 μl of HADase 200Units/mL was slowly introduced with air, and then the front part was recombined and sampled three at a time while reacting in an incubator at 37 ° C. Calculate the degradation rate by the following formula, and the result (average value of three times) is shown in Table 1 below.

formula

Decomposition rate =(AB)/C*100

A: Amount of decomposed liquid (including enzyme and decomposed HA)

B: Amount of enzyme added

C: Amount of cross-linked HA initially added

time Decomposition rate (%) Comparative Example 1 Example 2 2 10.60% 2.58% 4 19.23% 4.05% 9 63.24% 5.22% 12 82.38% 6.64% 24 95.14% 39.43% 36 100.00% 54.89% 48 76.52% 60 91.49% 72 100.0%

As shown in Table 1, the cross-linked hyaluronic acid of Comparative Example 1 was completely decomposed by the enzyme within 36 hours, whereas the hyaluronic acid cross-linked with the high-density network structure of Example 2 was completely decomposed after 72 hours. Therefore, it was confirmed that the hyaluronic acid crosslinked with a high-density network structure according to the present invention is a gel with excellent biocompatibility, which has enzyme resistance and can be completely decomposed in the body after a certain period of time.

Experimental Example 2: Complex Viscosity and Stability Test

The samples prepared in Examples 1 to 4 and Comparative Example 1 were stored for 6 months under accelerated conditions (45° C.±2° C., 75% RH±5% RH) at 1 Hz using a Rheometer (Kinexus Pro, Malvern). The complex viscosity was measured, and the results are shown in Table 2 below (unit: Pa.s).

0 months 3 months 6 months Example 1 48 45 42 Example 2 52 47 44 Example 3 52 47 44 Example 4 73 65 60 Comparative Example 1 31 20 15

As shown in Table 2 above, the hyaluronic acid cross-linked with a high-density network structure of Examples 1 to 4 according to the present invention has a higher complex viscosity than the cross-linked hyaluronic acid of Comparative Example 1, and stability carried out for 6 months under accelerated conditions It showed very good stability in the test.

Experimental Example 3: Scanning pressure measurement

The sample prepared in Example 2 was filled in a syringe, a needle was inserted into the syringe, and then extruded at a speed of 5 mm/min. From the data obtained using a tensile strength meter (JSV-1000, Jisco), the load on the sample ( After the load) was applied, the 5mm point was set as 1 point, and the point 5 mm before the point at the point where all samples were pulled out and the load rapidly increased, and the average value of the two points was calculated.

As a result, it was confirmed that the average injection pressure was 12N, which was significantly lower than about 32-35N measured using a commercially available product.

Experimental Example 4: Measurement of crosslinking rate

When HA is degraded by Streptomyces Hyaluronidase, in the case of linear HA, tetramers (2 HA molecules) and hexamers (3 HA molecules) are finally left, and cross-linking In the case of cross-linked HA, focusing on leaving more HA chains, check the RT of the peak by molecular weight by anion exchange HPLC, and the area of each peak The crosslinking rate was calculated through the value ratio. The peak at RT between each mer was viewed as a pendant link.

reaction conditions

- Enzyme solution: 100 U/mL Hyaluronate lyase from Streptomyces (Hyaluronate lyase from Streptomyces )

- Sample solution: 0.4% crosslinked HA solution in 100 mM NaOAc (pH 5.0)

After adding 40 μL of the enzyme solution to the 50 μL sample solution, 40 μL of the enzyme solution was added every 24 hours during incubation at 50° C. for 3 days (total 96 hours of reaction).

※ Incomplete digest: A standard for checking the RT of each mer. Add 100 μL of enzyme solution to 1 mL of 0.4% solution of low molecular weight linear HA, incubate at 50 ° C for 3 hours, add 100 μL of enzyme solution, incubate for 90 minutes, and terminate the reaction at 95 ° C for 5 minutes.

HPLC analysis conditions

1) mobile phase

A: distilled water

B: 0.4M sodium phosphate buffer (pH 5.8)

2) Injection volume: 10 μL

3) Gradient

hours (minutes) A B 0 100 0 5 90 10 55 20 80 57 100 0 60 100 0

As a result of measuring the crosslinking rate of the hyaluronic acid crosslinked with the high-density network structure of Example 2 as described above, the average crosslinking rate was 11.0%.

Experimental example 5: Resolution test ( in in vivo )

A 6-week-old male hairless mouse (hairless mouse, Skh:HR-a, central laboratory animal) was purchased, and after obtaining the animal, it was subjected to quarantine and a one-week acclimatization period. Before the test, group separation was performed into 3 groups of 10 animals each. The breeding environment of the experimental animals was maintained at temperature (23±2℃), humidity (55±10%), and a 12-hour light/dark cycle (light/dark cycle, lighted at 8am – lights out at 8pm). Solid feed (5L79; LabDiet, USA) and tap water that passed through a water purification device were freely fed. All experimental animals were handled according to the Guide for the Care and Use of Laboratory Animal (1996, USA) of the Institute of Laboratory Animal Resources.

Physiological saline, Example 2 and Example 3 administration groups were divided into 3 groups, each of 10 animals were tested. 20 μl of the sample was administered to the middle part of the back of the prepared hairless mouse subcutaneously using a syringe and observed. Before measurement, anesthesia was performed using Zoletil 50 (Virbac, France), and the administration site was measured for volume change using PRIMOS at 0, 2, and 6 months, respectively. At the last 6 months, mice were sacrificed and stained. (masson's trichrome) to observe the residual amount of hyaluronic acid and the degree of collagen formation. The results are shown in FIG. 1 .

As shown in FIG. 1, the hyaluronic acid of Examples 2 and 3 according to the present invention was maintained for 6 months in mice, and the amount of collagen formation was high, and there were no inflammatory reactions and side effects histopathologically.

Claims (13)

(i) dissolving the polymer hyaluronic acid in an aqueous alkali solution to linearize;
(ii) adding a crosslinking agent to the linearized aqueous solution of hyaluronic acid in an amount of 10 to 15 w/w% based on the hyaluronic acid unit and cross-linking to obtain a hydrogel;
(iii) cutting the hydrogel to a size of 1mm * 1mm to 20mm * 20mm;
(iv) swelling the cut hydrogel using a buffer of pH 6.8 or less; and
(v) a method for producing hyaluronic acid cross-linked into a high-density network comprising the step of pulverizing the swollen hydrogel. The method according to claim 1, wherein the intrinsic viscosity of the polymer hyaluronic acid in step (i) is 2.97 to 4.5 m ³ /kg. The method according to claim 1, wherein the polymer hyaluronic acid in step (i) has a concentration of 10 to 15 w/w% in an aqueous alkali solution. The method according to claim 1, wherein the crosslinking agent in step (ii) is a compound having a difunctional epoxy group. The method according to claim 1, wherein the crosslinking agent in step (ii) is 1,4-butanediol diglycidyl ether. delete delete The method according to claim 1, wherein the buffer in step (iv) is a phosphate buffer having a pH of 6.0 to 6.8. The method according to claim 1, further comprising, after step (v), (vi) homogenizing by mixing a soothing agent or a collagen-forming material. The method according to claim 1, further comprising sterilizing the homogenized hydrogel after step (v). 10. The method of claim 9, further comprising sterilizing the homogenized hydrogel after step (vi). The hyaluronic acid crosslinked into a high-density network structure prepared by the method according to any one of claims 1 to 5 and 8 to 11. A filler for removing wrinkles comprising hyaluronic acid cross-linked in a high-density network structure according to claim 12.

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