WO2017213404A1 - Hyaluronic acid crosslinked product having both monophasic and biphasic characteristics, preparation method therefor, and use thereof - Google Patents

Abstract

The present invention provides: a hyaluronic acid crosslinked product having both monophasic and biphasic characteristics; and, as a preparation method therefor, a method for preparing a granulated monophasic hyaluronic acid crosslinked product. The hyaluronic acid crosslinked product is characterized in that the ratio of viscosity and modulus of elasticity is 4:1 to 2:1. The hyaluronic acid cross-linked product of the present invention may be used as an injection in tissue.

Description

Hyaluronic acid crosslinked product having the characteristics of single phase and isomer phase at the same time, preparation method thereof and use thereof

The present invention relates to a hyaluronic acid crosslinked product.

Hyaluronic acid is a negatively charged polysaccharide present in connective tissues, subcutaneous tissues, nerve tissues or joint synovial fluid in vivo, and is extracted from microbial fermentation such as Streptococcus or chicken bran, and is used as fillers, joint cavity injections, anti-adhesion agents, ophthalmic drugs or It has been used as a raw material for cosmetics. However, as a problem has been reported that hyaluronic acid itself can be rapidly released from the body by decomposition by enzymes and free radicals, attempts to chemically crosslink to prolong the decomposition period in the body have begun.

In other words, in order to increase the chemical structural stability of hyaluronic acid to maintain the physical property retention period in vivo, a technique for stabilizing hyaluronic acid by adding a crosslinking agent has been developed, among which existing hyaluronic acid crosslinked body and hyaluronic acid derivative crosslinking The maximum in vivo retention time of water is reported to be about 6 to 24 months.

However, the high viscoelastic hyaluronic acid crosslinked body obtained by simply increasing the amount of crosslinking agent to increase the retention period of hyaluronic acid in vivo may cause an inflammatory reaction as the crosslinking agent component decomposed in vivo is recognized as a foreign substance. There is a need.

In summary, when the amount of the crosslinking agent is decreased in the preparation of the hyaluronic acid crosslinked product, the obtained material is exposed to the problem of low viscoelasticity and decomposition in a short time in the living body. In addition, although it may not be degraded for a long time in vivo, there is a problem that an inflammatory reaction is caused, and a hyaluronic acid crosslinkable that can maintain a proper balance is required.

In this regard, Korean Patent Publication No. 10-1239037 discloses hyaluronic acid in the above mixture which is used for crosslinking reaction in obtaining a hyaluronic acid crosslinked product by mixing hyaluronic acid, a crosslinking agent and water, and stirring and mixing under acidic or alkaline conditions. Adopt a high concentration of silver of at least 10 w / v% and a molecular weight of 100,000 or more, the concentration of the crosslinking agent in the mixture is a low concentration of 0.05 to 0.5 w / w%, the storage modulus of the mixture (G ', frequency 1 Hz) When it is 15,000 Pa or more, it has been suggested that hyaluronic acid chains can be partially crosslinked to exhibit excellent viscoelasticity even though a low crosslinking rate is constituted.

Subsequently, Korean Patent Publication No. 10-2015-0029578 discloses dissolving a high concentration of at least 10 w / w% in an aqueous alkali solution to form a long chain, followed by 0.5 to 1.0 w / w% of the linearized hyaluronic acid. After adding a small amount of crosslinking agent to crosslink the linearized hyaluronic acid into a high density network, the hydrogel formed of the high density network was cut into a size of 1mm * 1mm to 20mm * 20mm, and the cut hydrogel was buffered at a pH of 6.8 or less. When the residual crosslinking agent is swelled at swelling, it is possible to obtain a single-phase hyaluronic acid crosslinked product having a viscosity of 1,000 Pa · s or more and an elasticity of storage modulus (G ′, frequency 1 Hz) of 100 Pa or more. Post it.

Techniques for preparing a hyaluronic acid crosslinked body in the art are largely a method for producing a single phase using a certain amount of crosslinking agent and a method for producing an isomeric phase by mixing a certain amount of crosslinked hyaluronic acid and uncrosslinked hyaluronic acid. Here, the method for preparing the isophase includes mixing a low crosslinking hyaluronic acid crosslinked product in place of the uncrosslinked hyaluronic acid.

As another method for preparing a hyaluronic acid crosslinked product, a technique of dissolving and crosslinking hyaluronic acid having different molecular weights at the same time has been mentioned. However, this may allow the hyaluronic acid to maintain its in vivo physical properties only for a long time. However, there is a limit that does not have the characteristics of the crosslinked hyaluronic acid cross-linked single phase and isomer phase.

Briefly look at the single phase and isomer phase of the hyaluronic acid crosslinked product. The single-phase hyaluronic acid crosslinked body and the isomeric hyaluronic acid crosslinked body, such as the aforementioned Korean Patent Publication No. 10-1239037 or Korean Patent Application Publication No. 10-2015-0029578, show different characteristics.

For example, the single phase hyaluronic acid crosslinked material has high viscosity and low elasticity. On the contrary, the hyaluronic acid crosslinked product of the isotropic phase is low in viscosity and high in elasticity. The reason why the single phase and isomeric hyaluronic acid crosslinked body is different from the above is because the single phase hyaluronic acid crosslinked body is formed into a highly viscous homogeneous gel, whereas the isotropic hyaluronic acid crosslinked body is formed in the form of particles. .

Since the isomeric hyaluronic acid crosslinked product is manufactured in the form of particles as described above, the injection power is high when injected through the syringe needle, and it is known to use uncrosslinked hyaluronic acid as a lubricant to lower the injection power again.

Due to the different properties of the single-phase hyaluronic acid crosslinked body and the isomeric hyaluronic acid crosslinked body, the conventional hyaluronic acid crosslinked body may have other properties in the body. For example, the low-elasticity and high viscosity single phase hyaluronic acid crosslinked body is unlikely to escape from the injected site, but has a disadvantage in that it cannot maintain the injected form. On the contrary, the high elasticity but low viscosity isotropic hyaluronic acid crosslinked product In the case of having the advantage of maintaining the injected form for a long time, but shows a disadvantage that can be easily separated from the injected site.

The inventors of the present invention provide a hyaluronic acid crosslinked product having a form in which elasticity and viscosity have an intermediate characteristic between a single phase and an isomeric hyaluronic acid crosslinked product and a method for preparing the same, thereby maintaining the injected form for a long time, We tried to implement a property that is less likely to escape.

That is, an object of the present invention is to provide a hyaluronic acid crosslinked product having the characteristics of a single phase and an isomer phase at the same time. Another object of the present invention is to provide a method for producing a hyaluronic acid crosslinked product having both single phase and isomeric phase properties, and to provide a use of a hyaluronic acid crosslinked product having both single phase and isotropic phase properties.

The present invention has solved the above problems through the following means.

(1) A granulated single phase hyaluronic acid crosslinked product, wherein the ratio of the viscosity (unit Pa · s) and the elastic modulus (unit Pa) of the hyaluronic acid crosslinked product is 4: 1 to 2: 1 Crosslinked material.

(2) The hyaluronic acid crosslinked product according to (1), wherein the hyaluronic acid crosslinked product has a viscosity of 750 Pa · s or less and an elastic modulus of 325 Pa or less.

(3) The hyaluronic acid crosslinked product according to (1) or (2), wherein the average surface particle size of the hyaluronic acid crosslinked product is 100 to 700 micrometers.

(4) The hyaluronic acid crosslinked product according to any one of (1) to (3), wherein the final pH range of the hyaluronic acid crosslinked product is 7.2 to 7.4.

(5) The hyaluronic acid crosslinked product according to any one of (1) to (4), wherein the final concentration of the hyaluronic acid crosslinked product is 18 to 24 mg / ml.

(6) The hyaluronic acid crosslinked product according to any one of (1) to (5), wherein the hyaluronic acid crosslinked product is a BDDE crosslinked hyaluronic acid crosslinked product.

(7) crosslinking by stirring and mixing a mixture including hyaluronic acid, a crosslinking agent and purified water under acidic or alkaline conditions; A method for producing a granulated single phase hyaluronic acid crosslinked product by granulating the crosslinked product obtained in step (A).

(8) The production method according to (7), wherein the obtained hyaluronic acid crosslinked product is granulated so as to have an average surface particle size of 100 to 700 micrometers.

(9) A composition comprising the hyaluronic acid crosslinked product according to any one of (1) to (6) and a local anesthetic.

(10) The composition according to the above (10), wherein the composition is for filler injection.

(11) The composition according to (9) or (10), wherein the local anesthetic is lidocaine or a salt thereof.

The present invention provides a hyaluronic acid crosslinked product having the characteristics of a single phase and an isomer phase at the same time, and providing a method and a use thereof. Here, the characteristics of a single phase and an isomer phase simultaneously have the characteristics of a single phase in that the first product is made of only crosslinked hyaluronic acid, and secondly, unlike a single phase hyaluronic acid crosslinked product, the elastic force is different from that of the isotropic hyaluronic acid crosslinked product. Say something good enough to match.

Specifically, the hyaluronic acid crosslinked product according to the present invention has excellent viscosity compared to the conventional isotropic hyaluronic acid crosslinked body, and its elasticity is superior to the conventional single phase hyaluronic acid crosslinked body. In general, when used as a filler, a single phase hyaluronic acid crosslinked material having a low elasticity and a high viscosity is less likely to escape from the injected site, but has a disadvantage in that it cannot maintain the injected form. On the contrary, a high elasticity but low viscosity isomeric hyaluronic acid It is well known that the crosslinked body can maintain the injected form for a long time but can be easily released from the injected site. However, when the hyaluronic acid crosslinked product of the present invention is used as a filler, it is possible to maintain the injection form for a long time while minimizing departure from the injection site. This can be confirmed that the embodiment according to the present invention is left more than 6 months without being separated from the injection site in Figure 2, and that the injection shape in Figure 1 was well maintained and the wrinkle improvement effect was well maintained for 6 months. .

In addition, the present invention exhibits the same wrinkle improvement effect compared to the conventional single phase or isomer phase hyaluronic acid crosslinked when applied as a filler, it shows a markedly improved effect in terms of residual amount in the body.

In addition, the hyaluronic acid crosslinked product according to the present invention has a low content of a crosslinking agent, and thus there is little concern that inflammation may be caused by residual crosslinker components even when injected in vivo.

1 is a graph showing the wrinkle improvement effect of the hyaluronic acid crosslinked product.

2 is a graph showing the decomposition rate of the hyaluronic acid crosslinked body in the body.

3 is a graph showing the particle size of the hyaluronic acid crosslinked product.

4 is an optical micrograph showing the particle form of the hyaluronic acid crosslinked product.

The physical properties of hyaluronic acid in gel form have a significant impact on the effectiveness and duration of the filler, for example when applied as a filler. The physical properties of cross-linked hyaluronic acid can be measured by rheological methods, and the viscoelastic properties associated with the hardness and softness or flowability of the gel form a close relationship with the performance of the filler.

It has been understood that the viscosity and elasticity of the crosslinked hyaluronic acid may be affected by the concentration of the hyaluronic acid, the ratio of the crosslinked portion and the uncrosslinked portion, the degree of crosslinking, the molecular weight of the hyaluronic acid, or the manufacturing method. In view of the above-described conventional invention, Korean Patent Publication No. 10-1239037 may limit the concentration of hyaluronic acid, and Korean Patent Publication No. 10-2015-0029578 may limit the manufacturing method.

However, the present inventors have recognized that it is difficult to develop a hyaluronic acid crosslinked product having both viscosity and elasticity without breaking the conventional recognition, and surprisingly, it is possible to overcome the limitations of the prior art by controlling the final particle form of the crosslinked product. It was found through experiments, and came to complete the present invention. Examine the development of the present invention in more detail.

As described above, the hyaluronic acid crosslinked product has a single phase and an isomer phase. Single-phase hyaluronic acid cross-linked product is prepared using a homogeneous solution composed of cross-linked hyaluronic acid, and isophase hyaluronic acid cross-linked product is prepared by mixing two different phases of cross-linked hyaluronic acid particles and uncrosslinked hyaluronic acid. do.

The biggest difference between single phase hyaluronic acid crosslinked and isomeric hyaluronic acid crosslinked is in the manufacturing process. Since single-phase hyaluronic acid crosslinked products are prepared using a phase close to a homogeneous liquid phase rather than in the form of particles, elasticity is low and viscosity is high. In contrast, the isotropic filler is prepared by mixing a crosslinked hyaluronic acid particle body with a non-crosslinked hyaluronic acid close to a liquid phase, and generally has low viscosity and high elasticity.

Until now, the difficulties such as retention period of hyaluronic acid have been satisfactorily improved by means of crosslinking. However, the hyaluronic acid crosslinked material having low elasticity and high viscosity, or low viscosity and high elasticity as described above still exposed other problems when used as a filler. Specifically, according to the experiments of the present inventors, the hyaluronic acid crosslinked product in which the ratio of the viscosity and the elastic modulus does not satisfy 4: 1 to 2: 1 is easy to be separated from the injected site when used as a filler, even if it is not released. Insufficient to maintain the form for a long time.

Accordingly, the present inventors focused on the development of hyaluronic acid crosslinked material having excellent elasticity and viscosity, and focused on single-crosslinked hyaluronic acid crosslinked material in the form of particles to solve the present difficulty.

First, the inventors hypothesized that the conventionally developed single crosslinked hyaluronic acid crosslinked body, that is, single phase hyaluronic acid crosslinked body, would have a low elastic modulus because it was not in the form of particles. In other words, the present inventors have conducted numerous repeated experiments assuming that the reason why the isotropic hyaluronic acid filler has a high modulus of elasticity has been determined by the final form of the hyaluronic acid crosslinked product. As a result, it has been found that when the single-phase hyaluronic acid crosslinked product is prepared in the form of particles such as an isomeric hyaluronic acid crosslinked product, its elastic modulus can be increased.

Clearly, in the meantime, the viscosity and modulus of elasticity of hyaluronic acid crosslinked products have been commonly recognized as having inversely proportional properties. However, the present inventors have previously attempted to change the properties of the hyaluronic acid crosslinked body, the concentration of hyaluronic acid, the ratio of the crosslinked and uncrosslinked portions, the degree of crosslinking, the molecular weight of the hyaluronic acid, or the modification of the manufacturing method. is not; By controlling the final physical shape of the single-phase hyaluronic acid crosslinked product, the elastic modulus and viscosity of the hyaluronic acid crosslinked body were satisfactorily derived for the first time. Specifically, a hyaluronic acid crosslinked product of a novel substance having a ratio of viscosity and elastic modulus of 4: 1 to 2: 1 was obtained.

The present invention is characterized by granulating a single-phase hyaluronic acid crosslinked product. At this time, the average surface particle size is preferably 100 to 700 micrometers for the desired effect of the present invention. The average surface particle size may be measured by wet particle size measurement using a HELOS-OASIS device of Sympatec GmbH after diluting 0.5 g of the prepared cross-linked hyaluronic acid gel evenly in 10 g of distilled water.

On the other hand, as another property of the hyaluronic acid crosslinked body, it is well known in the art that the overall elastic modulus of the hyaluronic acid crosslinked body is determined by the amount of the crosslinking agent used in the crosslinking reaction or the concentration of the solution-like hyaluronic acid crosslinked body. Therefore, based on this, the hyaluronic acid crosslinked product of the present invention exhibiting a ratio of viscosity and elastic modulus of 4: 1 to 2: 1 may have a single phase hyaluronic acid crosslinked product depending on the amount of the crosslinking agent or the concentration of the hyaluronic acid crosslinked solution in solution. It can be prepared to have a range of elastic modulus values and viscosity values simultaneously having the properties of the isomeric hyaluronic acid crosslinked product.

In order to implement the present invention, the present inventors prepared a new hyaluronic acid crosslinked product having a different concentration by granulating it and diluting it in an aqueous solution while fixing the crosslinking rate of the hyaluronic acid crosslinked product. The characteristic constitution that can reproduce various viscoelastic ranges having the characteristics of the crosslinked body was found.

The hyaluronic acid crosslinked product having the characteristics of a single phase and an isomer phase at the same time includes a purification process and a sterilization process, and is nontoxic when administered in vivo. Therefore, the hyaluronic acid crosslinked product of the present invention can be injected into a tissue and can be used as a filler for face molding as a representative use purpose.

Further consideration is given to the characteristic constructions of the invention.

The inventors of the present invention found that when the ratio of the viscosity (unit: Pa.s) and the modulus of elasticity (unit: Pa) of the hyaluronic acid crosslinked product is in the range of 4: 1 to 2: 1, the single-phase hyaluronic acid crosslinked body and the isomeric hyaluronic acid crosslinked product. It was confirmed that the characteristics of the sieve at the same time. The ranges of the viscosity and the elastic modulus of the conventional single-phase hyaluronic acid crosslinked body and the isomeric hyaluronic acid crosslinked body are not included in the above range. For example, the single-phase hyaluronic acid crosslinked body is recognized that the elasticity can be increased by adjusting the longitudinal degree at the time of manufacture. However, even if the concentration is increased or decreased, the ratio of the viscosity: elastic coefficient is not 4: 1 to 2: 1. .

The hyaluronic acid crosslinked product of the present invention, in which the ratio of the viscosity and the elastic modulus satisfies the range of 4: 1 to 2: 1, does not deviate easily from the injected site when injected into the body, and has high elastic force at the injection site. Has an advantage.

In addition, the hyaluronic acid crosslinked product of the present invention may implement various ranges of viscosity and elastic modulus while maintaining a ratio of the viscosity and the elastic modulus at 4: 1 to 2: 1 according to its concentration. In general, the higher the concentration of the hyaluronic acid crosslinked product, the higher the viscosity and elastic modulus of the hyaluronic acid crosslinked product. However, when the amount of crosslinking agent included in the crosslinking reaction is the same, the viscosity of the hyaluronic acid crosslinked body may increase exponentially with increasing concentration, but the elastic modulus of the hyaluronic acid crosslinked body does not continuously increase and has a certain limit. .

The hyaluronic acid crosslinked product of the present invention can be adjusted to have physical properties of viscosity of 750 Pa · s or less and elastic modulus of 325 Pa or less depending on the characteristics of such a general hyaluronic acid crosslink. If the viscosity and the elastic modulus do not satisfy the upper limit, the injection force may be high, and thus use as a filler may be difficult.

The final pH range of the hyaluronic acid crosslinked product of the present invention is preferably 7.2 to 7.4 for commercialization.

As a non-limiting example, the final concentration of the hyaluronic acid crosslinked when the hyaluronic acid crosslinked product of the present invention is prepared by injection may be 18 to 24 mg / ml. In addition, when prepared by injection, for example, the hyaluronic acid crosslinked product of the present invention may include a local anesthetic. Lidocaine and salts thereof may be used as a type of local anesthetic, and the final content may be used within a range that does not exhibit toxicity, but in the case of lidocaine hydrochloride, 0.3% of the total weight is most suitable.

The method for preparing a hyaluronic acid crosslinked product having the characteristics of a single phase and an isomer phase at the same time is different from the method for preparing a hyaluronic acid crosslinked product, adding a granulation step to the method for producing a single phase hyaluronic acid crosslinked product. Is characteristic. There are two conventional methods for producing particles of a hyaluronic acid crosslinked product, including (1) a method of forming particles of several micrometers to several millimeters by filtering the hyaluronic acid crosslinked material, and (2) a homogenizer of the hyaluronic acid crosslinked material. By grinding to form particles of several hundred nanometers to several tens of micrometers in size. The present inventors confirmed that the hyaluronic acid crosslinked product of the present invention can be injected into the body when the average surface particle size is 100 to 700 micrometers, and has a high modulus of elasticity. Therefore, the hyaluronic acid crosslinked product of the present invention includes a method of manufacturing the hyaluronic acid crosslinked filter in order to have viscoelasticity to be implemented in the present invention.

In other words, the hyaluronic acid crosslinked body according to the present invention is mixed with hyaluronic acid, a crosslinking agent, and water, which is a conventional method for preparing a hyaluronic acid crosslinked product, and proceeds in the same manner until the process of stirring the acid or alkaline conditions, and then preparing the above particles It can be obtained by carrying out.

The hyaluronic acid in the above process may be used commercially available products, in the present invention may be used hyaluronic acid derivatives or salts. For example, as a derivative, the hydroxyl group or carboxyl group of hyaluronic acid, etc. can be considered esterified, etherified, amidated, acetylated, and ketalized. As a salt, metal salts, such as a sodium salt or potassium salt, can be considered. At this time, the molecular weight of hyaluronic acid should be 80 to 1.2 million Da, it is advantageous to achieve the desired physical properties of the present invention.

The crosslinking agent is not limited as long as it can crosslink the polymer chain of hyaluronic acid by chemical bonding, such as 1,3-butadiene diepoxide, 1,2,7,8-diepoxyoctane, 1,5-hexadiene diepoxy Alkyl diepoxy, such as dehydration; Diglycidyl ethers such as ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, and bisphenol A diglycidyl ether; Divinylsulfone; Epichlorohydrin and the like. Preferably, BDDE is adopted as the crosslinking agent.

As a component or content of hyaluronic acid and a crosslinking agent, a well-known thing can be employ | adopted suitably. Specifically, since the characteristics of the present invention are characterized by improving the elasticity and viscosity as desired by adjusting the physical shape of the final obtained hyaluronic acid crosslinked product, the component or content of the hyaluronic acid and the crosslinking agent in the intermediate stage is A well-known thing can be employ | adopted suitably within the range to do. Preferably, hyaluronic acid is dissolved in an alkali solution containing BDDE to 20 wt%.

A method for producing a gel by crosslinking may also be a known one. However, in the case of mixing by stirring stirring the blade by force, the molecular chain of the hyaluronic acid polymer in the process may be physically cut, so that the three-dimensional structure of the gel may not be obtained, it may be desirable to adjust appropriately.

The hyaluronic acid crosslinked product having the characteristics of the single phase and isomer phase of the present invention can be used as a filler used in subcutaneous tissue or intradermal tissue.

Hereinafter, the configuration of the present invention for producing a hyaluronic acid crosslinked product having both the characteristics of a single phase and an isomer phase will be described with reference to Examples and Experimental Examples, which illustrate the present invention, and the scope of the present invention is limited thereto. It doesn't happen.

Example 1

10 g of sodium hyaluronate was evenly dissolved in a sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (3 mol% of hyaluronic acid monomer), and then allowed to stand at 35 ° C for 24 hours to proceed with crosslinking reaction. This was dialyzed in distilled water to remove the unreacted crosslinking agent. After completion of dialysis, water was added to adjust the concentration of hyaluronic acid to a final weight of 500 g, which was then wet sterilized. Sterilized 55.5 g of 10-fold concentrated sodium phosphate buffer solution was added to the hyaluronic acid cross-linked product to adjust the pH to 7.2 to 7.4, and granulated using 180 μm.

Example 2

10 g of sodium hyaluronate was evenly dissolved in a sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (4 mol% of hyaluronic acid monomer), and then allowed to stand at 35 ° C for 24 hours to proceed with crosslinking reaction. This was dialyzed in distilled water to remove the unreacted crosslinking agent. After completion of dialysis, water was added to adjust the concentration of hyaluronic acid to a final weight of 450 g, which was then wet sterilized. Sterilized 50 g of a 10-fold concentrated sodium phosphate buffer solution was added to the hyaluronic acid crosslinked product to adjust the pH to 7.2 to 7.4 and granulated using 180 µm.

Example 3

10 g of sodium hyaluronate was evenly dissolved in a sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (4 mol% of hyaluronic acid monomer), and then allowed to stand at 35 ° C for 24 hours to proceed with crosslinking reaction. This was dialyzed in distilled water to remove the unreacted crosslinking agent. After completion of the dialysis, water was added to adjust the concentration of hyaluronic acid to a final weight of 375 g, which was then wet sterilized. Sterilized 41.7 g of 10-fold concentrated sodium phosphate buffer solution was added to the hyaluronic acid cross-linked product to adjust the pH to pH 7.2 to 7.4, and granulated using 180 μm.

Example 4

10 g of sodium hyaluronate was evenly dissolved in a sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (4 mol% of hyaluronic acid monomer), and then allowed to stand at 35 ° C for 24 hours to proceed with crosslinking reaction. This was dialyzed in distilled water to remove the unreacted crosslinking agent. After completion of dialysis, water was added to adjust the concentration of hyaluronic acid to a final weight of 450 g, which was then wet sterilized. Sterilized 50 g of 10-fold concentrated sodium phosphate buffer solution containing lidocaine hydrochloride was added to the hyaluronic acid crosslinked product to adjust the pH to 7.2 to 7.4, and granulated using 180 μm.

Comparative Example 1

After dissolving 10 g of sodium hyaluronate evenly in a sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (9 mol% of the hyaluronic acid monomer), the crosslinking reaction was allowed to stand for 24 hours at 35 ° C. This was dialyzed on a sodium phosphate buffer solution to remove the unreacted crosslinking agent. After adjusting the concentration of hyaluronic acid to adjust the final weight to 500 g after the type of dialysis, it was wet sterilized and homogenized using a homogeneous disperser.

Comparative Example 2

After dissolving 10 g of sodium hyaluronate evenly in a sodium hydroxide solution containing 1,4-butanediol diglycidyl ether (3 mol% of hyaluronic acid monomer), the crosslinking reaction was allowed to stand for 24 hours under 35 ° C conditions. This was dialyzed in sodium phosphate buffer to remove the unreacted crosslinking agent. After completion of dialysis, the concentration of hyaluronic acid was adjusted to a final weight of 500 g, which was then sterilized and granulated using 180 micrometers. 10 wt% of the uncrosslinked hyaluronic acid solution was dissolved therein.

Experimental Example 1

The viscosity of the hyaluronic acid crosslinked product of the present invention corresponding to Examples 1 to 3 and Comparative Examples 1 to 2 was measured under Shear rate 0.05 s ⁻¹ , Gap size 1 mm, 35 ° C., and the modulus of elasticity was 0.01 Hz. , Strain 0.01 (1%), was measured under 35 ° C conditions. The results are shown in Table 1. As a result, Examples 1 to 4 according to the present invention were included in the range of 4: 1 to 2: 1 in which the ratio of viscosity: elastic coefficient was in the middle range between the single phase and the isomeric hyaluronic acid crosslinked product.

Experimental Example 2

The average surface particle size was changed in Example 2, and the ratio of the viscosity and the elastic modulus was measured, and the results are shown in Table 2. As a result, when the average surface particle size is out of 700 micrometers, it can be seen that the ratio of viscosity and elastic modulus does not satisfy 4: 1 to 2: 1.

Experimental Example 3

Example 1, Example 3, Example 4 and Comparative Example 1 After the injection of the hyaluronic acid crosslinked body 200uL each into the rat subcutaneous tissue, the wrinkle improvement effect was measured and the results are shown in FIG. Experimental results Example 1, Example 3 and Example 4 according to the present invention showed an equivalent effect in the wrinkle improvement effect when compared with Comparative Example 1 prepared by the same method as the conventionally developed single-phase filler.

Experimental Example 4

Example 2, Comparative Example 1 and Comparative Example 2 After the injection of 200 μL each of the hyaluronic acid crosslinks into the rat subcutaneous tissue, the degree of degradation was measured 6 months later. The results are shown in FIG. 2. Experimental results Example 2 according to the present invention is 6 months after injection compared to Comparative Example 1 prepared by the same method as the conventionally developed single-phase filler and Comparative Example 2 prepared by the same method as the conventionally developed isotropic filler In terms of superiority.

Experimental Example 5

The average surface particle size of Example 2 was measured in a wet environment using a dynamic light scattering apparatus, and the results are shown in FIG. 3. As a result, it was confirmed that Example 2 according to the present invention was formed of particles having an average surface diameter within 700 micrometers, and was found to have a uniform particle size as the particle size showed a single distribution.

Experimental Example 6

Example 4, Comparative Example 1 and Comparative Example 2 were observed with an optical microscope to observe the particle shape, the results are shown in FIG. Experimental Results The hyaluronic acid crosslinked product having the characteristics of a single phase and an isomer phase simultaneously according to the present invention was compared with the single phase hyaluronic acid crosslinked product of Comparative Example 1 and the heterophasic hyaluronic acid crosslinked product of Comparative Example 2, It was confirmed that the prepared in the form of particles very similar to Comparative Example 2.

	Viscosity (Pas)	Modulus of elasticity (Pa)	Viscosity: Modulus of Elasticity
Example 1	275	85	3.23: 1
Example 2	482	173	2.79: 1
Example 3	575	175	3.28: 1
Example 4	400	198	2.02: 1
Comparative Example 1	1121	33	34.27: 1
Comparative Example 2	282	294	0.95: 1

	Average surface particle size	Viscosity (Pas)	Modulus of elasticity (Pa)	Viscosity: Modulus of Elasticity
Example 2	608.56	482	173	2.79: 1
In Example 2, when 850 μm was used instead of 180 μm	731.55	406	235	1.72: 1
In Example 2 in the case of using a 1000 μm instead of a 180 μm	742.60	408	220	1.85: 1

Claims (7)

1. A hyaluronic acid crosslinked granular single phase, wherein the ratio of the viscosity (unit Pa · s) and the modulus of elasticity (unit Pa) of the hyaluronic acid crosslinked product is 4: 1 to 2: 1.
2. The hyaluronic acid crosslinked product according to claim 1, wherein the hyaluronic acid crosslinked product has an average surface particle size of 100 to 700 micrometers.
3. The hyaluronic acid crosslinked product according to claim 1 or 2, wherein the hyaluronic acid crosslinked product is a BDDE crosslinked hyaluronic acid crosslinked product.
4. A composition comprising the hyaluronic acid crosslinked product according to claim 1 or 2 and a local anesthetic.
5. The composition of claim 4 wherein the composition is for filler injections.
6. (A) crosslinking a mixture comprising hyaluronic acid, a crosslinking agent and purified water by stirring and mixing under acidic or alkaline conditions; A method for producing the hyaluronic acid crosslinked product according to claim 1 or 2, wherein the crosslinked product obtained in step (A) is granulated.
7. The production method according to claim 6, wherein the obtained hyaluronic acid crosslinked product is granulated so that the average surface particle size is from 100 to 700 micrometers.

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