KR102320153B1 - Hyaluronic acid gel through double-crosslink and method for preparing the same - Google Patents

Abstract

The present invention comprises the steps of (a) preparing a first solution containing non-crosslinked hyaluronic acid; (b) preparing a second solution containing hyaluronic acid that is first crosslinked by a first crosslinking agent; and (c) adding a second crosslinking agent to the mixed solution of the first solution and the second solution, and then secondarily crosslinking the non-crosslinked hyaluronic acid and the first crosslinked hyaluronic acid. It relates to a gel, a method for producing the same, and a composition for a hyaluronic acid filler comprising the same.

Description

Hyaluronic acid gel and its manufacturing method through double crosslinking

The present invention relates to a hyaluronic acid gel through double crosslinking and a method for preparing the same.

Due to economic growth and rising interest in beauty, the facial beauty market, especially the filler market, is growing very rapidly, and is expected to grow to $2.7 billion in 2020. In particular, hyaluronic acid fillers occupy 90% of the total market due to their high safety and low side effects.

The hyaluronic acid filler includes a hyaluronic acid gel, and a conventional hyaluronic acid gel includes hyaluronic acid crosslinked by a crosslinking agent. Although hyaluronic acid has excellent biocompatibility, it has problems due to low physical properties and a short decomposition period in the body. In order to overcome these problems, crosslinking using a crosslinking agent is required. However, the high content of the crosslinking agent modifies the physical, chemical and biological properties of the hyaluronic acid gel, and when implanted in the body, attacks immune cells, etc., and increases the probability of causing side effects such as inflammation and granuloma formation. needs to be minimized.

On the other hand, the hyaluronic acid gel is divided into monophasic and biphasic according to the shape. Specifically, the monophasic hyaluronic acid gel refers to a soft sol-type gel, which has relatively high viscosity and low elasticity. . In addition, the biphasic hyaluronic acid gel refers to a mixture of a gel in the form of hard particles and a gel in the form of a soft sol, and has relatively high elasticity and low viscosity. It has the advantage that it can be implemented.

Therefore, while maintaining the same level of physical properties as the monophasic hyaluronic acid gel and the biphasic hyaluronic acid gel, there is a need for research to minimize the content of the final bound crosslinking agent.

The present invention is to prepare a hyaluronic acid gel having improved physical properties through double crosslinking, (a) preparing a first solution containing non-crosslinked hyaluronic acid; (b) preparing a second solution containing hyaluronic acid that has been first crosslinked by the first crosslinking agent; and (c) adding a second crosslinking agent to the mixed solution of the first solution and the second solution, and then secondarily crosslinking the non-crosslinked hyaluronic acid and the first crosslinked hyaluronic acid. An object of the present invention is to provide a method for preparing a gel, and the like.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention comprises the steps of (a) preparing a first solution containing non-crosslinked hyaluronic acid; (b) preparing a second solution containing hyaluronic acid that has been first crosslinked by the first crosslinking agent; and (c) adding a second crosslinking agent to the mixed solution of the first solution and the second solution, and then secondarily crosslinking the non-crosslinked hyaluronic acid and the first crosslinked hyaluronic acid. A method for preparing a gel is provided.

The hyaluronic acid concentration in the first solution in step (a) or the hyaluronic acid concentration in the second solution in step (b) may be 12 (w/w)% to 70 (w/w)%.

The first crosslinking agent in step (b) or the second crosslinking agent in step (c) is 1,4-butandiol diglycidyl ether, ethylene glycol diglycidyl ether diglycidyl ether), 1,6-hexanediol diglycidyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether (propylene glycol diglycidyl ether), polypropylene glycol It may include at least one selected from the group consisting of diglycidyl ether) and diglycerol polyglycidyl ether.

The first crosslinking agent in step (b) may include 1 part by weight to 10 parts by weight based on 100 parts by weight of hyaluronic acid in the second solution in step (b).

The second crosslinking agent in step (c) may include 1 part by weight to 10 parts by weight based on 100 parts by weight of hyaluronic acid in the mixed solution in step (c).

The primary crosslinking in step (b) may be performed at a temperature of 10° C. to 30° C. for 0.5 hours to 5 hours.

The secondary crosslinking in step (c) may be performed at a temperature of 20°C to 40°C for 20 hours to 30 hours.

In one embodiment of the present invention, a hyaluronic acid gel comprising hyaluronic acid crosslinked by a crosslinking agent and having an elastic strain of 1%·KPa ^{- 1} to 20%·KPa ^{-1 expressed by the following formula (1)} provides:

[Equation 1]

.

.

The hyaluronic acid gel may have a loss strain of 10% to 45% represented by Equation 2 below:

[Equation 2]

.

.

The hyaluronic acid gel may have a tackiness of 0.5 N to 1.2N in the axial direction measured through a rheometer in which the vertical ascending speed is controlled to 0.1 mm/sec.

The cross-linked hyaluronic acid may be cross-linked by a combination of the first cross-linking agent and the second cross-linking agent.

In another embodiment of the present invention, there is provided a composition for a hyaluronic acid filler comprising the hyaluronic acid gel.

The composition for a hyaluronic acid filler may further include a local anesthetic.

The method for producing a hyaluronic acid gel according to the present invention comprises the steps of (a) preparing a first solution containing non-crosslinked hyaluronic acid; (b) preparing a second solution containing hyaluronic acid that is first crosslinked by a first crosslinking agent; and (c) adding a second crosslinking agent to the mixed solution of the first solution and the second solution, and then secondarily crosslinking the non-crosslinked hyaluronic acid and the first crosslinked hyaluronic acid, The hyaluronic acid gel prepared according to the above method maintains the same level of elastic and viscoelastic properties as conventional monophasic hyaluronic acid gels and biphasic hyaluronic acid gels, and minimizes the content of the final bound crosslinking agent, thereby solving the problems. have an advantage

In addition, the hyaluronic acid gel prepared according to the above method has an excellent adhesive strength, and when a filler including the same is applied to the skin, it naturally adheres to the skin and has the advantage of minimizing a foreign body feeling.

1 is a flowchart showing a method for producing a hyaluronic acid gel through double crosslinking according to an embodiment of the present invention.

While the present inventors were studying a method for grafting the advantages of monophasic hyaluronic acid gel and biphasic hyaluronic acid gel, when the hyaluronic acid gel was prepared through double crosslinking of a high concentration hyaluronic acid solution, excellent elastic properties and viscoelastic properties While maintaining the , it was possible to minimize the content of the final bound crosslinking agent, as well as confirmed to have excellent adhesive strength, completed the present invention.

As used herein, the term “hyaluronic acid” refers to a high molecular compound composed of repeating units of N-acetylglucosamine and glucuronic acid, which is one of complex polysaccharides composed of amino acids and uronic acid, and hyaluronic acid and its pharmaceutical It is a concept that includes acceptable salts. Specifically, the hyaluronic acid can be prepared through various methods such as separation and synthesis, and it is preferable in terms of preventing biocompatibility of those derived from lactic acid bacteria, particularly, a Streptococcus sp. strain, but is not limited thereto. More specifically, the Streptococcus sp. strain is at least one selected from the group consisting of Streptococcus pyogenex, Streptococcus faecalis, Streptococcus dysgalactie, Streptococcus sepidemicur, Streptococcus equi, and Streptococcus equishimilis. can be used

As used herein, the term “gel” refers to a state in which a colloidal solution is thickened to a certain concentration or more and a strong network is formed and hardened. used as a concept.

As used herein, the term “hyaluronic acid gel” refers to a gel containing hyaluronic acid as an active ingredient, and the hyaluronic acid gel is classified into monophasic and biphasic according to its shape. .

In this case, the term “monophasic hyaluronic acid gel” refers to a soft sol-type gel, which has relatively high viscosity and low elasticity. In addition, "biphasic hyaluronic acid gel" refers to a mixture of a gel in the form of hard particles and a gel in the form of a soft sol, and has relatively high elasticity and low viscosity.

As used herein, the term “elasticity property” refers to elastic deformation when a force is applied to a fluid, and can be evaluated by measuring the storage modulus (G') using a rheometer at a specific frequency. have. In addition, as used herein, the term “viscocity property” refers to resistance to the flow of a fluid when a force is applied to the fluid, and a loss modulus (G”) using a rheometer at a specific frequency. It can be evaluated through measurement. In addition, as used herein, the term “viscoelasticity property” refers to both elastic and viscous properties, and a loss coefficient that means a loss modulus (G”) with respect to a storage modulus (G′). (loss tangent; tanδ) can be evaluated through calculation.

As used herein, the term “strain” refers to the ratio of the number of moles of a crosslinking agent bound to the number of moles of the hyaluronic acid repeating unit (disaccharide), and the strain is measured through NMR, which is a filler through the content of the final bound crosslinking agent. As a unit for evaluating the efficacy of the composition for use, it may be expressed by the following Equation 3:

[Equation 3]

.

.

At this time, if the deformation rate is too low, there is a problem that the efficacy of the filler composition cannot be properly exhibited due to the deterioration of the physical properties of the hyaluronic acid gel and a short decomposition period in the body, and if the deformation rate is too high, the physical, chemical and There is a problem in that the biological properties are modified and the probability of causing side effects such as inflammation and granuloma formation by attacking immune cells when transplanted into the body is increased.

In addition, the term "elastic strain" in the present specification refers to the strain with respect to the storage modulus (G'), which is used to evaluate the efficacy of the composition for fillers through the content of the final bonded crosslinking agent compared to the elastic properties. As a unit, it can be expressed by the following Equation 1:

[Equation 1]

.

.

In addition, the term "loss strain" in the present specification refers to the strain with respect to the loss coefficient (loss tangent; tanδ), which is a unit for evaluating the efficacy of the composition for filler through the content of the final bonded crosslinking agent compared to the viscoelastic properties As, it can be expressed by the following Equation 2:

[Equation 2]

.

.

As used herein, the term “filler” may refer to a supplementary material that is directly injected or inserted into skin cells to fill in wrinkles or pitted scars. The filler may be used for various purposes, such as relieving skin curvature and replenishing moisture, and there is no limitation. The filler may be applied over the entire skin area, and specifically, may include the face, neck, and the like.

Hereinafter, the present invention will be described in detail.

Method for producing hyaluronic acid gel

The present invention comprises the steps of (a) preparing a first solution containing non-crosslinked hyaluronic acid; (b) preparing a second solution containing hyaluronic acid that has been first crosslinked by the first crosslinking agent; and (c) adding a second crosslinking agent to the mixed solution of the first solution and the second solution, and then secondarily crosslinking the non-crosslinked hyaluronic acid and the first crosslinked hyaluronic acid. A method for preparing a gel is provided.

1 is a flowchart showing a method for producing a hyaluronic acid gel through double crosslinking according to an embodiment of the present invention.

As shown in FIG. 1 , according to an embodiment of the present invention, a first solution containing non-crosslinked hyaluronic acid is prepared. Then, a second solution containing hyaluronic acid that has been crosslinked primarily by the first crosslinking agent is prepared. Then, a mixed solution is prepared by mixing the first solution and the second solution, and then a second crosslinking agent is added to the mixed solution, followed by secondary crosslinking to prepare a hyaluronic acid gel.

First, the method for producing a hyaluronic acid gel according to the present invention includes a step [step (a)] of preparing a first solution containing non-crosslinked hyaluronic acid.

The hyaluronic acid in the first solution is characterized in that it has a high concentration, specifically, it is preferably 12 (w/w)% to 70 (w/w)%, and 15 (w/w)% to 40 (w/w)%. It is more preferably w)%, and even more preferably 15 (w/w)% to 25 (w/w)%, but is not limited thereto. That is, by maintaining a high concentration of hyaluronic acid in the first solution, the content of the final bound crosslinking agent can be minimized, and thus the strain rate can be lowered. In addition, the weight average molecular weight (Mw) of the hyaluronic acid in the first solution is preferably 500,000 to 3,000,000, but is not limited thereto.

At this time, when the concentration or weight average molecular weight (Mw) of hyaluronic acid in the first solution is too low, there is a problem due to low physical properties and a short decomposition period in the body, and the concentration or weight average molecular weight (Mw) of hyaluronic acid in the first solution ( Mw) is too high, there is a problem that the hyaluronic acid gel is not properly formed.

Next, the method for producing a hyaluronic acid gel according to the present invention includes a step [step (b)] of preparing a second solution containing hyaluronic acid that is first cross-linked by a first cross-linking agent.

It is characterized in that the hyaluronic acid in the second solution also has a high concentration, specifically, it is preferably 12 (w/w)% to 70 (w/w)%, and 15 (w/w)% to 40 (w/w)% It is more preferably w)%, and even more preferably 15 (w/w)% to 25 (w/w)%, but is not limited thereto. That is, by maintaining a high concentration of hyaluronic acid in the second solution as well, the content of the final bound crosslinking agent can be minimized, and thus the strain rate can be lowered. In addition, the weight average molecular weight (Mw) of the hyaluronic acid in the second solution is also preferably 500,000 to 3,000,000, but is not limited thereto.

At this time, when the concentration or weight average molecular weight (Mw) of hyaluronic acid in the second solution is too low, there is a problem due to low physical properties and a short decomposition period in the body, and the concentration or weight average molecular weight (Mw) of hyaluronic acid in the second solution ( Mw) is too high, there is a problem that the hyaluronic acid gel is not properly formed.

The second solution includes hyaluronic acid that is primarily crosslinked by the first crosslinking agent, and the problem of hyaluronic acid itself in the second solution can be overcome due to the primary crosslinking using the first crosslinking agent.

The first crosslinking agent is for primary crosslinking, and specifically, for primary crosslinking and bonding of hyaluronic acid in the second solution to each other. Specifically, the first crosslinking agent is 1,4-butandiol diglycidyl ether (1,4-butandiol diglycidyl ether), ethylene glycol diglycidyl ether (ethylene glycol diglycidyl ether), 1,6-hexanediol diglycidyl ether Cydyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, and diglycerol polyglycidyl ether ( It is preferably at least one selected from the group consisting of diglycerol polyglycidyl ether), and more preferably 1,4-butandiol diglycidyl ether (1,4-butandiol diglycidyl ether), but is not limited thereto.

In addition, the first cross-linking agent, based on 100 parts by weight of hyaluronic acid in the second solution, preferably includes 1 to 10 parts by weight, more preferably 1 to 5 parts by weight, but is not limited thereto. does not At this time, when the content of the first crosslinking agent is too low, there is a problem in that the physical properties of the hyaluronic acid itself in the second solution cannot be improved, and when the content of the second crosslinking agent is too high, there is a problem due to the high content of the crosslinking agent There is this. That is, since the hyaluronic acid in the second solution has a high concentration, even if a small amount of the first crosslinking agent is used, the physical properties of the hyaluronic acid itself in the second solution can be sufficiently increased.

The primary crosslinking may be treated as preliminary crosslinking, and is preferably performed at a temperature of 10° C. to 30° C. for 0.5 hours to 5 hours, and is preferably performed at a temperature of 20° C. to 30° C. for 0.5 hours to 2 hours. More preferably, but not limited thereto. At this time, when the first crosslinking is performed at a temperature higher than the temperature range or for a time longer than the time range, the hyaluronic acid in the second solution is completely gelled and mixing with the first solution to be described later becomes impossible. Not only there is a problem, there is also a problem that secondary cross-linking, which will be described later, is also impossible.

Next, in the method for producing a hyaluronic acid gel according to the present invention, a second crosslinking agent is added to the mixed solution of the first solution and the second solution, and then the non-crosslinked hyaluronic acid and the first crosslinked hyaluronic acid Secondary crosslinking step [(c) step] is included.

The mixed solution is prepared by mixing the first solution and the second solution with each other, and the weight ratio of the first solution and the second solution may be 1:9 to 9:1, and 1:5 to 5: It is preferably 1, but is not limited thereto. At this time, as the content of the first solution increases compared to the second solution, a hyaluronic acid gel having the same level of physical properties as the monophasic hyaluronic acid gel can be finally prepared, and the first solution compared to the second solution As the content decreases, a hyaluronic acid gel having the same level of physical properties as the ideal hyaluronic acid gel can be finally prepared.

The second crosslinking agent is for secondary crosslinking, and may be for bonding the non-crosslinked hyaluronic acid and the primary crosslinked hyaluronic acid. Specifically, the second crosslinking agent is for bonding the hyaluronic acid in the first solution and the hyaluronic acid in the second solution to each other by secondary crosslinking. A portion of the second crosslinking agent may be for secondary crosslinking and bonding of hyaluronic acids in the first solution, or secondary crosslinking of hyaluronic acids in the second solution to each other. Specifically, the second crosslinking agent may be the same as or different from the first crosslinking agent, 1,4-butandiol diglycidyl ether (1,4-butandiol diglycidyl ether), ethylene glycol diglycidyl ether ( ethylene glycol diglycidyl ether), 1,6-hexanediol diglycidyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether (propylene glycol diglycidyl ether), polypropylene glycol diglycidyl ether ( Preferably, at least one selected from the group consisting of polypropylene glycol diglycidyl ether and diglycerol polyglycidyl ether, and 1,4-butandiol diglycidyl ether More preferably, but not limited thereto.

In addition, the second crosslinking agent, based on 100 parts by weight of hyaluronic acid in the mixed solution, preferably includes 1 to 10 parts by weight, more preferably 1 to 5 parts by weight, but is not limited thereto. . At this time, when the content of the second crosslinking agent is too low, there is a problem in that the physical properties of hyaluronic acid itself in the mixed solution cannot be improved, and when the content of the second crosslinking agent is too high, there is a problem due to the high content of the crosslinking agent have. That is, since the hyaluronic acid in the mixed solution has a high concentration, even if a small amount of the second crosslinking agent is used, the physical properties of the hyaluronic acid itself in the mixed solution can be sufficiently increased.

The secondary crosslinking is preferably performed at a temperature of 20°C to 40°C for 20 hours to 30 hours, and more preferably performed at a temperature of 30°C to 40°C for 20 hours to 25 hours, but is not limited thereto. . Compared to the conventional crosslinking conditions, the secondary crosslinking is performed at a low temperature, so that after the secondary crosslinking of the hyaluronic acid in the mixed solution, it has the advantage of preventing it from decomposing itself at a high temperature again.

The hyaluronic acid gel prepared according to the above method was prepared through double crosslinking using a high concentration of hyaluronic acid and a low content of a crosslinking agent, and contains hyaluronic acid crosslinked by a crosslinking agent, and the elastic deformation rate represented by the following Equation 1 is 1 It may be characterized as %·KPa ^-1 to 20%·KPa ^-1:

[Equation 1]

.

.

In addition, it may be characterized in that the loss strain represented by the following Equation 2 is 10% to 45%:

[Equation 2]

.

.

In addition, it may be characterized in that the tackiness in the axial direction measured through a rheometer in which the vertical ascending speed is controlled to 0.1 mm/sec is 0.5 N to 1.2 N.

Therefore, the hyaluronic acid gel prepared according to the above method maintains the same level of elastic and viscoelastic properties as conventional monophasic hyaluronic acid gels and biphasic hyaluronic acid gels, and minimizes the content of the final bound crosslinking agent, thereby solving the problem. have the advantage of being able to

In addition, the hyaluronic acid gel prepared according to the above method has an excellent adhesive strength, and when a filler including the same is applied to the skin, it naturally adheres to the skin and has the advantage of minimizing a foreign body feeling.

hyaluronic acid gel

The present invention provides a hyaluronic acid gel comprising hyaluronic acid crosslinked by a crosslinking agent and having an elastic strain of 1%·KPa ^-1 to 20%·KPa ^{-1 expressed by the following formula:}

[Equation 1]

.

.

First, the hyaluronic acid gel according to the present invention includes hyaluronic acid crosslinked by a crosslinking agent, and the crosslinking is a double crosslinking. . Specifically, the hyaluronic acid gel includes hyaluronic acid cross-linked by a combination of the first cross-linking agent and the second cross-linking agent, and in addition, hyaluronic acid cross-linked only by the first cross-linking agent and hyaluronic acid cross-linked only by the second cross-linking agent can do.

Next, the hyaluronic acid gel according to the present invention is characterized in that the elastic strain represented by the following formula (1) is 1%·KPa ^{- 1} to 20%·KPa ^-1 :

[Equation 1]

.

.

The hyaluronic acid gel preferably has an elastic strain of 1%·KPa ^{- 1} to 15%·KPa ^-1 , but is not limited thereto.

Specifically, the hyaluronic acid gel may have a strain rate of 1% to 7%, preferably 1% to 5%, but is not limited thereto. In addition, the hyaluronic acid gel preferably has a storage modulus (G') of 0.01 KPa to 2.0 KPa measured using a rheometer in a frequency range of 0.1 Hz to 10 Hz, but is not limited thereto.

That is, the hyaluronic acid gel maintains an elastic strain of 1%·KPa ^-1 to 20%·KPa ^-1 , so that the hyaluronic acid gel has the same level of elasticity as conventional monophasic hyaluronic acid gels and biphasic hyaluronic acid gels. It is expected that this problem can be solved by minimizing the content of the final bound crosslinking agent while maintaining it.

In addition, the hyaluronic acid gel according to the present invention may be characterized in that the loss strain represented by the following Equation 2 is 10% to 45%:

[Equation 2]

.

.

Specifically, the hyaluronic acid gel may have a strain rate of 1% to 7%, preferably 1% to 5%, but is not limited thereto. In addition, in the frequency range of 0.1 Hz to 10 Hz, the hyaluronic acid gel may have a loss factor (tanδ) measured and calculated using a rotary rheometer of 0.05 to 1.0, preferably 0.10 to 0.50, but limited thereto doesn't happen

That is, the hyaluronic acid gel maintains a loss strain of 10% to 45%, so that the hyaluronic acid gel maintains the same level of viscoelastic properties as the conventional monophasic hyaluronic acid gel and the biphasic hyaluronic acid gel, while maintaining the final combined crosslinking agent. It is expected that this problem can be solved by minimizing the content.

In addition, the hyaluronic acid gel according to the present invention may be characterized in that the tackiness to the adherend measured through a rotary rheometer is 0.5 N to 1.2 N:

The hyaluronic acid gel maintains an adhesive force of 0.5 N to 1.2 N, so that when a hyaluronic acid filler containing the hyaluronic acid is applied to the skin, it naturally adheres to the skin, thereby minimizing the feeling of foreign body.

Composition for hyaluronic acid filler comprising hyaluronic acid gel

The present invention provides a composition for a hyaluronic acid filler comprising the hyaluronic acid gel.

Since the hyaluronic acid gel has been described above, a redundant description thereof will be omitted.

The composition for filler includes the hyaluronic acid as an active ingredient, and the total concentration of the hyaluronic acid in the composition for filler is 1 mg/ml to 70 mg/ml (0.1 (w/v)% to 7 (w/w) v) %), preferably 10 mg/ml to 60 mg/ml (1 (w/v)% to 6 (w/v)%), but is not limited thereto.

Optionally, the composition for filler may include a local anesthetic, and the concentration of the local anesthetic in the composition for filler may be an amount effective to relieve pain experienced when the composition for filler is injected. The concentration of the local anesthetic in the composition for filler may be 0.1 (w/v)% to 5 (w/v)%, preferably 0.2 (w/v)% to 1.0 (w/v)%, and 0.3 It is more preferably (w/v)%, but is not limited thereto.

Specifically, the local anesthetic is ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine (biphenamine), bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloro Procaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diferrodone ( diperodon), dicyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprosine (euprocin), phenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate), levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine , naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phena phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, procaine Panocaine, propanocaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol ( salicyl alcohol), tetracaine, tolycaine, trimecaine, zolamine, and salts thereof may be selected from the group consisting of, preferably lidocaine, lidocaine HCl It is more preferably, but is not limited thereto.

In addition, the composition for fillers may further include additives such as buffers, antioxidants, bacteriostats, diluents, dispersants, surfactants, binders, and lubricants.

The filler composition is intended to be administered to an individual, and the individual may refer to any animal, including humans. In this case, the animal is preferably not only a human, but also a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, an antelope, a dog, and a cat that requires tissue repair, similar to this, but is not limited thereto. In addition, administration may refer to parenteral administration rather than oral administration. In this case, parenteral administration is preferably subcutaneous or intradermal administration, but is not limited thereto. In addition, the frequency of administration may be once a day, or several times by dividing the dose.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example 1]

Example 1: Preparation of hyaluronic acid gel (1) through double crosslinking

A first solution was prepared by mixing 10 g of hyaluronic acid (weight average molecular weight (Mw) = 1,000,000) and 55 g of 0.24N sodium hydroxide solution. 10 g of hyaluronic acid (weight average molecular weight (Mw) = 1,000,000), 55 g of 0.24N sodium hydroxide solution, and 0.2 g of 1,4-butanediol diglycidyl ether were mixed, and then primary cross-linking at 25° C. for about 1 hour to prepare a second solution.

After mixing the prepared first solution and the second solution in a weight ratio of about 1:1, 0.2 g of 1,4-butanediol diglycidyl ether was further mixed, followed by secondary crosslinking at 37° C. for about 24 hours. , neutralized with equimolar HCl and washed with PBS to finally prepare a hyaluronic acid gel.

Example 2: Preparation of hyaluronic acid gel (2) through double crosslinking

A first solution was prepared by mixing 16 g of hyaluronic acid (weight average molecular weight (Mw) = 1,000,000) and 90 g of 0.24N sodium hydroxide solution. 4 g of hyaluronic acid (weight average molecular weight (Mw) = 1,000,000), 25 g of 0.24N sodium hydroxide solution, and 0.1 g of 1,4-butanediol diglycidyl ether were mixed, and then primary cross-linking at 25° C. for about 1 hour to prepare a second solution.

After mixing the prepared first solution and the second solution in a weight ratio of about 4:1, 0.4 g of 1,4-butanediol diglycidyl ether was further mixed, followed by secondary crosslinking at 37°C for about 24 hours. After neutralization with equimolar HCl, it was washed with PBS to finally prepare a hyaluronic acid gel.

Example 3: Preparation of hyaluronic acid gel (3) through double crosslinking

A first solution was prepared by mixing 16 g of hyaluronic acid (weight average molecular weight (Mw) = 1,000,000) and 70 g of 0.24N sodium hydroxide solution. 4 g of hyaluronic acid (weight average molecular weight (Mw) = 1,000,000), 25 g of 0.24N sodium hydroxide solution, and 0.05 g of 1,4-butanediol diglycidyl ether were mixed, and then primary cross-linking at 25° C. for about 1 hour to prepare a second solution.

After mixing the prepared first solution and the second solution in a weight ratio of about 4:1, 0.1 g of 1,4-butanediol diglycidyl ether was further mixed, followed by secondary crosslinking at 37°C for about 24 hours. After neutralization with equimolar HCl, it was washed with PBS to finally prepare a hyaluronic acid gel.

Comparative Example 1: Preparation of hyaluronic acid gel through single crosslinking

A solution of 20 g of hyaluronic acid (weight average molecular weight (Mw) = 1,000,000) mixed with 105 g of 0.24N sodium hydroxide solution and 1.2 g of 1,4-butanediol diglycidyl ether was prepared, and then at 50° C. for about 3 hours. crosslinked. Thereafter, after neutralization with equimolar HCl and washed with PBS, a biphasic hyaluronic acid gel was finally prepared.

.

Comparative Example 2: Preparation of monophasic hyaluronic acid gel

As a monophasic hyaluronic acid gel, a commercialized Juvederm Voluma product of Allergan was prepared.

Comparative Example 3: Preparation of biphasic hyaluronic acid gel

As a biphasic hyaluronic acid gel, a commercially available Restylene SubQ product of Galderma was prepared.

Experimental Example: Evaluation of viscous and elastic properties, strain and adhesion

First, the viscous and elastic properties and strain of the hyaluronic acid gel prepared in Examples 1 to 3 and the hyaluronic acid gel prepared in Comparative Examples 1 to 3 were measured, and based on this, the elastic strain and loss strain were derived and compared.

Specifically, the hyaluronic acid gel prepared in Examples 1 to 3 and the hyaluronic acid gel prepared in Comparative Examples 1 to 3 were stored using a rheometer (product name: Kinexus Lab+, Malven) in a frequency range of 1 Hz. The elastic modulus (G') and the loss modulus (G”) were measured, and the loss coefficient (tanδ) was derived based thereon, and the results are shown in Table 1 below.

In addition, using the hyaluronic acid gel prepared in Examples 1 to 3 and the hyaluronic acid gel prepared in Comparative Examples 1 to 3 as samples, Hdase (500 IU/ml, Sigma-aldrich, USA) enzyme was used under 37 ° C. Thus, the sample was decomposed to a concentration of 10 mg/ml. At this time, the unbound crosslinking agent was removed by washing before decomposition through filtration using a 0.22 μm filter. Then, the number of moles of the hyaluronic acid repeating unit and the number of moles of the bound crosslinking agent were measured through NMR, and the strain was calculated through the following Equation 3, and based on this, the elastic strain represented by the following Equation 1 and the following Equation 2 The indicated loss strain was derived, and the results are shown in Table 1 below:

[Equation 1]

,

,

[Equation 2]

,

,

[Equation 3]

.

.

storage modulus
(G’)(KPa) loss modulus
(G”) (KPa) loss factor
(tanδ) strain
(MoD) (%) elastic strain
(% KPa ^-One ) viscoelastic
strain
(%) Example 1 0.430 0.069 0.16 4.4 10.23 27.5 Example 2 0.382 0.033 0.10 4.4 11.51 44 Example 3 0.610 0.060 0.10 2.9 4.75 29.48 Comparative Example 1 0.177 0.033 0.18 8.2 21.58 45.55 Comparative Example 2 0.347 0.028 0.08 9.1 26.22 113.75 Comparative Example 3 0.949 0.088 0.09 9.1 109.58 101.11

As shown in Table 1, the hyaluronic acid gels prepared in Examples 1 to 3 were prepared through double crosslinking using a high concentration of hyaluronic acid and a low content of a crosslinking agent, and the monophasic hyaluronic acid gel prepared in Comparative Example 2 and It is confirmed that the elastic deformation rate is lower than that of the biphasic hyaluronic acid gel prepared in Comparative Examples 1 and 3.

In particular, the hyaluronic acid prepared in Example 3 uses a higher concentration of hyaluronic acid and a lower content of the crosslinking agent, compared to the hyaluronic acid gel prepared in Example 2, It is confirmed that the elastic deformation rate can be further lowered.

From these results, it is expected that the hyaluronic acid gels prepared in Examples 1 to 3 can solve the problems by minimizing the content of the final bound crosslinking agent while maintaining excellent elastic properties.

In addition, the hyaluronic acid gel prepared in Examples 1 to 3 was due to the double crosslinking of high concentration of hyaluronic acid and the high content of the crosslinking agent, the monophasic hyaluronic acid gel prepared in Comparative Example 2 and the ideality prepared in Comparative Examples 1 to 3 Compared to the hyaluronic acid gel, it is confirmed that the loss strain is low. At this time, the biphasic hyaluronic acid gel prepared in Comparative Example 1 also has a low loss strain, which can be seen as a result of using a high concentration of hyaluronic acid.

On the other hand, using the hyaluronic acid gel prepared in Examples 1 to 3 and the hyaluronic acid gel prepared in Comparative Examples 1 to 3 as samples, the tackiness in the axial direction through a rheometer (product name: Kinexus Lab+, Malven) was measured. Specifically, after placing the sample on the measuring plate, lowering the cone to a height of 0.5 mm, removing the sample that comes out of the cone, and controlling the formula rising speed of the cone to 0.1 mm/sec. The maximum force was measured, and the results are shown in Table 2 below.

tackiness (N) Example 1 0.75 Example 2 0.80 Example 3 0.60 Comparative Example 1 0.10 Comparative Example 2 0.60 Comparative Example 3 0.09

As shown in Table 2 above, the hyaluronic acid gels prepared in Examples 1 to 3 had significantly better adhesion in the axial direction than the biphasic hyaluronic acid gels prepared in Comparative Examples 1 and 3, and in Comparative Example 2 Compared to the prepared monophasic hyaluronic acid gel, the adhesive force in the axial direction is maintained at an equal or high level, and when a hyaluronic acid filler containing it is applied to the skin, it naturally adheres to the skin and has the advantage of minimizing the feeling of foreign body. .

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (13)

A method for producing a hyaluronic acid gel, characterized in that the elastic strain represented by the following formula (1) is 1%·KPa ^-1 to 20%·KPa ^-1,
[Equation 1]

,
(a) preparing a first solution comprising non-crosslinked hyaluronic acid;
(b) preparing a second solution containing hyaluronic acid that is first crosslinked by a first crosslinking agent; and
(c) adding a second crosslinking agent to the mixed solution of the first solution and the second solution, and then secondarily crosslinking the non-crosslinked hyaluronic acid and the first crosslinked hyaluronic acid,
Method for producing hyaluronic acid gel.
According to claim 1,
The hyaluronic acid concentration in the first solution in step (a) or the hyaluronic acid concentration in the second solution in step (b) is 12 (w/w)% to 70 (w/w)%,
Method for producing hyaluronic acid gel.
According to claim 1,
The first crosslinking agent in step (b) or the second crosslinking agent in step (c) is 1,4-butandiol diglycidyl ether, ethylene glycol diglycidyl ether diglycidyl ether), 1,6-hexanediol diglycidyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether (propylene glycol diglycidyl ether), polypropylene glycol containing at least one selected from the group consisting of diglycidyl ether) and diglycerol polyglycidyl ether (diglycerol polyglycidyl ether)
Method for producing hyaluronic acid gel.
According to claim 1,
The first crosslinking agent in step (b) contains 1 to 10 parts by weight based on 100 parts by weight of hyaluronic acid in the second solution in step (b).
Method for producing hyaluronic acid gel.
According to claim 1,
The second crosslinking agent in step (c) contains 1 part by weight to 10 parts by weight based on 100 parts by weight of hyaluronic acid in the mixed solution in step (c)
Method for producing hyaluronic acid gel.
According to claim 1,
The primary crosslinking in step (b) is performed at a temperature of 10°C to 30°C for 0.5 hours to 5 hours.
Method for producing hyaluronic acid gel.
According to claim 1,
The secondary crosslinking in step (c) is performed at a temperature of 20°C to 40°C for 20 hours to 30 hours.
Method for producing hyaluronic acid gel.
Containing hyaluronic acid crosslinked by a crosslinking agent,
Characterized in that the elastic strain represented by the following formula (1) is 1%·KPa ^-1 to 20%·KPa ^-1,
Hyaluronic acid gel:
[Equation 1]

.
9. The method of claim 8,
Characterized in that the loss strain represented by the following Equation 2 is 10% to 45%
Hyaluronic acid gel:
[Equation 2]

.
9. The method of claim 8,
Characterized in that the tackiness in the axial direction measured through a rheometer in which the vertical ascent rate is controlled to 0.1 mm/sec is 0.5 N to 1.2 N
Hyaluronic acid gel:
9. The method of claim 8,
The cross-linked hyaluronic acid is cross-linked by a combination of the first cross-linking agent and the second cross-linking agent
Hyaluronic acid gel.
A composition comprising the hyaluronic acid gel according to any one of claims 8 to 11,
A composition for a hyaluronic acid filler.
13. The method of claim 12,
further comprising a local anesthetic
A composition for a hyaluronic acid filler.

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