KR20230166560A - Method of prepairing crosslinked hyaluronic acid gel - Google Patents

Abstract

The present invention relates to a method for producing a cross-linked hyaluronic acid gel and a cross-linked hyaluronic acid gel produced by the method. The method for producing a cross-linked hyaluronic acid gel according to one aspect of the present invention can provide a biocompatible and safe cross-linked hyaluronic acid gel, and the cross-linked hyaluronic acid gel produced by the method has excellent resistance to degrading enzymes and a high elastic modulus. has

Description

Method for producing crosslinked hyaluronic acid gel {METHOD OF PREPAIRING CROSSLINKED HYALURONIC ACID GEL}

This specification relates to a method of preparing cross-linked hyaluronic acid gel.

Hyaluronic acid is a linear polymer composed of β-N-acetyl-D-glucosamine and β-D-glucuronic acid alternately bonded, and is known to be an effective moisturizing ingredient. This type of hyaluronic acid is very slippery and sticky, so it has limitations in that it is difficult to use at high concentrations in cosmetic formulations. To overcome this problem, a hyaluronic acid gel was prepared and used by chemical crosslinking.

Conventionally, BDDE (1,4-butanediol diglycidyl ether), PEGDE (polyethylene glycol diglycidyl ether), and DVS (Divinyl sulfones) were used as chemical crosslinking agents to crosslink hyaluronic acid, which combine the OH group of hyaluronic acid and epoxide. ) induces ether bonds between the cross-linking agents of the series.

However, these chemical crosslinking agents all have the potential risk of causing skin irritation, so they require multiple washings during the manufacturing process. If the washing is not sufficient, skin irritation may occur, so caution is required when using them.

In addition, cross-linked hyaluronic acid gel is generally formed by stirring and mixing hyaluronic acid and a cross-linking agent in an aqueous solution and chemically bonding the hyaluronic acid polymer chains with the cross-linking agent. When this cross-linked hyaluronic acid gel is administered to the skin or into the body, After the gel is decomposed, the remaining cross-linking agent component may be recognized as a foreign substance in the living body, causing adverse effects such as causing an inflammatory reaction. Accordingly, there is a need to develop a method for producing cross-linked hyaluronic acid gel with a low cross-linking rate so that it is biocompatible.

Korean Patent Publication No. 10-2020-0048476

In one aspect, the present specification seeks to provide a method for producing a safe, biocompatible cross-linked hyaluronic acid gel.

In one aspect, the present disclosure provides a method comprising: homogenizing a mixture comprising hyaluronic acid or a salt thereof, a cross-linking agent, and water to form a homogenate; And cross-linking the homogenate to form a gel, wherein the hyaluronic acid or a salt thereof has a concentration of more than 100 mg/g in the mixture, and the cross-linking agent includes trimetaphosphate. A manufacturing method is provided.

In an exemplary embodiment, the trimetaphosphate salt is one or more selected from the group consisting of monovalent metal cation salts of trimetaphosphoric acid, divalent metal cation salts of trimetaphosphoric acid, and trivalent metal cation salts of trimetaphosphoric acid. It can be included.

In an exemplary embodiment, the trimetaphosphate may include sodium trimetaphosphate.

In an exemplary embodiment, the stirring may be performed under alkaline conditions.

In an exemplary embodiment, the mixture may include 3 to 90 parts by weight of the crosslinking agent based on 100 parts by weight of the hyaluronic acid or its salt.

In an exemplary embodiment, the crosslinking reaction may be performed at 20 to 70° C. for 0.5 to 36 hours.

In an exemplary embodiment, the weight average molecular weight of the hyaluronic acid or its salt may be 500,000 to 4,000,000 Da.

In an exemplary embodiment, the method for producing the cross-linked hyaluronic acid gel may further include swelling the gel with purified water.

In one exemplary embodiment, the method for producing the cross-linked hyaluronic acid gel may further include the step of pulverizing the swollen gel.

In one aspect, the present specification provides a cross-linked hyaluronic acid gel prepared by the method for producing the cross-linked hyaluronic acid gel.

In one aspect, the present specification is a cross-linked hyaluronic acid gel, wherein the gel includes hyaluronic acid cross-linked with sodium trimetaphosphate, and the weight ratio of the hyaluronic acid and sodium trimetaphosphate is 1:0.03 to 0.9. provides.

In one aspect, the method for producing a cross-linked hyaluronic acid gel disclosed herein can provide a safe, biocompatible cross-linked hyaluronic acid gel.

In one aspect, the cross-linked hyaluronic acid gel prepared according to the method for producing the cross-linked hyaluronic acid gel disclosed herein has high resistance to degrading enzymes.

In one aspect, a cross-linked hyaluronic acid gel prepared according to the method for producing a cross-linked hyaluronic acid gel disclosed herein has a high elastic modulus and excellent flow properties.

Figure 1 shows the form of a hyaluronic acid gel according to an embodiment of the present disclosure.
Figure 2 shows the form of a swollen hyaluronic acid gel according to one embodiment of the present disclosure.
Figure 3 is a graph showing the enzymatic degradation resistance of a hyaluronic acid gel according to an embodiment of the present disclosure.

The terms used in this specification are general terms that are currently widely used as much as possible while considering the function in the present invention, but this may vary depending on the intention of a technician working in the field, precedents, or the emergence of new technology. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in this specification should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the present invention pertains. Generally understood terms should be interpreted as having the same meaning as the meaning they have in the context of the related technology, and unless clearly defined in the present invention, they should not be interpreted in an idealized or excessively formal sense.

Numerical ranges include the numerical values defined herein. Every maximum numerical limit given throughout this specification includes all lower numerical limits as if the lower numerical limit were explicitly written out. Every minimum numerical limit given throughout this specification includes every higher numerical limit as if such higher numerical limit was clearly written. All numerical limits given throughout this specification will include all better numerical ranges within the broader numerical range, as if the narrower numerical limits were clearly written.

As used herein, the words “comprising,” “having,” and “comprising” are inclusive or open-ended and do not exclude additional unstated elements or method steps. As used herein, the term “or combination thereof” refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or any combination thereof" means A, B, C, AB, AC, BC, or ABC, and, if the order is important in a particular context, BA, CA, CB, CBA, BCA. , it is intended to include at least one of ACB, BAC or CAB. Along with this example, combinations containing repetitions of one or more items or terms may be included, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise apparent from the context.

Hereinafter, the present invention will be described in detail.

In one aspect, the present disclosure includes the steps of homogenizing a mixture comprising hyaluronic acid or a salt thereof, a cross-linking agent, and water to form a homogenate; and crosslinking the homogenate to form a gel.

As used herein, “salt” refers to a salt according to one aspect of the present invention that is acceptable in medicine, cosmetics and food and has the desired activity of the parent compound. The salts are (1) formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) Benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-Toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert -acid addition salts formed with organic acids such as butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; or (2) a salt formed when an acidic proton present in the parent compound is replaced. Additionally, the salt may be a pharmaceutically acceptable salt.

As used herein, “pharmaceutically acceptable” means approval by the government or equivalent regulatory body that it can be used in animals, more specifically in humans, by avoiding significant toxic effects when used in normal medicinal dosages. means available, approved, listed in a pharmacopoeia, or otherwise recognized by a general pharmacopoeia.

In one embodiment, the cross-linking agent may include trimetaphosphate. The trimetaphosphate may include, for example, one or more selected from the group consisting of monovalent metal cation salts of trimetaphosphoric acid, divalent metal cation salts of trimetaphosphoric acid, and trivalent metal cation salts of trimetaphosphoric acid. You can. For example, the monovalent metal cation salt of trimetaphosphate may include sodium trimetaphosphate, potassium trimetaphosphate, etc., and the divalent metal cation salt of trimetaphosphate may include calcium trimetaphosphate and barium trimetaphosphate. The trivalent metal cation salt of trimetaphosphoric acid may include aluminum trimetaphosphate, etc.

Sodium trimetaphosphate (STMP) is a substance whose safety has been confirmed. In addition to the main reaction of hyaluronic acid forming a phosphodiester bond with the phosphate group of STMP under strong base conditions, a side reaction with NaOH occurs. Through the process, an incompletely cross-linked form (Pg) and pyrophosphate (ppi) are generated (see Chemical Formula 1 below).

[Formula 1]

This reaction process is the cause of the cross-linking efficiency of hyaluronic acid and STMP being lowered compared to epoxide-based cross-linking agents, and conventional technologies attempted to solve this problem by increasing the amount of STMP used in the cross-linking reaction process or mixing additional cross-linking agents. . However, the inventors of the present invention have completed a method of producing a cross-linked hyaluronic acid gel that exhibits excellent viscoelasticity while minimizing the amount of cross-linking agent used by controlling the concentration of hyaluronic acid.

In one embodiment, the concentration of hyaluronic acid or its salt in the mixture may be greater than 100 mg/g. If the concentration of hyaluronic acid or its salt is 100 mg/g or less, the viscoelasticity of the cross-linked hyaluronic acid gel may decrease. Specifically, the hyaluronic acid or its salt has a concentration in the mixture of more than 100 mg/g, more than 110 mg/g, more than 120 mg/g, more than 130 mg/g, more than 140 mg/g, more than 150 mg/g. , 160 mg/g or more, 170 mg/g or more, 180 mg/g or more, 190 mg/g or more, 200 mg/g or more, 210 mg/g or more, 220 mg/g or more, 230 mg/g or more, 240 mg/g or more, 250 mg/g or more, 260 mg/g or more, 270 mg/g or more, 280 mg/g or more, 290 mg/g or more, or 300 mg/g or more and 450 mg/g or less, 440 mg /g or less, 430 mg/g or less, 420 mg/g or less, 410 mg/g or less, 400 mg/g or less, 390 mg/g or less, 380 mg/g or less, 370 mg/g or less, 360 mg/g It may be less than or equal to 350 mg/g.

In one embodiment, the stirring may be performed under alkaline conditions. For example, the alkaline condition may have a pH of 8 or more, and specifically, the pH may be 8 to 14, 8 to 13.5, 8.5 to 13.5, 8.5 to 13, or 9 to 13.

In one embodiment, the mixture may include 3 to 90 parts by weight of the crosslinking agent based on 100 parts by weight of the hyaluronic acid or its salt. If the content of the cross-linking agent is outside the above range, the physical properties of the cross-linked hyaluronic acid gel may deteriorate. For example, the crosslinking agent is used in an amount of 3 parts by weight or more, 5 parts by weight or more, 7 parts by weight or more, 10 parts by weight or more, 13 parts by weight or more, 15 parts by weight or more, 17 parts by weight or more, based on 100 parts by weight of hyaluronic acid or a salt thereof. or more, 20 parts by weight or more, 23 parts by weight or more, 25 parts by weight or more, 27 parts by weight or more, 30 parts by weight or more, 33 parts by weight or more, 35 parts by weight or more, 37 parts by weight or more, or 40 parts by weight or more, and 90 parts by weight. parts by weight or less, 87 parts by weight or less, 85 parts by weight or less, 83 parts by weight or less, 80 parts by weight or less, 77 parts by weight or less, 75 parts by weight or less, 73 parts by weight or less, 70 parts by weight or less, 67 parts by weight or less, 65 parts by weight It may be included in the mixture in an amount of 63 parts by weight or less, 60 parts by weight or less, 57 parts by weight or less, 55 parts by weight or less, 53 parts by weight or less, or 50 parts by weight or less.

In one embodiment, the crosslinking reaction may be performed for 0.5 to 36 hours. If the crosslinking reaction is performed for less than 0.5 hours, crosslinking may not sufficiently occur. For example, the crosslinking reaction is carried out for 0.5 hours or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more. hour or more, 11 hours or more, or 12 hours or more, but not more than 36 hours, not more than 35 hours, not more than 34 hours, not more than 33 hours, not more than 32 hours, not more than 31 hours, not more than 30 hours, not more than 29 hours, not more than 28 hours, not more than 27 hours It may be performed for less than 25 hours or less than 24 hours.

In one embodiment, the crosslinking reaction may be performed at 20 to 70°C. For example, the crosslinking reaction is performed at 20℃ or higher, 21℃ or higher, 22℃ or higher, 23℃ or higher, 24℃ or higher, 25℃ or higher, 26℃ or higher, 27℃ or higher, 28℃ or higher, 29℃ or higher, or 30℃. or higher, and can be performed at 70°C or lower, 69°C or lower, 68°C or lower, 67°C or lower, 66°C or lower, 65°C or lower, 64°C or lower, 63°C or lower, 62°C or lower, 61°C or lower, or 50°C or lower. there is.

In one embodiment, the weight average molecular weight of the hyaluronic acid or its salt may be 500,000 to 4,000,000 Da. If the weight average molecular weight of hyaluronic acid or its salt is outside the above range, the physical properties of the cross-linked hyaluronic acid gel may deteriorate. For example, the weight average molecular weight of the hyaluronic acid or its salt is 500,000 Da or more, 550,000 Da or more, 600,000 Da or more, 650,000 Da or more, 700,000 Da or more, 750,000 Da or more, 800,000 Da or more, 850,000 Da or more, 900,000 Da or more, More than 950,000 Da or more than 1,000,000 Da, but less than 4,000,000 Da, less than 3,900,000 Da, less than 3,800,000 Da, less than 3,700,000 Da, less than 3,600,000 Da, less than 3,500,000 Da, less than 3,400,000 Da, 3,30 0,000 Da or less, 3,200,000 Da or less, 3,100,000 Da or less, or 3,000,000 It may be less than Da.

In one embodiment, the method for producing a cross-linked hyaluronic acid gel according to an aspect of the present disclosure may further include swelling the gel with purified water. The step of swelling the gel with purified water may be performed by placing the gel in a washing net or dialysis net and using purified water for 12 to 36 hours. For example, the swelling step may be performed for at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, or at least 22 hours and at least 36 hours, at most 34 hours, at most 32 hours, at most 30 hours, 28 hours. It may be performed for less than an hour or less than 26 hours.

In one embodiment, the method for producing a cross-linked hyaluronic acid gel according to an aspect of the present disclosure may further include the step of pulverizing the swollen gel. In the pulverizing step, the swollen gel can be pulverized to a size of 50 to 300 um.

In one aspect, the present disclosure provides a cross-linked hyaluronic acid gel prepared by the method for producing a cross-linked hyaluronic acid gel according to one aspect of the present disclosure.

In one aspect, the present disclosure provides a cross-linked hyaluronic acid gel comprising hyaluronic acid cross-linked with sodium trimetaphosphate.

In one embodiment, the weight ratio of the hyaluronic acid and sodium trimetaphosphate may be 1:0.03 to 0.9. For example, the weight ratio of hyaluronic acid and sodium trimetaphosphate is 1: 0.03 to 0.9, 1: 0.05 to 0.9, 1: 0.05 to 0.8, 1: 0.05 to 0.7, 1: 0.1 to 0.7, 1: 0.1 to 0.6. , 1: 0.3 to 0.6 or 1: 0.5 to 0.6.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

[Preparation Example 1] Preparation of cross-linked hyaluronic acid gel

Sodium hyaluronate (weight average molecular weight: 1-2M Da) and a cross-linking agent with the composition shown in Table 1 were added to the NaOH aqueous solution and stirred for 3 hours to homogenize. The homogenized mixture was placed in a dryer at 35°C and a crosslinking reaction was performed for 24 hours to prepare a gel mixture. HCl was added to the gel mixture to neutralize the pH to about 7, and then the neutralized gel was placed in a washing net and washed and swollen in purified water for 24 hours. Afterwards, the swollen gel was pulverized to a size of 100 to 200 um using an extruder equipped with a 100 um sieve and autoclaved at 121°C to prepare a cross-linked hyaluronic acid gel.

Sodium Hyaluronate
(mg/g) Cross-linking agent (part by weight relative to hyaluronic acid) NaOH (N) 1,4-butanediol diglycidyl ether
(BDDE) polyethylene glycol diglycidyl ether
(PEGDE) sodium trimetaphosphate
(STMP) Example 1 200 - - 30 0.24 Example 2 200 - - 50 0.24 Example 3 200 - - 60 0.24 Example 4 200 - - 60 0.48 Example 5 200 - - 60 One Example 6 250 - - 50 0.24 Example 7 300 - - 5 0.24 Example 8 300 - - 10 0.24 Example 9 300 - - 20 0.24 Comparative Example 1 100 - - 100 0.24 Comparative Example 2 100 - - 200 0.24 Comparative Example 3 200 5 - - 0.24 Comparative Example 4 200 - 10 - 0.24

[Experimental Example 1] Measurement of physical properties of cross-linked hyaluronic acid gel

The viscoelasticity of Examples 1 to 9 and Comparative Examples 1 to 4 was measured using a rheometer (KINEXUS Pro+, USA). The measurement conditions were elasticity (storage modulus, G') and viscosity (loss modulus, G") at a frequency range of 0.1 to 10 Hz, 1 Pa shear stress, and 1 mm gap using a 20 mm plate at 25°C. Results is shown in Table 2 by comparing the G', G", and Tanδ values at 1Hz, and the Tanδ value was calculated according to the formula: Tanδ=G"/G'. Examples 1 to 9 and Comparative Examples 1 to 4 The concentration of hyaluronic acid was measured using a carbazole assay and is shown in Table 2. In addition, the shape of the gel was observed with the naked eye after the crosslinking reaction of Example 8 and Comparative Examples 3 to 4, and the results are shown in Figure 1. The shapes of the swollen gels of Examples 1 to 9 were observed with the naked eye and are shown in Figure 2.

HA concentration (mg/g) 1Hz - G'(Pa) 1Hz - G"(Pa) 1Hz - Tanδ Before sterilization After sterilization Before sterilization After sterilization Before sterilization After sterilization Example 1 3.0 3 0.08 4 0.15 1.33 1.88 Example 2 4.8 25 16 5 6 0.21 0.39 Example 3 5.2 59 46 15 18 0.25 0.40 Example 4 10.7 517 495 169 152 0.33 0.31 Example 5 10.5 181 133 54 43 0.30 0.32 Example 6 15.2 543 521 204 202 0.38 0.39 Example 7 10.8 504 488 158 149 0.31 0.30 Example 8 24.1 1634 1534 522 486 0.31 0.31 Example 9 37.4 4532 4325 1426 1395 0.31 0.32 Comparative Example 1 14.2 276 152 105 87 0.38 0.57 Comparative Example 2 15.5 329 178 122 97 0.37 0.54 Comparative Example 3 22.3 1598 1438 476 413 0.29 0.28 Comparative Example 4 24.9 1425 1373 459 439 0.32 0.31

As shown in Table 2, in the case of Examples 1 to 9, it was confirmed that the loss of viscoelasticity did not occur during the sterilization process and the properties of the gel were stably maintained compared to the Comparative Example. In addition, as can be seen from the results in Figure 1, Comparative Examples 3 to 4 using BDDE or PEGDE as a crosslinking agent were discolored under strong base conditions, but Example 8 using STMP did not discolor.

[Experimental Example 2] Evaluation of enzyme degradation resistance of cross-linked hyaluronic acid gel

To evaluate the enzymatic degradation resistance of the cross-linked hyaluronic acid gel, 1 g of each of Examples 4 to 8 and Comparative Examples 1 to 2 was injected into the Membrane (Float A lyzer, 50 KDa, 1 ml, Spectra). Afterwards, the membrane injected with the cross-linked hyaluronic acid gel was placed in a hyaluronidase mixed solution (concentration: 500 IU) and well sealed with parafilm. While reacting in a shaking incubator at 37°C and 60rpm, the remaining amount of hyaluronic acid gel in the membrane was measured at 8.24 and 48 hours, and the weight compared to the initial weight was converted into percentage and shown in Figure 3.

As shown in Figure 3, it was confirmed that Examples 4 to 8 had higher resistance to hyaluronidase, an enzyme that decomposes hyaluronic acid, compared to Comparative Examples 1 to 2.

[Experimental Example 3] Stability evaluation of cross-linked hyaluronic acid gel

To confirm the stability of the cross-linked hyaluronic acid gel, the degree of loss of viscoelasticity due to heat was measured. Specifically, 5 mL of the cross-linked hyaluronic acid gels of Examples 4, 6 to 8, and Comparative Examples 1 to 2 were placed in tubes, sealed, and stored at 80°C for 50 hours. Afterwards, the storage modulus (G') of each cross-linked hyaluronic acid gel was measured using a rheometer (KINEXUS Pro+, USA) at 25°C and a frequency of 1 Hz, and the results are shown in Table 3. .

Initial G' G' after 50 hours Survival rate (%) Example 4 517 425 82.3 Example 6 543 505 93.1 Example 7 511 467 91.4 Example 8 1589 1421 89.4 Comparative Example 1 276 192 69.6 Comparative Example 2 329 257 78.1

As shown in Table 3, compared to Comparative Examples 1 to 2, it was confirmed that Examples 4 and 6 to 8 had less loss of storage modulus even after being stored at 80°C for 50 hours, showing excellent stability against heat.

Claims (11)

Homogenizing a mixture comprising hyaluronic acid or a salt thereof, a cross-linking agent, and water to form a homogenate; and
Comprising: forming a gel by cross-linking the homogenate,
The hyaluronic acid or its salt has a concentration of more than 100 mg/g in the mixture,
A method for producing a cross-linked hyaluronic acid gel, wherein the cross-linking agent includes trimetaphosphate. According to paragraph 1,
The method wherein the trimetaphosphate salt includes at least one selected from the group consisting of monovalent metal cation salts of trimetaphosphoric acid, divalent metal cation salts of trimetaphosphoric acid, and trivalent metal cation salts of trimetaphosphoric acid. According to paragraph 1,
The method of claim 1, wherein the trimetaphosphate salt includes sodium trimetaphosphate. According to paragraph 1,
The method of claim 1, wherein the homogenization is performed under alkaline conditions. According to paragraph 1,
The mixture includes 3 to 90 parts by weight of the crosslinking agent based on 100 parts by weight of the hyaluronic acid or salt thereof. According to paragraph 1,
The method wherein the crosslinking reaction is performed at 20 to 70° C. for 0.5 to 36 hours. According to paragraph 1,
A method wherein the weight average molecular weight of the hyaluronic acid or its salt is 500,000 to 4,000,000 Da. According to paragraph 1,
The method further comprising swelling the gel with purified water. According to clause 8,
The method further comprising crushing the swollen gel. A cross-linked hyaluronic acid gel prepared by the method according to any one of claims 1 to 9. As a cross-linked hyaluronic acid gel,
The gel comprises hyaluronic acid cross-linked with sodium trimetaphosphate,
A cross-linked hyaluronic acid gel wherein the weight ratio of the hyaluronic acid and sodium trimetaphosphate is 1:0.03 to 0.9.