JP5734536B1 - Carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof and a method for producing the same - Google Patents

Abstract

The method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid includes a step of maintaining a solution of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof at −200 ° C. or higher and 10 ° C. or lower.

Description

  The present invention relates to a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof and a method for producing the same, a water-swellable gel containing the crosslinked product and a method for producing the gel, and a medical material and a cosmetic containing the crosslinked product , And cosmetic materials.

  Hyaluronic acid is widely distributed in living tissues such as chicken crown, umbilical cord, skin, cartilage, vitreous body, and joint fluid, and is widely used, for example, as a component of cosmetics, pharmaceuticals, and foods.

  Patent Document 1 (Japanese Patent Laid-Open No. 5-58881) describes a hyaluronic acid gel and a method for producing the same. However, hyaluronic acid is easily degraded by hyaluronidase in vivo.

JP-A-5-58881

  The present invention relates to a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof having excellent enzyme degradation resistance and moderate thermal stability, a method for producing the same, a water-swellable gel containing the crosslinked product, and A manufacturing method thereof, and a medical material, a cosmetic, and a cosmetic material containing the crosslinked product are provided.

  The inventor of the present application has obtained a cross-linked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof obtained by maintaining at a predetermined temperature in a solution. It was found to have water swellability.

  1. In the method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to one embodiment of the present invention, the solution of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is adjusted to −200 ° C. or higher and 10 ° C. or lower. Holding.

  2. 2. The method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to the above 1, wherein the concentration of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof in the solution is 1% by mass or more and 30% by mass. % Or less.

  3. In the method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof as described in 2 above, the pH of the solution may be 3 or less.

  4). 4. The method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of 1 to 3, wherein the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has an average molecular weight of 30. The carboxymethylation rate for the disaccharide units constituting hyaluronic acid can be 1% or more.

  5. In the method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof according to any one of 1 to 4 above, a composition containing the crosslinked product is obtained by the holding step, and the composition Washing with water and washing the composition with water to remove impurities other than the cross-linked product.

  6). The crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to one embodiment of the present invention is the crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of 1 to 5 above. Obtained by the manufacturing method.

  7). The crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof described in 6 above can have water swellability.

  8). The crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof having water swellability according to one embodiment of the present invention has a degree of swelling with respect to water of 10 to 250 times (mass ratio).

  9. The cross-linked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to 8 above has an average molecular weight of 300,000 or more and a carboxymethylation ratio of 1% or more with respect to a disaccharide unit constituting hyaluronic acid. The carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof may be included.

  10. The carboxymethyl group-containing modified hyaluronic acid and / or a cross-linked product thereof according to any one of 6 to 9 above can have reversible water swellability.

  11. The cross-linked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of 6 to 10 above is the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of 6 to 10 above. The residual ratio after the mixture prepared by dispersing in a physiological saline so that the concentration of the cross-linked product is 1% by mass (solid content) can be stored at 50 ° C. for 72 hours can be 20% or more. .

  12 The cross-linked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of 6 to 11 above is the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of 6 to 11 above. A mixture prepared by dispersing in a 50 mM phosphate buffer so that the concentration of the cross-linked product was 0.1% by mass (solid content) was heated to 40 ° C. in the presence of hyaluronidase (5 units / mg of cross-linked product). The residual rate after storage for 24 hours can be 1% or more.

  13. A water-swellable gel according to an aspect of the present invention includes the carboxymethyl group-containing modified hyaluronic acid and / or salt salt thereof according to any one of the above 6 to 12, and water, and the carboxymethyl group The content of the contained modified hyaluronic acid and / or salt thereof is 0.4% by mass or more and 10% by mass or less in terms of solid content.

  14 The water-swellable gel as described in 13 above can have a residual rate of 20% or more after being stored at 50 ° C. for 72 hours.

  15. The water-swellable gel described in the above 13 or 14 may have a residual ratio of 1% or more after being stored at 40 ° C. for 24 hours in the presence of hyaluronidase (5 units / per mL of gel).

  16. The medical material which concerns on 1 aspect of this invention contains the crosslinked material of the carboxymethyl group containing modified hyaluronic acid in any one of said 6 thru | or 12, and / or its salt.

  17. The medical material described in 16 above can be used as at least one selected from a knee joint injection, an anti-adhesion agent, a subcutaneous injection, and a drug sustained-release agent.

  18. A cosmetic according to one embodiment of the present invention includes the crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof according to any one of 6 to 12 above.

  19. A cosmetic material according to one embodiment of the present invention includes the crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof according to any one of 6 to 12 above.

  According to the method for producing a crosslinked product, by including a step of maintaining a solution of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof at −200 ° C. or higher and 10 ° C. or lower, the enzyme degradation resistance is excellent, and A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof having appropriate thermal stability and water swellability can be obtained. The crosslinked product can be used, for example, as a component of medical materials, cosmetic materials, and cosmetics.

  Moreover, the crosslinked product has water swellability, and can form a stable water-swellable gel when the degree of swelling with respect to water is 10 to 250 times (mass ratio).

  Further, the water-swellable gel contains a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof, and water, and the content of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is solid. When the content is 0.4% by mass or more and 10% by mass or less in terms of minutes, the enzyme is excellent in enzyme degradation resistance and has an appropriate thermal stability.

  Hereinafter, the present invention will be described in detail with reference to the drawings. In the present invention, “parts” means “parts by mass” and “%” means “mass%” unless otherwise specified.

[Method for producing crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof]
The carboxymethyl group-containing modified hyaluronic acid and / or cross-linked product (hereinafter, also simply referred to as “cross-linked product”) according to an embodiment of the present invention. The production method includes a step of maintaining a solution of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof at −200 ° C. or higher and 10 ° C. or lower.

In the present invention, the term "carboxymethyl group" refers to the group represented by _{^"- -} CH ₂ -CO 2 H" or _"-CH 2 -CO _2". Moreover, the raw material modified hyaluronic acid and / or salt thereof used in the production method according to the present embodiment can be produced by the method described later.

<Mechanism of manufacturing method according to this embodiment>
In the production method according to this embodiment, the step of holding reduces the distance between sugar chains constituting the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof, and as a result, the functional groups constituting the sugar chain ( For example, hydrogen bonds are generated between carboxyl groups, between hydroxyl groups, between N-acetyl groups, between carboxyl groups and hydroxyl groups, hydroxyl groups and amino groups, and amino groups and carboxyl groups. As a result, it is presumed that the thermal stability is enhanced by the sugar chains constituting the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof being strongly bonded through hydrogen bonds.

  Further, it is presumed that a sugar chain constituting the carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof is bonded to each other through a hydrogen bond to form a three-dimensional network structure. The crosslinked product according to the present embodiment can form a water-swellable gel, which will be described later, by incorporating water into the three-dimensional network structure.

  In particular, the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has more carboxymethyl groups, so that more carboxymethyl group-containing modified hyaluronic acid and / or salt thereof in one structural unit of the hyaluronic acid skeleton than hyaluronic acid and / or salt thereof. Has a group. That is, since the number of carboxyl groups that can participate in hydrogen bonding in the one structural unit is greater than that of hyaluronic acid and / or its salt, more hydrogen bonds can be formed in the holding step. Guessed.

  Therefore, the crosslinked product according to the present embodiment has excellent thermal stability described later at room temperature. That is, the hydrogen bond of the cross-linked product according to this embodiment is not easily broken at room temperature. In the present invention, “room temperature” means a temperature of 25 ° C. or higher and 30 ° C. or lower.

<Holding temperature>
In the holding step, the hardness of the water-swellable gel obtained using the crosslinked product according to the present embodiment can be adjusted by adjusting the temperature of the solution. For example, in the production method according to the present embodiment, the solution is retained in that a hydrogen-bonded product can be obtained, and a cross-linked product can be obtained as a raw material for a water-swellable gel that is harder and has higher thermal stability. The step is more preferably performed at -1 ° C or lower, further preferably performed at -10 ° C or lower, and is preferably performed at -30 ° C or higher. Moreover, it is more preferable that the step of holding the solution is performed at 0 ° C. or higher and 10 ° C. or lower, in that a crosslinked product as a raw material of a water-swellable gel having flexibility can be obtained. More preferably, it is performed as follows. In addition, the water-swellable gel tends to have higher resistance to enzymatic degradation as it is harder (that is, the degree of swelling of the gel is lower). Therefore, by carrying out the step of holding the solution at 10 ° C. or lower, a water-swellable gel having a low degree of swelling and high resistance to enzymatic degradation can be obtained.

<Retention time>
In the manufacturing method according to this embodiment, the holding step is performed for 400 seconds or more depending on the holding temperature of the solution in the holding step and the type of apparatus for holding the solution at a predetermined holding temperature. In order to increase the heat stability and the enzyme degradation resistance, it is preferable to carry out the reaction for 30 hours or more, while it may be 300 hours or less, and 96 hours. The following times can be done.

<Concentration of raw material modified hyaluronic acid and / or salt thereof in solution>
In the production method according to this embodiment, the concentration of the raw material-modified hyaluronic acid and / or salt thereof in the solution is 1% by mass or more in that hydrogen bonds can be more reliably formed and thermal stability can be further increased. It is preferably 30% by mass or less, more preferably 3% by mass or more, and more preferably 25% by mass or less.

<PH of solution>
In the production method according to the present embodiment, the pH of the solution of the raw material-modified hyaluronic acid and / or salt thereof is 3 or less (0) in that hydrogen bonds can be formed more reliably and thermal stability can be further increased. The range is preferably 3 or less, more preferably 2 or less, and may be 1 or less.

In the holding step, when the pH of the solution of the raw material modified hyaluronic acid and / or salt thereof is 3 or less, the carboxyl group contained in the raw material modified hyaluronic acid and / or salt thereof is converted to the acid form (—CO ₂ H). Therefore, it is presumed that the thermal stability is more excellent because hydrogen bonds are more easily constructed between the carboxyl groups and between the carboxyl group and other functional groups (for example, a hydroxyl group, an amino group, etc.). .

  In addition, as long as the acid used in order to adjust the pH of the said solution to acidity (less than pH7) can adjust the pH of this solution to acidity, any acid can be used. From the viewpoint of reducing the amount of acid used, the acid may be, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid, or an organic acid such as acetic acid or citric acid.

<Solvent>
In the production method according to this embodiment, the solvent for dissolving the raw material-modified hyaluronic acid and / or salt thereof can be a mixture of water and a water-soluble organic solvent that is miscible with water.

  Examples of the water-soluble organic solvent include alcohol solvents such as methanol, ethanol, 1-propanol, and 2-propanol, ketone solvents such as acetone and methyl ethyl ketone, tetrahydrofuran, acetonitrile, and the like. Can be used in combination.

<Swelling dissolution>
Dissolve raw material modified hyaluronic acid and / or its salt in a solvent in order to suppress a decrease in molecular weight of the raw material modified hyaluronic acid and / or its salt solution and to keep the raw material modified hyaluronic acid and / or its salt hygienic. When preparing the solution, it is preferable to swell and dissolve the raw material-modified hyaluronic acid and / or a salt thereof, for example, by holding at a temperature of 0 ° C. or higher and 20 ° C. or lower for 5 hours or more and 48 hours or less.

<Washing process>
In the production method according to the present embodiment, the step of holding by the point that by-products can be removed (particularly, when the solution contains an acid, the pH can be increased by removing the acid). Thus, a composition containing the crosslinked product can be obtained, and the method can further include a step of washing the composition with water to obtain the crosslinked product (isolation of the crosslinked product).

  As the cleaning liquid used in the cleaning step, for example, water and a mixed liquid of water and a water-soluble organic solvent can be used. Here, the water-soluble organic solvent only needs to have low solubility of the modified hyaluronic acid and / or salt thereof. In the step of cleaning, the temperature of the cleaning liquid is 30 ° C. or lower (usually 0 ° C. or higher and 25 ° C. or lower, preferably 3 ° C. or higher, more preferably 4 ° C. or higher). Therefore, it is possible to maintain the properties (thermal stability, water swellability) of the composition according to this embodiment finally obtained.

<Repetition processing>
In the manufacturing method according to the present embodiment, the step of holding and the composition are washed with water in order to more reliably form hydrogen bonds and further improve the thermal stability, and other than the cross-linked product. The combination with the step of removing impurities is preferably repeated one or more times, and can usually be repeated two to five times.

[Raw material modified hyaluronic acid and / or salt thereof]
<Molecular weight of raw material modified hyaluronic acid and / or salt thereof>
The raw material modified hyaluronic acid and / or salt thereof according to this embodiment has a molecular weight of usually 4,000 to 4,000,000. In the present invention, the molecular weight of the raw material-modified hyaluronic acid and / or salt thereof can be measured by the following method.

  Using a gel filtration column, a plurality of (purified) hyaluronic acids (reference substances) with known molecular weights are analyzed by liquid chromatography, and a calibration curve is created from their retention times. Similarly, the molecular weight of the raw material-modified hyaluronic acid can be determined by performing liquid chromatography analysis on the raw material-modified hyaluronic acid to be measured and determining the molecular weight using the calibration curve.

  Examples of the liquid chromatography analyzer that can be used for the liquid chromatography analysis include Waters Alliance 2690 HPLC Separations Module (manufactured by Waters), Waters Alliance 2695 HPLC Separations Module (manufactured by Waters), 1200 Series Manufactured). In addition, as a column that can be used for liquid chromatography analysis, for example, a column for ligand exchange chromatography (ligand exchange mode + size exclusion mode) manufactured by shodex, model name “SUGAR KS-801”, "SUGAR KS-802", "SUGAR KS-803", "SUGAR KS-804", "SUGAR KS-805", "SUGAR KS-806", "SUGAR KS-807", and a size exclusion chromatography column made by TOSOH Model name “TSKgel GMPW”.

  In the production method according to the present embodiment, the average molecular weight of the raw material-modified hyaluronic acid and / or salt thereof is preferably 300,000 or more, more preferably 500,000 or more, in that hydrogen bonds can be obtained more reliably. Is more preferable (usually 3 million or less and 2 million or less).

In the present invention, the carboxymethylation rate (hereinafter also simply referred to as “carboxymethylation rate”) of the disaccharide unit constituting the hyaluronic acid of the modified hyaluronic acid and / or salt thereof having a carboxyl group is ¹ H-NMR. In the spectrum, the carboxymethyl group (—CH ₂ ) relative to the integrated value of the peak (expressed in the vicinity of 2 ppm) indicating the proton of the methyl group (—CH ₃ ) of the N-acetyl group bonded to the C-2 position in the hyaluronic acid skeleton. Ratio of integrated value of peak (expressed in a range of 3.8 ppm or more and 4.2 ppm or less) indicating a proton of a methylene group (—CH ₂ —) in —CO ₂ H or —CH ₂ —CO ₂ ^— ) It is represented by

  In the present invention, the “disaccharide unit constituting hyaluronic acid” refers to one unit composed of disaccharides (glucuronic acid and N-acetylglucosamine) adjoining to constitute hyaluronic acid. “Carboxymethylation rate with respect to disaccharide units constituting an acid” is the number of carboxymethyl groups contained in one unit relative to the one unit, and more specifically, when the unit is 100%. The ratio (%) of the number of carboxymethyl groups contained in one unit relative to the one unit.

<Carboxymethylation rate of raw material modified hyaluronic acid and / or salt thereof>
In the production method according to this embodiment, the carboxymethylation rate of the raw material-modified hyaluronic acid and / or a salt thereof is 1% or more in that hydrogen bonds can be more reliably formed and thermal stability can be further increased. It is preferably 10% or more, more preferably 50% or more, while it is usually 400% or less and can be 200% or less.

<Yield>
In the manufacturing method according to the present embodiment, the composition containing the crosslinked product is obtained by the holding step, and the composition further includes a step of obtaining the crosslinked product by washing the composition with water. The mass of the cross-linked product with respect to the mass of is preferably 30% or more, more preferably 40% or more, and further preferably 50% or more. The yield can be calculated by the method shown in the examples of the present application.

[Method for producing raw material-modified hyaluronic acid and / or salt thereof]
In the production method according to this embodiment, the raw material carboxymethyl group-containing modified hyaluronic acid and / or salt thereof (hereinafter also referred to as “raw material modified hyaluronic acid and / or salt thereof”) has a temperature of, for example, 30 ° C. It can be obtained by reacting the raw material hyaluronic acid and / or its salt with haloacetic acid and / or its salt in the following hydrous solvent.

  In the reaction step, at least a part of the raw material hyaluronic acid and / or its salt (preferably all or most of hyaluronic acid and / or its salt) and haloacetic acid and / or its salt in the reaction solution (hydrous solvent). The hyaluronic acid and / or salt thereof and the haloacetic acid and / or salt thereof can be reacted in the state where the salt is dissolved. In this case, the reaction solution may be visually transparent in that hyaluronic acid and / or a salt thereof and haloacetic acid and / or a salt thereof are dissolved.

<Raw material hyaluronic acid and / or salt thereof>
In the present invention, “hyaluronic acid” refers to a polysaccharide having one or more repeating structural units composed of disaccharides of glucuronic acid and N-acetylglucosamine. The “hyaluronic acid salt” is not particularly limited, but is preferably a food or pharmaceutically acceptable salt, for example, sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, ammonium salt, etc. Is mentioned.

  Hyaluronic acid is basically a disaccharide or higher sugar containing at least one disaccharide unit in which the 1-position of β-D-glucuronic acid and the 3-position of β-D-N-acetyl-glucosamine are combined, and β It is basically composed of -D-glucuronic acid and β-DN-acetyl-glucosamine and is a combination of a plurality of disaccharide units. The sugar may be an unsaturated sugar, and examples of the unsaturated sugar include non-reducing terminal sugars, usually those having unsaturated carbon atoms between positions 4 and 5 of glucuronic acid.

  Hyaluronic acid and / or its salt (raw hyaluronic acid and / or its salt) used to produce raw material-modified hyaluronic acid and / or its salt are natural products such as animals (eg chicken crown, umbilical cord, skin, joint fluid) May be extracted from living tissues such as, or obtained by culturing microorganisms, animal cells or plant cells (for example, fermentation using bacteria of the genus Streptococcus), chemical or enzymatic Those synthesized in the above can be used.

  As the raw material hyaluronic acid and / or a salt thereof, either the crude extract or the purified product may be used, but the purity is 90% in that the purified product, specifically, carboxymethylation can proceed smoothly. It is preferable to use raw material hyaluronic acid and / or a salt thereof having a (mass ratio) above.

<Average molecular weight of raw material hyaluronic acid and / or salt thereof>
The average molecular weight of the raw material hyaluronic acid and / or a salt thereof is usually preferably 4,000 or more and 4 million or less, more preferably 3 million or less, from the viewpoint that carboxymethylation can be carried out smoothly. The average molecular weight of the raw material hyaluronic acid and / or salt thereof can be measured by the following method.

<Measurement method of molecular weight>
That is, about 0.05 g of (purified) hyaluronic acid (this product) was accurately weighed and dissolved in 0.2 mol / L sodium chloride solution to make exactly 100 mL, and 8 mL, 12 mL, and 16 mL of this solution were accurately measured. A sample solution is prepared by adding 0.2 mol / L sodium chloride solution to each to make exactly 20 mL. This sample solution and a 0.2 mol / L sodium chloride solution were 30.0 ± 0.1 ° C. according to the viscosity measurement method (first method capillary viscometry method) of the Japanese Pharmacopoeia (16th revision) general test method. The specific viscosity is measured by (Equation (A)), and the reduced viscosity at each concentration is calculated (Equation (B)). A graph is drawn with the reduced viscosity on the vertical axis and the concentration (g / 100 mL) of the product converted to dry matter on the horizontal axis, and the intrinsic viscosity is determined from the intersection of the straight line connecting the points and the vertical axis. The intrinsic viscosity calculated here is substituted into Laurent's formula (formula (C)), and the average molecular weight is calculated (Torvar C Laurent, Marion Ryan, and Adolph Pietroszkiewicz, "Fractionation of hydronic acid ac. , 42, 476-485 (1960), Chikako Yokata, “Molecular weight evaluation of sodium hyaluronate preparation by SEC-MALLS”, National Institute of Health, 121, 030-033 (2003)).
(Formula A) Specific viscosity = {(required flow time of sample solution) / (required flow time of 0.2 mol / L sodium chloride solution)}-1
(Formula B) Reduced viscosity (dL / g) = specific viscosity / (concentration g / 100 mL with respect to the dried product converted to this product))
(Formula C) Intrinsic viscosity (dL / g) = 3.6 × 10 ⁻⁴ M ^0.78
M: average molecular weight

<Content of raw material hyaluronic acid and / or salt thereof>
In the raw material hyaluronic acid and / or its salt, the content of hyaluronic acid and / or its salt is an indicator of the purity of the raw material hyaluronic acid and / or its salt, and the higher the content of hyaluronic acid and / or its salt, It can be said that the raw material hyaluronic acid and / or a salt thereof has high purity.

  In the present invention, the content of hyaluronic acid in the raw material hyaluronic acid and / or its salt is a value calculated from the glucuronic acid quantitative value measured by the carbazole sulfate method (for example, Japanese Pharmacopoeia).

  In the carbazole sulfate method, an aqueous solution of hyaluronic acid is added and mixed in a sodium borate / sulfuric acid solution, the hyaluronic acid is decomposed by heating, cooled, mixed with a carbazole / ethanol solution, heated and left to cool. This is a method for measuring absorbance (530 nm). A calibration curve is prepared using D-glucuronolactone processed in the same manner, and a D-glucuronolactone conversion value is calculated, and then multiplied by 1.102 to obtain a glucuronic acid quantitative value. The obtained glucuronic acid quantitative value is multiplied by (molecular weight of hyaluronic acid / molecular weight of glucuronic acid) to calculate the content of hyaluronic acid.

<Carboxymethylation>
In the present invention, “modified hyaluronic acid and / or salt thereof” refers to hyaluronic acid and / or a salt thereof in which an organic group is introduced at least partially, and is different from hyaluronic acid and / or a salt thereof. It has a structure. In the present invention, the “organic group” refers to a group having a carbon atom.

  Accordingly, in the present invention, “carboxymethyl group-containing modified hyaluronic acid and / or salt thereof” refers to hyaluronic acid and / or a salt thereof into which a carboxymethyl group has been introduced at least partially.

More specifically, in the raw material modified hyaluronic acid and / or its salt, for example, a hydroxyl group constituting hyaluronic acid (see the following formula (1)) (in the following formula (1), N-acetylglucosamine constituting the hyaluronic acid Hydrogen atoms of at least some of the hydroxyl groups of C-4 position, C-6 position, and C-2 position and C-3 position of glucuronic acid constituting hyaluronic acid are -CH ₂ -CO ₂ H and And / or a group represented by —CH ₂ —CO ₂ ^— ). That is, in the raw material-modified hyaluronic acid and / or a salt thereof, the hydrogen atom of the hydroxyl group at one or more of the hydroxyl groups at these positions is —CH ₂ —CO ₂ H and / or —CH ₂ —CO. ₂ ^- may be substituted with a group represented by.

（式中、ｎは１以上７，５００以下の数を示す。）

(In the formula, n represents a number of 1 to 7,500.)

  The raw material modified hyaluronic acid and / or salt thereof can be, for example, a compound represented by the following formula (2).

（式中、Ｒ^１〜Ｒ^５は独立して、水酸基、−ＣＨ_２−ＣＯ_２Ｈ、または−ＣＨ_２−ＣＯ_２ ⁻で表される基を表し（ただし、Ｒ^１〜Ｒ^５がいずれも水酸基を表す場合を除く。）ｎは１以上７，５００以下の数を示す。）

(In the formula, R ^{1 to} R ⁵ independently represent a group represented by a hydroxyl group, —CH ₂ —CO ₂ H, or —CH ₂ —CO ₂ ^— (wherein R ^{1 to} R ⁵ are all Except for the case of representing a hydroxyl group.) N represents a number of 1 to 7,500.)

<PH>
In the production method according to this embodiment, the reaction of the raw material hyaluronic acid and / or salt thereof with haloacetic acid and / or salt thereof is carried out under basic conditions in that the nucleophilicity of the hydroxyl group can be enhanced. The pH of the reaction solution (hydrous solvent) is preferably 9 or more (9 or more and 14 or less, preferably 10 or more and 14 or less, more preferably 11 or more and 14 or less).

  In this case, a basic electrolyte can be used in the reaction solution in order to adjust the reaction solution to basic. Examples of the basic electrolyte include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and alkaline earth metal hydroxides such as calcium hydroxide, magnesium hydroxide and barium hydroxide. The concentration of the basic electrolyte in the reaction solution is, for example, 0.2 mol / L or more and 10 mol / L or less, preferably 0 in that the raw material-modified hyaluronic acid and / or salt thereof can be obtained efficiently. .5 mol / L or more and 8 mol / L or less.

  In this case, the concentration of the hyaluronic acid in the water-containing solvent is preferably 0.05 g / mL or more and 0.5 g / mL or less.

<Haloacetic acid and / or salt thereof>
In the method for producing a raw material-modified hyaluronic acid and / or salt thereof according to this embodiment, haloacetic acid and / or a salt thereof is used for introducing a carboxymethyl group into the raw material hyaluronic acid and / or a salt thereof.

  The haloacetic acid can be, for example, monohaloacetic acid and / or a salt thereof, and more specifically, chloroacetic acid and / or a salt thereof, or bromoacetic acid or a salt thereof is preferable. The salt of haloacetic acid is preferably, for example, an alkali metal salt of chloroacetic acid and / or an alkali metal salt of bromoacetic acid, and more preferably sodium chloroacetate and / or sodium bromoacetate.

<Reaction temperature>
When bromoacetic acid and / or a salt thereof is used as haloacetic acid and / or a salt thereof, the temperature of the reaction solution is 10 ° C. or less (for example, more than 0 ° C. It is preferable to carry out the reaction in the following).

  For example, to produce a raw material modified hyaluronic acid and / or a salt thereof having a high molecular weight (for example, 800,000 or more) and a high carboxymethylation rate (for example, 50% or more, preferably 50% or more and 200% or less). The reaction may be carried out using bromoacetic acid and / or a salt thereof as a haloacetic acid and / or a salt thereof at a reaction solution temperature of 10 ° C. or lower (for example, more than 0 ° C. and 10 ° C. or lower). preferable.

<Amount of haloacetic acid and / or salt thereof>
The amount of haloacetic acid and / or its salt used is usually 10% or more and 500% or less (mass ratio) of the amount of raw material hyaluronic acid and / or its salt used, and 50% or more and 200% or less (mass ratio). Is preferred.

<Water-containing solvent>
In the method for producing a raw material-modified hyaluronic acid and / or salt thereof according to this embodiment, the water-containing solvent is water, or a water-soluble organic solvent and water, because the raw material hyaluronic acid and / or salt thereof is highly soluble. A mixed solution of

  When the water-containing solvent is a mixed solution of a water-soluble organic solvent and water, that is, when the water-containing solvent contains both water and a water-soluble organic solvent, the mixed solution can be improved in that the solubility of hyaluronic acid can be increased. The ratio of the water-soluble organic solvent in the solvent is preferably 60 v / v% or less (over 0 v / v% to 60 v / v%), and 40 v / v% or less (over 0 v / v% to 40 v / v % Or less) is more preferable.

  Examples of the water-soluble organic solvent include alcohol solvents such as methanol, ethanol, 1-propanol, and 2-propanol, ketone solvents such as acetone and methyl ethyl ketone, tetrahydrofuran, acetonitrile, and the like. Can be used in combination. Of these, lower alcohols having 1, 2 or 3 carbon atoms such as isopropanol and ethanol are preferred.

<Reaction temperature>
In the reaction, it is preferable that the temperature of the reaction solution is usually 30 ° C. or lower (preferably higher than 0 ° C. and 30 ° C. or lower) from the viewpoint that carboxylation can proceed smoothly and a decrease in molecular weight can be suppressed. More preferably, it is 10 ° C. or lower (preferably higher than 0 ° C. and 30 ° C. or lower). In particular, by setting the temperature of the reaction solution to 10 ° C. or lower, a high molecular weight (800,000 or higher) raw material-modified hyaluronic acid and / or salt thereof can be easily obtained.

  For example, when chloroacetic acid and / or a salt thereof are used as haloacetic acid and / or a salt thereof, carboxymethylation can proceed smoothly and browning of the resulting raw material-modified hyaluronic acid and / or salt thereof can be suppressed. In that respect, the temperature of the reaction solution in the reaction can be normal (preferably higher than 0 ° C. and 30 ° C. or lower), and preferably 1 ° C. or higher and 30 ° C. or lower.

  For example, when bromoacetic acid and / or a salt thereof are used as haloacetic acid and / or a salt thereof, carboxymethylation can proceed smoothly, and the resulting raw material-modified hyaluronic acid and / or salt thereof is browned and the molecular weight is decreased. The temperature of the reaction solution in the reaction is usually 10 ° C. or lower (preferably higher than 0 ° C. and 10 ° C. or lower), and preferably 1 ° C. or higher and 10 ° C. or lower.

<Reaction time>
In the reaction, the reaction time is usually preferably 30 minutes or longer and 100 hours or shorter, more preferably 60 minutes or longer and 60 hours or shorter from the viewpoint that the carboxylation can proceed smoothly and the decrease in molecular weight can be suppressed. preferable.

[Crosslinked product]
The cross-linked product according to one embodiment of the present invention is obtained by the method for producing a cross-linked product according to the above embodiment. The cross-linked product according to the present embodiment can have water swellability.

  In the present invention, the “water swellability” means a property of taking up water and swelling, and generally means a property of taking up water and becoming a gel. The cross-linked product according to this embodiment has a three-dimensional network structure formed by hydrogen bonding between functional groups generated by the holding step, and swells by incorporating water into the three-dimensional network structure. Can constitute a gel.

  The cross-linked product according to this embodiment has the same chemical structural formula, average molecular weight and carboxymethylation as the raw material modified hyaluronic acid and / or salt thereof (carboxymethyl group-containing modified hyaluronic acid and / or salt thereof). However, due to the above-described formation of hydrogen bonds, it has different physical properties from the raw material-modified hyaluronic acid and / or its salt (water expansibility and thermal stability described later).

  Therefore, the crosslinked product (carboxymethyl group-containing modified hyaluronic acid and / or salt thereof) according to this embodiment can have an average molecular weight of 300,000 or more and a carboxymethylation rate of 1% or more. Moreover, the crosslinked product (carboxymethyl group-containing modified hyaluronic acid and / or salt thereof) according to the present embodiment has a structure represented by the above-described formula (2).

<Swelling degree>
The cross-linked product according to one embodiment of the present invention has water swellability, and the degree of swelling with respect to water is 10 to 250 times (mass ratio).

  In the present invention, “gel” refers to a polymer having a three-dimensional network structure, and “water-swellable gel” refers to a swollen body that takes in and holds water inside the three-dimensional network structure. Okay, more specifically, filter paper No. for Kiriyama funnel. A polymer having the property that water does not flow out of the filter paper when placed on 707 for 1 hour. In the present invention, the “swelling degree” means the mass of water (the mass ratio of the crosslinked product to water) relative to the mass of the crosslinked product in the water-swellable gel.

  The degree of swelling of the cross-linked product according to the present embodiment is more preferably 20 times or more (mass ratio), and on the other hand, it is 300 times or less (mass ratio) in that water can be retained more stably. More preferably, it is more preferably 250 times or less (mass ratio), may be 200 times or less (mass ratio), and may be 100 times or less (mass ratio) when it is a hard gel, It is preferably 50 times or less (mass ratio).

<Reversible water swellability>
One of the features of the cross-linked product according to this embodiment is that it has reversible water swellability. In the present invention, the term “reversible water swellability” means that when water is added, it swells into a gel, and when the gel is dried to remove water, it becomes a solid, and the gel is converted into a solid. It refers to the property of being able to repeat changes and changes from solid to gel. The reversible water swellability of the cross-linked product according to this embodiment can be maintained unless the cross-linked product is heated or the cross-linked product is exposed to basic conditions.

  Since the cross-linked product according to this embodiment has reversible water swellability, the change from gel to solid and the change from solid to gel can be repeated many times.

<Storage stability>
Since the crosslinked product according to this embodiment is a solid, it can be stored as a solid. Polysaccharides such as hyaluronic acid and the cross-linked product according to this embodiment are usually more resistant to degradation when stored in a solid state than when stored in the presence of water (for example, in an aqueous solution or gel state). Can do. For this reason, when the crosslinked product according to the present embodiment is stored as a solid, the storage stability is excellent.

<Enzyme degradation resistance>
The cross-linked product according to this embodiment has an enzyme-resistant (hyaluronidase) degradability. More specifically, a mixture prepared by dispersing in a 50 mM phosphate buffer so that the concentration of the cross-linked product according to the present embodiment is 0.1% by mass (solid content) is treated with hyaluronidase (5 units / unit). The residual ratio after storage for 24 hours at 40 ° C. in the presence of 1 mg of the crosslinked product is preferably 1% or more, and more preferably in the range of 10% or more and 100% or less. The enzyme degradation resistance can be measured, for example, by the method shown in the examples of the present application.

  One of the causes that the cross-linked product according to the present embodiment is excellent in enzyme degradation resistance is that the cross-linked product according to the present embodiment contains a carboxymethyl group. When the cross-linked product according to this embodiment contains a carboxymethyl group, hyaluronidase is difficult to recognize the hyaluronic acid skeleton contained in the cross-linked product according to this embodiment, and thus it is estimated that the enzyme degradation resistance is improved. .

In addition, it can identify that the crosslinked material which concerns on this embodiment contains a carboxymethyl group by presence of the peak which appears in 3.8 ppm or more and 4.2 ppm or less in ¹ H-NMR spectrum analysis of a crosslinked material, for example. it can.

<Thermal stability>
The crosslinked product according to this embodiment is excellent in thermal stability. More specifically, after the mixture prepared by dispersing in physiological saline so that the concentration of the cross-linked product according to this embodiment is 1% by mass (solid content) is stored at 50 ° C. for 72 hours. The residual rate is preferably 20% or more, and the residual rate after storage for 96 hours is more preferably 20% or more (more preferably, the residual rate after storage for 168 hours is 20% or more). The thermal stability can be measured by, for example, the method shown in the examples of the present application.

  According to the cross-linked product according to the present embodiment, the inclusion of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof includes more carboxyl groups that can participate in hydrogen bonding than hyaluronic acid and / or a salt thereof. Therefore, it is more excellent in thermal stability.

  In addition, since the thermal stability of the cross-linked product according to the present embodiment is due to the hydrogen bond described above, the thermal stability gradually decreases with time. More specifically, the cross-linked product according to the present embodiment has a property that hydrogen bonds are gradually dissociated by heat existing in the living body (for example, around 37 ° C. to 40 ° C.), and thus has an appropriate thermal stability. .

[Water-swellable gel]
<Content of cross-linked product>
The water-swellable gel according to an embodiment of the present invention includes a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof, and water, and the content of the crosslinked product is 0.00 on a solids basis. 4 mass% or more (may be 0.5 mass% or more) and 10 mass% or less.

  The ratio of the water contained in the water-swellable gel according to this embodiment is the ratio of the water-swellable gel according to this embodiment to the mass difference before and after removing the water by drying the water-swellable gel according to this embodiment. It can be calculated from the mass before drying. In addition, drying (removal of water) of the water-swellable gel according to the present embodiment can be performed by, for example, vacuum drying, reduced pressure drying, or freeze drying.

  It is preferable that the pH of the water-swellable gel according to the present embodiment is 3 or more and 8 or less because it is more excellent in thermal stability.

<Thermal stability>
The water-swellable gel according to this embodiment is excellent in thermal stability. More specifically, the water-swellable gel according to this embodiment preferably has a residual rate of 20% or more after being stored at 50 ° C. for 72 hours, and has a residual rate of 20% after being stored for 96 hours. More preferably (the remaining ratio after storage for 168 hours is 20% or more).

<Enzyme degradation resistance>
The water-swellable gel according to this embodiment is excellent in enzyme degradation resistance. More specifically, the water-swellable gel according to the present embodiment preferably has a residual ratio of 1% or more after storage for 24 hours at 40 ° C. in the presence of hyaluronidase (5 units / per mL of gel). More preferably, it is in the range of 10% or more and 100% or less.

[Medical materials]
The medical material which concerns on one Embodiment of this invention contains the crosslinked material (for example, water-swellable gel) which concerns on the said embodiment. By including the cross-linked product according to the above embodiment, the medical material according to the present embodiment is excellent in enzyme degradation resistance and thermal stability, and can be stored in a solid state, so that it has excellent storage stability. Yes.

  In particular, when the medical material according to the present embodiment includes the water-swellable gel according to the above-described embodiment, the medical material according to the above-described embodiment results from the reversible water-swellability of the water-swellable gel according to the above-described embodiment. Since the water-swellable gel can be prepared by adding water to the crosslinked product according to the form, the handling property is excellent.

  The medical material according to the present embodiment can be used for applications that generally require in-vivo compatibility. Examples of the use include knee joint injections, anti-adhesion agents, subcutaneous injections, drug sustained-release agents, pharmacologically active substance carriers, wound dressings, artificial skin, surgical sutures, hemostatic agents, artificial organs, Examples include medical tools and medical devices.

<Knee joint injection>
The medical material according to the present embodiment can be used as, for example, a knee joint injection. When the medical material which concerns on this embodiment contains the water-swellable gel which concerns on the said embodiment, it is excellent in moderate elasticity, moderate thermal stability, and the outstanding enzyme degradability. For this reason, when the medical material according to this embodiment is used as, for example, a knee joint injection, the water-swellable gel according to the above embodiment remains in the living body without being decomposed for a certain period of time, and then in vivo Since it is decomposed, adhesion between tissues can be prevented and it is excellent in safety.

  For example, the water-swellable gel according to the above embodiment has, for example, a carboxymethyl group-containing modified hyaluronic acid having an average molecular weight of 300,000 to 2,000,000 and a carboxymethylation rate of 1% or more and / or its In the case of containing salt, it has an appropriate flexibility, so that it can provide a good feel when injected into the knee joint.

<Molded body>
Since the medical material according to the present embodiment includes the cross-linked product according to the above-described embodiment, it can be formed according to the application site, for example, gel, sheet, film, particle, fiber, sponge, etc. Can be tubular, can be used for surgery. More specifically, the medical material according to the present embodiment in the form of a sheet or film can be attached to the affected area. Moreover, when the medical material which concerns on this embodiment contains the water-swellable gel which concerns on the said embodiment, for example, in endoscopic surgery, the medical material which concerns on this gel-like embodiment is affected using an endoscope. Therefore, it can be suitably used as an anti-adhesion agent.

<Anti-adhesion agent / subcutaneous injection>
The cross-linked product (for example, water-swellable gel) according to the above-described embodiment included in the medical material according to the present embodiment is excellent in moderate thermal stability and excellent enzyme degradation resistance. For this reason, when the medical material according to the present embodiment is used as, for example, an antiadhesive agent or a subcutaneous injection, the water-swellable gel according to the above embodiment remains without being decomposed for a certain period of time in vivo, Since it is decomposed in vivo, it can prevent adhesion between tissues and is excellent in safety.

<Slow drug release agent>
The medical material which concerns on this embodiment can be used as a drug sustained release agent, for example. When the medical material which concerns on this embodiment contains the water-swellable gel which concerns on the said embodiment, it is excellent in water retention, moderate thermal stability, and the outstanding enzymatic degradation resistance. For this reason, when the medical material according to this embodiment is used as, for example, a drug sustained-release agent, the water-swellable gel according to the above embodiment remains in the living body without being decomposed for a certain period of time, and then in vivo Since it is decomposed, it has an effect of assisting the sustained release of the drug and is excellent in safety.

  In addition, the medical material according to the present embodiment is blended with a bulking agent, a binder, a lubricant, a preservative, an antioxidant, a fragrance, a sweetener, an acidulant, an excipient, and the like as necessary. Can do. In addition, vitamins such as vitamin C, vitamin B2, vitamin B12, and vitamin E, various nutritional components such as nutritional components such as nucleic acid, chondroitin sulfate, and collagen, and mineral components such as iron and zinc can also be blended.

[Cosmetics]
A cosmetic according to an embodiment of the present invention includes a cross-linked product (for example, a water-swellable gel) according to the above-described embodiment.

<Water retention effect>
In addition, the crosslinked product according to the present embodiment has a high water retention effect due to the carboxyl group constituting the crosslinked product. More specifically, since the carboxyl group contained in the cross-linked product according to the present embodiment forms a hydrogen bond with water, it is presumed that the water retention power is excellent due to the carboxyl group. For this reason, it has a high water retention effect in biological tissues such as skin. Therefore, a high moisturizing effect can be achieved by using the crosslinked product according to the present embodiment as, for example, a cosmetic component.

  Therefore, the cross-linked product according to the above embodiment included in the cosmetic according to the present embodiment is excellent in high water retention effect, moderate thermal stability and excellent enzyme degradation resistance, and therefore when used as a cosmetic, Highly moisturizing action lasts long because it decomposes gradually in the body. In addition, when the cosmetic according to the present embodiment includes the water-swellable gel according to the above-described embodiment, the gel has an appropriate elasticity, and therefore, when blended in the cosmetic, a gel-specific tactile sensation can be generated. Moreover, the active ingredient can be released gradually by blending the active ingredient in the gel.

  The cross-linked product (for example, water-swellable gel) according to this embodiment may be applied to or ingested on the surface of a living tissue, and particularly applied to skin such as the face, arms, fingers, feet, and joints. Or it is preferable to contact.

  Although the aspect of the cosmetics which concern on this embodiment is not specifically limited, For example, skin cosmetics are mentioned. By using the cross-linked product according to the above embodiment for skin cosmetics, it has an appropriate viscosity and has a high water retention effect, so it can moisturize the skin and improve the feeling of bulkiness of the skin. .

  Examples of the cosmetic for skin according to the present embodiment include, for example, face wash, cleanser, lotion (for example, whitening lotion), cream (for example, vanishing cream, cold cream), milky lotion, cosmetic liquid, pack ( For example, jelly-type peel-off type, paste-type wipe-off type, powder-type wash-out type), cleansing, foundation, lipstick, lip balm, lip gloss, lip liner, blusher, shaving lotion, after sun lotion, deodorant lotion, body lotion (hand care lotion) Body oils, soaps, and bath additives.

  The cosmetics according to this embodiment may further contain the following components. Examples of the component include cationized polysaccharides (eg, cationized hyaluronic acid, cationized hydroxyethyl cellulose, cationized guar gum, cationized starch, cationized locust bean gum, cationized dextran, cationized chitosan, and cationized honey). ), Anionic surfactants (eg, alkylbenzene sulfonates, polyoxyalkylene alkyl ether sulfates, alkyl sulfates, olefin sulfonates, fatty acid salts, dialkylsulfosuccinates, etc.), nonionic surfactants (eg , Polyoxyethylene fatty acid esters, polyoxyethylene hydrogenated castor oil derivatives, etc.), cationic surfactants (eg, alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkylpyridines) Um salt, stearyltrimethylammonium chloride, etc.), amphoteric surfactant (eg, alkylbetaine, alkylamidopropylbetaine, imidazolinium betaine, egg yolk lecithin, soybean lecithin, etc.), oil (eg, silicone, silicone derivatives, liquid paraffin, Squalane, beeswax, carnauba wax, olive oil, avocado oil, camellia oil, jojoba oil, horse oil, etc.), moisturizer (eg, sodium hyaluronate, hydrolyzed hyaluronic acid, acetylated hyaluronic acid, dimethylsilanol hyaluronate, ceramide, lauroyl glutamic acid) Diphytosteryl octyldodecyl, phytoglycogen, hydrolyzed eggshell membrane, trehalose, glycerin, atelocollagen, sorbitol, maltitol, 1,3-butylene glycol, etc.), higher fat (Eg, lauric acid, behenic acid, palmitic acid, stearic acid, isostearic acid, oleic acid, etc.), higher alcohols (eg, cetyl alcohol, stearyl alcohol, behenyl alcohol, isostearyl alcohol, batyl alcohol, etc.), polyhydric alcohols (eg, Glycerin, diglycerin, 1,3-propanediol, propylene glycol, polyethylene glycol, pentylene glycol, etc.), thickener (eg, cellulose ether, carboxyvinyl polymer, xanthan gum, dextrin palmitate, etc.), amphoteric polymer resin Compound (for example, betanated dialkylaminoalkyl acrylate copolymer), cationic polymer resin compound (for example, vinylpyrrolidone / dimethylaminoethyl methacrylate copolymer) Combined cationized products, polydimethyldiallylammonium halide type cationic polymers, etc.), preservatives (eg, methylparaben, ethylparaben, butylparaben, propylparaben, phenoxyethanol, etc.), antioxidants (eg, tocophenol, BHT, etc.), metals Blocking agents (for example, edetate, etidronate, etc.), UV absorbers (for example, benzophenone derivatives, paraaminobenzoic acid derivatives, methoxycinnamic acid derivatives, etc.), UV reflectors (for example, titanium oxide, zinc oxide, etc.), Protein hydrolyzate (eg, keratin peptide, collagen peptide, soybean peptide, wheat peptide, milk peptide, silk peptide, egg white peptide, etc.), amino acid (eg, arginine, glutamic acid, glycine, alanine, hydroxyproline) Cysteine, serine, L-theanine, etc.), natural product extracts (cudin extract, kazil extract, seaweed extract, eucalyptus extract, royal jelly extract, rosemary extract, beech tree extract, etc.), other functional ingredients (coenzyme Q10, arbutin, polyquaternium 51 , Elastin, platinum nanocolloid, retinol palmitate, panthenol, allantoin, lysine dilauroyl glutamate, ascorbyl magnesium phosphate, 2-glucoside L-ascorbate, ellagic acid, kojic acid, linoleic acid, tranexamic acid, etc.), phosphorus A lipid polymer, a fragrance | flavor, and a pigment | dye are mentioned.

[Beauty materials]
The cosmetic material according to one embodiment of the present invention includes the cross-linked product (for example, a water-swellable gel) according to the above-described embodiment. By injecting the cosmetic material according to the present embodiment into, for example, the face, head, neck, chest, abdomen, buttocks, back, waist, upper limbs, and lower limbs, cosmetic effects (for example, breast augmentation, facial beauty, legs) Etc. for improving the appearance). The cosmetic material according to the present embodiment has excellent enzyme degradation resistance and moderate thermal stability.

[Example]
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to an Example.

<Example 1: Preparation of carboxymethyl group-containing modified hyaluronic acid (raw material modified hyaluronic acid)>
After weighing 1.04 g of sodium hydroxide in a 30 ml sample bottle, 12 ml of water was added and dissolved. Next, 2.0 g of hyaluronic acid having a molecular weight of 1,750,000 was added and dissolved, then 3.62 g of monobromoacetic acid was added and dissolved, and the mixture was allowed to stand at 1 ° C. for 16 hours. Thereafter, 80 ml of ethanol was placed in a 200 ml beaker, and the reaction solution was added with stirring. Thereafter, the precipitate was collected with a 400-mesh filter cloth, and 40 ml of 10% sodium chloride aqueous solution was added to dissolve the precipitate. Further, after adjusting the pH with an 8% aqueous hydrochloric acid solution, washing with 100 ml of ethanol three times, filtering under reduced pressure, and drying under reduced pressure at 55 ° C. for 3 hours, the carboxymethyl group-containing modified hyaluronic acid of Example 1 was obtained. It was.

  The carboxymethyl group-containing modified hyaluronic acid obtained in Example 1 had a molecular weight of 1.06 million and a carboxymethylation rate of 99%. In Examples 1 to 3, the carboxymethylation rate was measured and calculated by the method described later.

<Example 2: Preparation of carboxymethyl group-containing modified hyaluronic acid (raw material modified hyaluronic acid)>
Using a 1000 mL beaker, 42.6 g of sodium hydroxide was weighed, 240 mL of water was added and dissolved, 40.0 g of hyaluronic acid having a molecular weight of 1.8 million was added and dissolved, and then monobromoacetic acid was added. 72.4 g was added and dissolved, and dissolved at 4 ° C. for 16 hours. Thereafter, 10% saline was added to dilute the reaction solution until the concentration of hyaluronic acid reached 3% by mass, and then the pH of the reaction solution was adjusted to 6.3 using hydrochloric acid. Double the amount of ethanol was added to form a precipitate. This precipitate was washed twice with 70% ethanol and once with 90% ethanol, and the residue obtained was dried under reduced pressure at room temperature for 24 hours, whereby the carboxymethyl group-containing modified hyaluronic acid of Example 2 was obtained.

  The carboxymethyl group-containing modified hyaluronic acid obtained in Example 2 had a molecular weight of 1,220,000 and a carboxymethylation rate of 99%.

<Example 3: Preparation of carboxymethyl group-containing modified hyaluronic acid (raw material modified hyaluronic acid)>
The carboxymethyl group-containing modified hyalurone of Example 3 was used in the same manner as in Example 2 except that the reaction scale was 1/4 of the reaction scale used in Example 2 and the reaction time was 3 hours. The acid was obtained.

  The carboxymethyl group-containing modified hyaluronic acid obtained in Example 3 had a molecular weight of 1.26 million and a carboxymethylation rate of 59%.

<Measurement and calculation of carboxymethylation rate>
The carboxymethylation rate of the carboxymethyl group-containing modified hyaluronic acid obtained in Examples 1 to 3 was determined from the integrated value of ¹ H-NMR spectrum by the following method.
(Sample preparation)
7 mg of sample and 1 mg of sodium 4,4-dimethyl-4-silapentanesulfonate (DSS) as an internal standard substance were dissolved in 0.7 ml of heavy water, transferred to an NMR sample tube, and capped.
(Measurement condition)
Apparatus: Varian NMR system 400NB type (Varian Technologies Japan Limited)
Observation frequency: 400 MHz
Temperature: 30 ° C
Standard: DSS (0 ppm)
Integration count: 64 times (Analysis method)
The peak of the N-acetyl group (CH ₃ ) of hyaluronic acid appearing in the vicinity of 2.0 ppm of the ¹ H-NMR spectrum and the methylene group (—CH ₂ —) of the carboxymethyl group appearing in the range of 3.8 ppm to 4.2 ppm. ) Peak was integrated. From the integrated value, the number of carboxymethyl groups bonded to each disaccharide repeating unit of hyaluronic acid constituting the modified hyaluronic acid was determined from the following formula to determine the carboxymethylation rate (CM conversion rate).
CM conversion rate = (integral value of peak appearing in the range of 3.8 ppm to 4.2 ppm / 2) / (integral value of 2.0 ppm peak / 3)

<Example 4: Preparation of crosslinked product of carboxymethyl group-containing modified hyaluronic acid>
The carboxymethyl group-containing modified hyaluronic acid obtained in Example 1 was treated under the conditions shown in Table 1. First, the amount of the modified hyaluronic acid and each additive is adjusted so that the carboxymethyl group-containing modified hyaluronic acid has the concentrations shown in Table 1, and stored at 4 ° C. for 20 hours to swell and dissolve. A solution containing the group-containing modified hyaluronic acid was prepared. This solution was held at −18 ° C. for 16 hours. Subsequently, this solution was washed with running water at 25 ° C. for 10 minutes to obtain a composition (Experiment Nos. 1 to 4) containing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid. The pH of the solution in Experiment No. 3 is 0.1, the pH of the solution in Experiment Nos. 14 to 17 is 0.2, and the pH of the solution in Experiment Nos. 1, 2, 5 to 9, 23 and 25 is The pH of the solution in Experiment No. 4 was 0.9.

In addition, the yield shown in Table 1 is a value measured by the following method. That is, a gel was prepared by dispersing the composition obtained under each condition shown in Table 1 in pure water so that the concentration was 1% by mass, and the gel was allowed to stand at 1 ° C. for 24 hours. Then, the solution was neutralized to pH 6 with a 1% by mass aqueous sodium hydroxide solution. Next, the residue (gel) is collected on a filter paper by suction filtration, washed with distilled water, spread on a weighed petri dish, dried under reduced pressure, then measured the petri dish mass, and the change in the petri dish mass From the minute, the mass (solid content) of the residue (crosslinked product) was calculated. The yield was calculated by the following formula (3).
Yield (%) = Mass of residue (crosslinked product) / Mass of composition (solid content) × 100 (3)

  Further, using the carboxymethyl group-containing modified hyaluronic acid or hyaluronic acid (unmodified) of Examples 1 to 3, carboxymethyl under conditions different from the above (retention temperature, concentration of additive, concentration of raw material hyaluronic acid) A composition (Experiment Nos. 5 to 25) containing a cross-linked product of group-containing modified hyaluronic acid was obtained, and a gel was prepared using the composition by the same method as described above. In addition, a thermal stability test and an enzyme degradation resistance test were performed using the crosslinked product by the methods described later. The results are shown in Table 1. In Experiment No. 8, the solution was kept at 4 ° C. for 20 hours, and then kept at 25 ° C. for 47 hours. In Experiment No. 9, the solution was held at −18 ° C. for 20 hours and then further held at 25 ° C. for 47 hours.

<Test Example 1: Thermal stability test>
A mixture is prepared by dispersing the crosslinked product of carboxymethyl group-containing modified hyaluronic acid obtained under each condition shown in Table 1 in physiological saline so that the concentration is 1% by mass (solid content). Was allowed to stand at 50 ° C. for 7 days. The stored material was collected by suction filtration using a 420 mesh nylon strainer, washed with distilled water, then spread on a weighed petri dish and dried under reduced pressure. Thereafter, the mass of the petri dish was measured, and the mass of the residue was calculated from the change in the mass of the petri dish. From the mass of the crosslinked product and the mass of the residue, the residual ratio in the thermal stability test was calculated by the following formula (4).
Residual rate in thermal stability test (%) = mass of residue / mass of crosslinked product (solid content) × 100 (4)

<Test Example 2: Enzymatic degradation resistance test>
10 mg of each of the crosslinked products of carboxymethyl group-containing modified hyaluronic acid obtained under each condition shown in Table 1 was dispersed in 50 mM phosphate buffer to prepare a 9 mL mixture, and hyaluronidase (derived from Sigma, bovine tests) 50 Unit (1 mL) was added to make a 10 mL mixture containing 0.1% by mass (solid content), 5 units / cross-linked 1 mg hyaluronidase, and the mixture was allowed to stand at 40 ° C. for 24 hours. . Next, the preserved material was collected by suction filtration with a 420 mesh nylon strainer, washed with distilled water, then spread on a weighed petri dish and dried under reduced pressure. Thereafter, the mass of the petri dish was measured, and the mass of the residue was calculated from the change in the mass of the petri dish. The residual ratio in the enzyme degradation resistance test was calculated by the following formula (5).
Residual rate (%) in enzyme degradation resistance test = mass of residue / mass of cross-linked product (solid content) × 100 (5)

  According to Experiment Nos. 1 to 24 in Table 1, the crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof has a property of forming a water-swellable gel, and the degree of swelling with respect to water is 10 times. It can be understood that it is 250 times or less (mass ratio). As shown in Table 1, the mixture of Experiment No. 23 disappeared in 5 days in the thermal stability test.

  Moreover, according to the experiment numbers 1 to 24 of Table 1, it contains the crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof, and water, and the content of the crosslinked product is 0. 0 in terms of solid content. It can be understood that the water-swellable gel of 4% by mass or more and 10% by mass or less has excellent enzyme degradation resistance and moderate thermal stability.

  On the other hand, a commercially available crosslinked hyaluronic acid Synvisc (Genzyme) and Restylane (Q-Med) were subjected to an enzyme degradation resistance test. More specifically, Synvisc was prepared to be 9 mL by dispersing 1.25 g of gel (containing 10 mg of hyaluronic acid) and Restylane into 0.5 mL of gel (containing 10 mg of hyaluronic acid) in 50 mM phosphate buffer. 50 units (1 mL) of hyaluronidase dissolved in an acid buffer was added to prepare 10 mL of the mixture, and the enzyme degradation resistance of the mixture was measured by the same method as in Test Example 2 above. Was 0%, and it was confirmed that the enzyme degradation resistance was poor. Moreover, the residual rate of the crosslinked material of experiment number 25 (Comparative Example 1) was 0%. Since the crosslinked product of Experiment No. 25 (Comparative Example 1) is a crosslinked product of hyaluronic acid, it can be understood that the enzyme degradation resistance is poor.

<Formulation example 1: lotion>
In this blending example, a lotion containing the cross-linked product obtained in Experiment No. 1 of Example 1 was prepared according to the formulation described below.
Cross-linked product 0.2%
Sodium hyaluronate 0.1%
Hydrolyzed hyaluronic acid 0.1%
Hydrolyzed alkyl hyaluronate (C12-13) glyceryl 0.1%
Collagen peptide 0.1%
1,3-butylene glycol 5.0%
Glycerol 3.0%
Isostearyl alcohol 0.1%
Tocopherol acetate 0.1%
POE (20) sorbitan monolaurate 0.5%
POE (15) lauryl alcohol ether 0.5%
Zinc pyrrolidonecarboxylate 0.1%
Ethylparaben 0.1%
Methylparaben 0.15%
Ethanol 5.0%
Perfume Appropriate amount Purified water Remaining

<Formulation Example 2: Latex>
In this blending example, an emulsion was prepared by blending the cross-linked product obtained in Experiment No. 1 of Example 1 with the formulation described below.
Cross-linked product 0.3%
Pentylene glycol 5.0%
Glycerol 3.0%
Squalane 5.0%
Stearic acid 0.5%
Stearyl alcohol 2.0%
Vaseline 4.0%
Sorbitan stearate 1.0%
POE (10) monostearate 1.0%
Carboxy vinyl polymer 0.5%
Polyquaternium-51 0.1%
Methylparaben 0.15%
Propylparaben 0.1%
Potassium hydroxide 0.1%
BHT 0.02%
EDTA-2 sodium 0.02%
Perfume Appropriate amount Purified water Remaining

<Formulation Example 3: Cream>
In this blending example, a cream (emollient cream) was prepared by blending the cross-linked product obtained in Experiment No. 2 of Example 1 with the formulation described below.
Cross-linked product 0.5%
Polyethylene glycol 4.0%
1,3-propanediol 6.0%
Squalane 11.0%
Dimethicone 1.0%
Cetanol 6.0%
Stearic acid 2.0%
Hydrogenated cocoglyceryl 4.0%
Tricaprylin 8.0%
Glycerol monostearate 3.0%
POE (20) cetyl alcohol ether 2.0%
Coenzyme Q10 0.03%
Ceramide 0.1%
Dilauroyl glutamate ricin sodium 0.1%
EDTA-2 sodium 0.02%
Propylparaben 0.1%
Methylparaben 0.15%
Perfume Appropriate amount Purified water Remaining

<Formulation example 4: essence>
In this blending example, a cosmetic liquid (whitening moisturizing essence) was prepared by blending the cross-linked product obtained in Experiment No. 2 of Example 1 with the formulation described below.
Cross-linked product 0.8%
Sodium hyaluronate 0.2%
Hydrolyzed hyaluronic acid 0.1%
1,3-butylene glycol 5.0%
Glycerin 1.5%
POE sorbitan monostearate 1.0%
Sorbitan monostearate 0.5%
Xanthan gum 0.2%
Sodium alginate 0.2%
Carboxyvinyl polymer 0.2%
Potassium hydroxide 0.1%
Olive oil 0.2%
Tocopherol 0.1%
EDTA-2 sodium 0.02%
Arginine 0.15%
Dipotassium glycyrrhizinate 0.05%
Arbutin 0.2%
Retinol palmitate 0.2%
Cousin extract 0.2%
Seaweed extract 0.2%
Tranexamic acid 0.1%
Elastin 0.1%
Collagen 0.1%
Magnesium phosphate ascorbate 0.1%
Sodium citrate 1.0%
Citric acid 0.1%
Propylparaben 0.1%
Methylparaben 0.15%
Perfume Appropriate amount Purified water Remaining

<Formulation example 5: serum pack>
In this blending example, a cosmetic liquid pack (paste peel-off type) was prepared by blending the cross-linked product obtained in Experiment No. 1 of Example 1 with the formulation described below.
Cross-linked product 0.5%
Polyvinyl acetate emulsion 17.0%
Polyvinyl alcohol 11.0%
Sorbitol 5.0%
Polyethylene glycol 400 5.0%
Squalane 2.5%
POE sorbitan monostearate 1.0%
Titanium oxide 4.0%
Talc 8.0%
Ethanol 8.0%
Methylparaben 0.15%
Perfume Appropriate amount Purified water Remaining

<Formulation Example 6: Face wash>
In this blending example, a face wash (cleansing foam) was prepared by blending the cross-linked product obtained in Experiment No. 1 of Example 1 with the formulation described below.
Cross-linked product 0.2%
Cationized hyaluronic acid 0.1%
(Hypo Veil, manufactured by QP Corporation)
Glycerin 10.0%
Polyethylene glycol 400 15.0%
Dipropylene glycol 10.0%
Sodium lauroyl glutamate 20.0%
POE (2) monostearate 5.0%
Palm fatty acid sodium glutamate 8.0%
Alkyl betaine 2.0%
EDTA-2 sodium 0.02%
Propylparaben 0.1%
Methylparaben 0.15%
Perfume Appropriate amount Purified water Remaining

<Formulation example 7: Sunscreen>
In this blending example, a sunscreen (milky lotion) blended with the crosslinked product obtained in Experiment No. 1 of Example 1 was prepared according to the formulation described below.
Cross-linked product 0.2%
1,3-butylene glycol 3.0%
Dipropylene glycol 3.0%
Cyclomethicone 5.0%
Dimethicone 5.0%
Cetanol 1.0%
Vaseline 1.0%
Octyl methoxycinnamate 5.0%
Titanium oxide 2.0%
Zinc oxide 2.0%
Sorbitan stearate 1.0%
POE (20) sorbitan monostearate 1.0%
Phenoxyethanol 0.8%
Methylparaben 0.1%
Perfume Appropriate amount Purified water Remaining

<Formulation Example 8: Lip Balm>
In this blending example, a lip balm blended with the cross-linked product obtained in Experiment No. 1 of Example 1 was prepared according to the formulation described below.
Cross-linked product 0.1%
Microcrystalline wax 1.5%
Ceresin 12.0%
Squalane 10.0%
Decamethyltetrasiloxane 10.0%
Diisostearyl malate 5.0%
Candelilla wax 2.0%
Vaseline 8.0%
Glyceryl hydroxystearate 2.0%
Menthol 0.05%
Liquid paraffin 1.0%
Tocopherol acetate 0.1%
Tocopherol 0.05%
Propylparaben 0.1%
Perfume Appropriate amount Purified water Remaining

<Formulation example 9: shampoo>
In this blending example, a shampoo was prepared by blending the cross-linked product obtained in Experiment No. 1 of Example 1 with the formulation described below.
Cross-linked product 0.2%
Cationized hyaluronic acid 0.1%
(Hypo Veil, manufactured by QP Corporation)
POE (20) sodium lauryl ether sulfate 11.0%
Lauroyl sodium aspartate 10.0%
Coconut oil fatty acid amidopropyl betaine 4.0%
Coconut oil fatty acid monoethanolamide 2.0%
EDTA-2 sodium 0.1%
Sodium benzoate 0.2%
Phenoxyethanol 0.8%
Methylparaben 0.1%
Perfume Appropriate amount Purified water Remaining

<Formulation example 10: hair conditioner>
In this blending example, a hair conditioner was prepared by blending the cross-linked product obtained in Experiment No. 1 of Example 1 with the formulation described below.
Cross-linked product 0.3%
Cationized hyaluronic acid 0.2%
(Hypo Veil, manufactured by QP Corporation)
Stearyl alcohol 4.0%
Cetanol 1.5%
Hydroxyethyl urea 1.0%
Aminopropyl dimethicone 1.5%
Dimethicone 0.5%
Hydrolyzed silk 1.0%
1,3-butylene glycol 1.0%
Glycerol 3.0%
Cetyl 2-ethylhexanoate 2.0%
Isocetyl myristate 0.4%
L-Arginine 0.1%
Trehalose 0.1%
Sorbitol 0.1%
Keratin peptide 0.1%
POE (4) stearyl ether 1.0%
Stearic acid dimethylaminopropylamide 3.0%
Distearyldimethylammonium chloride 0.2%
Sodium benzoate 0.3%
Phenoxyethanol 0.8%
Methylparaben 0.1%
Perfume Appropriate amount Purified water Remaining

<Formulation Example 11: Soft capsule>
In this blending example, a soft capsule was prepared by blending the cross-linked product obtained in Experiment No. 1 of Example 1 with the formulation described below.
Cross-linked 20%
Olive oil 35%
Beeswax 5%
Medium chain triglyceride 5%
Gelatin 25%
Glycerin 10%

<Formulation example 12: Powder>
In this blending example, a powder (granule) was prepared by blending the cross-linked product obtained in Experiment No. 1 of Example 1 with the formulation described below.
Cross-linked product 10%
Lactose 60%
Corn starch 25%
Hypromellose 5%

<Formulation example 13: soft capsule>
In this blending example, a tablet was prepared by blending the cross-linked product obtained in Experiment No. 1 of Example 1 with the formulation described below.
Cross-linked product 25%
Lactose 24%
20% crystalline cellulose
Corn starch 15%
Dextrin 15%
Silicon dioxide 1%

<Formulation example 14: jelly beverage>
In this blending example, a white peach jelly beverage containing a spout pouch was prepared by blending the cross-linked product obtained in Experiment No. 1 of Example 1 with the formulation described below.
Cross-linked product 0.20%
Xanthan gum 1.00%
Carrageenan 0.5%
Dextrin alcohol 3.0%
Sucralose 1%
4-fold concentrated white peach juice 5.00%
Citric acid 0.60%
Sodium citrate 0.20%
L-ascorbic acid 0.10%
Peach flavoring 0.20%
Purified water remaining

<Example 3: Anti-adhesion agent>
The cross-linked product obtained in Experiment No. 2 of Example 1 was rolled into a 1 mm-thick film and molded. After sterilization, a sheet-shaped adhesion inhibitor was obtained.

<Example 4: Subcutaneous injection>
The dried product of the crosslinked product (1% in terms of solid content) obtained in Experiment No. 2 of Example 1 was swollen with water for injection containing 0.9% NaCl, and aseptically filled into a 1 mL syringe. After sterilization, A subcutaneous injection was obtained.

<Example 5: Drug sustained-release agent>
The dried product of the crosslinked product obtained in Experiment No. 2 of Example 1 (2% in terms of solid) was swollen with water for injection containing 0.9% NaCl and 0.001% prostaglandin E1, and after sterilization treatment A 3 mL syringe was aseptically filled to obtain a sustained-release drug.

<Example 6: Knee joint injection>
The dried product of the crosslinked product (0.8% in terms of solids) obtained in Experiment No. 2 of Example 1 was swollen with water for injection containing 0.9% NaCl, sterilized, and aseptically filled into a 2 mL syringe. Thus, a knee joint injection was obtained.

Claims (18)

A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof, comprising a step of maintaining a solution of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof at −200 ° C. or higher and 10 ° C. or lower. ,
The crosslinked product has the following properties: After storing a mixture prepared by dispersing in a physiological saline solution so that the concentration of the crosslinked product is 1% by mass (solid content) at 50 ° C. for 72 hours. A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof, wherein the residual ratio is 20% or more . In claim 1,
The manufacturing method of the crosslinked product of the carboxymethyl group containing modification hyaluronic acid and / or its salt whose density | concentration of the said carboxymethyl group containing modification hyaluronic acid and / or its salt in the said solution is 1 mass% or more and 30 mass% or less. In claim 1 or 2,
A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof, wherein the solution has a pH of 3 or less. In any one of Claims 1 thru | or 3,
The carboxymethyl group-containing modification wherein the average molecular weight of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is 300,000 or more, and the carboxymethylation ratio to the disaccharide unit constituting the hyaluronic acid is 1% or more. A method for producing a crosslinked product of hyaluronic acid and / or a salt thereof. In any one of Claims 1 thru | or 4,
By the holding step, a composition containing the crosslinked product is obtained,
A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof, further comprising the step of washing the composition with water to remove impurities other than the crosslinked product.   A cross-linked product of a carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof obtained by the production method according to any one of claims 1 to 5. In claim 6,
A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof having water swellability. A carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof having a water swelling property, having a swelling degree with respect to water of 10 to 250 times (mass ratio) ,
The cross-linked product has a residual ratio of 20% after a mixture prepared by dispersing in a physiological saline solution so that the concentration of the cross-linked product is 1% by mass (solid content) at 50 ° C. for 72 hours. A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof as described above . In claim 8,
Carboxymethyl group-containing, including the above-mentioned carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof having an average molecular weight of 300,000 or more and a carboxymethylation rate of 1% or more with respect to a disaccharide unit constituting hyaluronic acid A crosslinked product of modified hyaluronic acid and / or a salt thereof. In any one of Claims 6 thru | or 9,
A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof having reversible water swellability. The carboxymethyl group-containing modified hyaluronic acid according to any one of claims 6 to 10 and / or a salt thereof in a 50 mM phosphate buffer so that the concentration of the crosslinked product is 0.1% by mass (solid content). the mixture prepared by dispersing, hyaluronidase (5 units / crosslinked product per 1 mg) after storage for 24 hours at 40 ° C. in the presence of residual ratio is 1% or more, any one of claims 6 to 10 2. A crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to item 1. A crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of claims 6 to 11 , and water,
A water-swellable gel in which the content of the crosslinked product is 0.4% by mass or more and 10% by mass or less in terms of solid content. In claim 12 ,
A water-swellable gel having a residual rate of 20% or more after storage at 50 ° C. for 72 hours. In claim 12 or 13 ,
A water-swellable gel having a residual ratio of 1% or more after storage at 40 ° C. for 24 hours in the presence of hyaluronidase (5 units / mL of gel). A medical material comprising the crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of claims 6 to 11 . In claim 15 ,
A medical material used as at least one selected from a knee joint injection, an anti-adhesion agent, a subcutaneous injection, and a drug sustained-release agent. A cosmetic comprising the crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of claims 6 to 11 . A cosmetic material comprising the crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of claims 6 to 11 .