TW201601732A - Crosslinked product of carboxymethyl-group-containing modified hyaluronic acid and/or salt thereof, and method for producing same - Google Patents

Abstract

This method for producing a crosslinked product of a carboxymethyl-group-containing modified hyaluronic acid includes a step for keeping a solution comprising a carboxymethyl-group-containing modified hyaluronic acid and/or a salt thereof between -200 DEG C and 10 DEG C, inclusive.

Description

Crosslinked product of modified hyaluronic acid containing carboxymethyl group and/or its salt and preparation method thereof

The present invention relates to a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof, a method for producing the same, a water-swellable colloid comprising the crosslinked product, a method for producing the same, and a medical treatment comprising the crosslinked product Materials, cosmetics, and materials for beauty.

Hyaluronic acid is widely distributed in biological tissues such as cockscombs, umbilical cords, skin, cartilage, translucent bodies, and joint fluids, and is widely used as a component of, for example, cosmetics, pharmaceuticals, and foods.

A hyaluronic acid colloid and a method for producing the same are described in Patent Document 1 (JP-A-H05-58881). However, hyaluronic acid is easily decomposed by hyaluronan in the living body.

[Previous Technical Literature] [Patent Document]

Patent Document 1: Japanese Patent Laid-Open No. Hei 5-58881

The present invention provides a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof which is excellent in enzyme decomposition resistance and has moderate thermal stability, a method for producing the same, and water swellability including the crosslinked product. A colloid, a method for producing the same, and a medical material, a cosmetic, and a cosmetic material comprising the crosslinked product.

The inventors of the present invention have found that a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof obtained by maintaining a specific temperature in a solution has excellent enzyme decomposition resistance, moderate thermal stability, and water swellability.

A method for producing a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to the present invention, which comprises maintaining a solution of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof - The steps of 200 ° C or more and 10 ° C or less.

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

3. The method for producing a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to the above 2, wherein the pH of the solution is 3 or less.

4. The modification containing a carboxymethyl group according to any one of the above 1 to 3 A method for producing a crosslinked product of hyaluronic acid and/or a salt thereof, wherein the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof has an average molecular weight of 300,000 or more, and is carboxymethyl with respect to 2 saccharide units constituting hyaluronic acid. The basis ratio can be 1% or more.

5. The method for producing a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of items 1 to 4 above, wherein the method further comprises the step of maintaining the crosslinked product After the composition, the composition is washed with water, and the composition is washed with water to remove impurities other than the crosslinked product.

A crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of the above 1 to 5, which is a modified hyaluronic acid containing carboxymethyl group and/or The method for producing a cross-linking of salt.

7. The crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to the above 6, which is capable of having water swellability.

A crosslinked product of a water-swellable modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to one aspect of the present invention, which has a water swellability of 10 times or more and 250 times or less (mass ratio).

9. The crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to the above 8, which can comprise the above modified hyaluronic acid containing carboxymethyl group and/or a salt thereof, the modified hyaluronic acid containing carboxymethyl group and/or The average molecular weight of the salt thereof or the salt thereof is 300,000 or more, and the carboxymethylation ratio is 1% or more with respect to the two saccharide units constituting hyaluronic acid.

10. Repair of carboxymethyl group according to any one of the above 6 to 9 A crosslinked product of hyaluronic acid and/or a salt thereof, which is capable of reversible water swellability.

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

12. The crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of the above items 6 to 11, wherein the mixture is in the presence of hyaluronic acid (5 units/1 mg per cross-linking) The residual rate after storage for 24 hours at 40 ° C can be 1% or more, and the mixture is a crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or its salt as in any one of the above 6 to 11 in 50 mM. The concentration in the phosphate buffer solution was adjusted to 0.1% by mass (solid content).

A water-swellable colloid according to one aspect of the present invention, which comprises a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of items 6 to 12 above, and water, and the aforementioned The content of the modified hyaluronic acid and/or its salt containing a carboxymethyl group is 0.4% by mass or more and 10% by mass or less in terms of solid content.

14. The water-swellable colloid of the above 13, which can be stored at 50 ° C for 72 hours and has a residual ratio of 20% or more.

15. The water-swellable colloid of 13 or 14 above, which is maintained at 40 ° C in the presence of hyaluronidase (5 units per colloid 1 mL) The residual rate after 24 hours of storage can be 1% or more.

A medical material according to one aspect of the present invention, which comprises a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of items 6 to 12 above.

17. The medical material according to the above 16 which can be used as at least one selected from the group consisting of a knee joint injection, a post-adhesion preventive agent, a subcutaneous injection, and a drug depot.

A cosmetic according to one aspect of the invention, which comprises a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of items 6 to 12 above.

A cosmetic material according to one aspect of the present invention, which comprises a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of items 6 to 12 above.

In the method for producing a crosslinked product, the solution containing the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof is maintained at -200 ° C or higher and 10 ° C or lower, whereby the enzyme decomposition resistance is excellent, and Moderate thermal stability and cross-linking of water-expandable modified hyaluronic acid and/or its salt containing carboxymethyl groups. The crosslinked product can be used as a component of, for example, a medical material, a cosmetic material, and a cosmetic.

Further, the crosslinked product can form a stable water-swellable colloid by having water swellability and having a water swellability of 10 times or more and 250 times or less (mass ratio).

Further, the water-swellable colloid comprises a crosslinked product containing a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof, and water, and the content of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof is converted into solid content. When it is 0.4% by mass or more and 10% by mass or less, it is excellent in enzyme decomposition resistance and has moderate heat stability.

[Formation of the Invention]

Hereinafter, the present invention will be described in detail. Further, in the present invention, "parts" means "parts by mass" and "%" means "mass%" without particular hindrance.

[Method for Producing Crosslinked Compound of Modified Hyaluronic Acid Containing Carboxymethyl Group and/or Its Salt]

A method for producing a modified hyaluronic acid containing carboxymethyl group and/or a crosslinked product (hereinafter also referred to simply as "crosslinked product") according to an embodiment of the present invention comprises a modified hyaluronic acid containing carboxymethyl group and/or The salt solution is maintained at a temperature above -200 ° C and below 10 ° C.

In the present invention, "carboxymethyl" means a group represented by "-CH ₂ -CO ₂ H" or "-CH ₂ -CO ₂ ^- ". Further, the raw material modified hyaluronic acid and/or its salt used in the production method according to the present embodiment can be produced by a method described later.

<Mechanism of Manufacturing Method According to the Present Embodiment>

In the production method according to the embodiment, the sugar chain containing the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof is mutually constituted by the above-described holding step The distance is shortened, and as a result, the functional groups constituting the sugar chain are mutually different (for example, a carboxyl group, a hydroxyl group, a N-acetyl group, a carboxyl group and a hydroxyl group, a hydroxyl group and an amine group, an amine group and a carboxyl group). There will be hydrogen bonds between them. As a result, it is estimated that the sugar chains constituting the modified hyaluronic acid containing carboxymethyl group and/or its salt are strongly bonded to each other via hydrogen bonding, whereby the thermal stability is improved.

Further, it is presumed that the sugar chains constituting the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof are bonded to each other via a hydrogen bond, and the three-dimensional network structure is constructed. In the crosslinked product according to the present embodiment, the water-expandable colloid described later can be formed by absorbing water in the three-dimensional network structure.

In particular, the portion having a carboxymethyl group in the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof has a larger number of carboxyl groups in one constituent unit of the hyaluronic acid skeleton than hyaluronic acid and/or a salt thereof. In other words, it is presumed that since the carboxyl group capable of hydrogen bonding is more than the hyaluronic acid and/or its salt in the constituent unit, a large number of hydrogen bonds can be formed in the step of holding.

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 crosslinked product according to the present embodiment is hard to be broken at room temperature. Further, in the present invention, "room temperature" means a temperature of 25 ° C or more and 30 ° C or less.

<Keep temperature>

In the above-described holding step, the hardness of the water-swellable colloid obtained by 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, water which can form more hydrogen bonds and is harder and has higher heat stability can be obtained. From the viewpoint of the crosslinked product of the raw material of the expansive colloid, the step of maintaining the solution is preferably carried out at -1 ° C or lower, more preferably at -10 ° C or lower, and more preferably at -30 ° C or higher. . Further, from the viewpoint of obtaining a crosslinked product which is a raw material of a water-swellable colloid having flexibility, the step of holding the solution is more preferably 0 ° C or more and 10 ° C or less, and is 1 ° C or more. It is even better to perform below 10 °C. Further, the harder the water-swellable colloid (i.e., the lower the degree of expansion of the colloid), the higher the resistance to decomposition of the enzyme. Therefore, by carrying out the step of maintaining the solution at 10 ° C or lower, it is possible to obtain a water-swellable colloid having a low degree of swelling and high enzyme decomposition resistance.

<hold time>

In the manufacturing method according to the embodiment, the holding step can be 1 second or longer depending on the type of the holding temperature of the solution in the holding step or the type of the device for holding the solution at a specific holding temperature. In order to further improve the thermal stability and the enzyme decomposition resistance, it is preferable to carry out the time of 30 hours or longer, and on the other hand, it is possible to carry out the time of 300 hours or less and to perform the time of 96 hours or less. .

<Concentration of hyaluronic acid and/or its salt modified in the solution>

In the production method according to the present embodiment, the concentration of the raw material-modified hyaluronic acid and/or its salt in the solution is 1% by mass or more and 30%, in view of the fact that the hydrogen bond can be formed more reliably and the thermal stability is further improved. quality% The following is more preferably 3 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 its salt is 3 or less (0 or more, from the viewpoint of more reliably forming a hydrogen bond and further improving thermal stability. The range of 3 or less is more preferably 2 or less, and may be 1 or less.

In the step of maintaining, when the pH of the solution of the raw material modified hyaluronic acid and/or its salt is 3 or less, the carboxyl group contained in the raw material modified hyaluronic acid and/or its salt may become an acid form (-CO ₂ H). It is presumed that between the carboxyl groups and the carboxyl group and other functional groups (for example, a hydroxyl group or an amine group), hydrogen bonding is more easily constructed, and thermal stability is further improved.

Further, an acid used to adjust the pH of the solution to an acidity (pH less than 7) may be any acid as long as it can adjust the pH of the solution to an acidity. The acid can be 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, from the viewpoint of reducing the amount of acid used.

<solvent>

In the production method according to the embodiment, the solvent in which the raw material modified hyaluronic acid and/or its salt is dissolved may be a mixture of water and a water-soluble organic solvent mixed with water.

Examples of the water-soluble organic solvent include methanol and B. An alcohol solvent such as an alcohol, 1-propanol or 2-propanol, a ketone solvent such as acetone or methyl ethyl ketone, tetrahydrofuran or acetonitrile may be used alone or in combination.

<Expansion and dissolution>

In order to suppress the decrease in the molecular weight of the solution of the raw material-modified hyaluronic acid and/or its salt, and to maintain the raw material-modified hyaluronic acid and/or its salt hygienically, and to dissolve the raw material-modified hyaluronic acid and/or its salt in a solvent to prepare a solution, for example, The temperature of 0 ° C or more and 20 ° C or less is maintained for 5 hours or more and 48 hours or less, and it is preferred to swell and dissolve the raw material modified hyaluronic acid and/or its salt.

<washing step>

In the production method according to the present embodiment, in view of the ability to remove by-products (in particular, when the solution contains an acid, the acid can be removed and the pH is increased), the following steps can be further included: In the step of maintaining, a step of obtaining the composition of the crosslinked product and washing the composition with water to obtain the crosslinked product (the separation of the crosslinked product) is obtained.

For the washing liquid used in the washing 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 may be one in which the solubility of the modified hyaluronic acid and/or its salt is low. In the washing step, the temperature of the washing liquid is set to 30 ° C or lower (usually 0 ° C or higher and 25 ° C or lower, preferably 3 ° C). In the above, more preferably 4° C. or more, since the hydrogen bond formed in the holding step can be maintained, the properties (thermal stability and water swellability) of the finally obtained composition according to the embodiment can be maintained.

<Repeat processing>

In the production method according to the present embodiment, the step of maintaining the hydrogen bond is more reliably formed and the thermal stability is further improved, and the step of maintaining the composition is repeated once or more, and the composition is washed with water. The combination of the steps of removing impurities other than the crosslinked product is preferable, and it is usually possible to repeat two or more times and five or less times.

[Material modified hyaluronic acid and / or its salt] <Molecular weight of raw material modified hyaluronic acid and/or its salt>

The raw material-modified hyaluronic acid and/or its salt according to the present embodiment has a molecular weight of usually 4,000 or more and 4,000,000 or less. In the present invention, the molecular weight of the raw material-modified hyaluronic acid and/or its salt can be measured by the following method.

The calibration curve is prepared by colloidal filtration through a column and performing liquid chromatography analysis on a plurality of (purified) hyaluronic acid (reference material) having a known molecular weight. 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 above-described calibration curve.

As a liquid chromatography layer analyzer which can be used for the liquid chromatography analysis described above, for example, Waters Alliance 2690 HPLC Separations Module (Waters Corporation), Waters Alliance 2695 HPLC Separations Module (manufactured by Waters Co., Ltd.) and 1200 Series (manufactured by Agilent Co., Ltd.). Further, as a column that can be used for liquid chromatography analysis, for example, a column for ligand exchange chromatography by a shodex company (coordination exchange mode + size exclusion mode), the model name "SUGAR KS-801" , "SUGAR KS-802", "SUGAR KS-803", "SUGAR KS-804", "SUGAR KS-805", "SUGAR KS-806", "SUGAR KS-807", or TOSOH size exclusion color layer Analyze the column, the name is "TSKgel GMPW".

In the production method according to the present embodiment, the average molecular weight of the raw material-modified hyaluronic acid and/or its salt is preferably 300,000 or more, and more preferably 500,000 or more, from the viewpoint of obtaining a hydrogen bond more reliably (generally capable of Less than 3 million, less than 2 million).

In the present invention, the carboxymethylation rate (hereinafter referred to simply as "carboxymethylation rate") of the saccharide unit of hyaluronic acid constituting the modified hyaluronic acid having a carboxyl group and/or its salt. It is the total of the peaks of the protons (expressed in the vicinity of 2 ppm) of the methyl group (-CH ₃ ) of the N-acetyl group bonded to the C-2 position in the hyaluronic acid skeleton in the ¹ H-NMR spectrum. The value indicates the peak of the proton of the alkenyl group (-CH ₂ -) in the carboxymethyl group (-CH ₂ -CO ₂ H or -CH ₂ -CO ₂ ^- ) (in the range of 3.8 ppm or more and 4.2 ppm or less) The ratio (%) of the integrated value of the performance) is expressed.

In the present invention, the "two-saccharide unit constituting hyaluronic acid" means one unit composed of two sugars (glucuronic acid and N-acetylglucosamine) constituting hyaluronic acid and adjacently bonded, "relative to the composition of hyaluronic acid 2 carboxymethylation rate of a sugar unit" means that the unit is 1 unit with respect to the unit The amount of the carboxymethyl group contained in the one unit is more specifically the ratio (%) of the carboxymethyl group contained in the one unit to the unit.

<Carboxymethylation rate of raw material modified hyaluronic acid and/or its salt>

In the production method according to the present embodiment, the carboxymethylation ratio of the raw material-modified hyaluronic acid and/or its salt is preferably 1% or more from the viewpoint of more reliably forming a hydrogen bond and further improving thermal stability. More preferably, it is 10% or more, more preferably 50% or more, and on the other hand, it is usually 400% or less, and can be 200% or less.

<yield>

In the production method according to the embodiment, the quality of the crosslinked product relative to the mass of the composition is preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more. In this step, the composition containing the crosslinked product is obtained by the above-described holding step, and the composition is washed with water to obtain the crosslinked product. Further, the above yield can be calculated by the method shown in the examples of the present invention.

[Method for producing raw material modified hyaluronic acid and/or its salt]

In the production method according to the embodiment, the modified hyaluronic acid containing a carboxymethyl group and/or a salt thereof (hereinafter also referred to as "raw material-modified hyaluronic acid and/or a salt thereof") can be obtained, for example, by the following steps. The step is to use the hyaluronic acid and/or its salt in an aqueous solvent having a temperature of 30 ° C or lower. Reacts with halogen-containing acetic acid and/or its salt.

In the step of reacting, in the reaction liquid (aqueous solvent), at least a part of the raw material hyaluronic acid and/or a salt thereof (preferably all or a majority of hyaluronic acid and/or a salt thereof) and haloacetic acid and/or The salt can react the hyaluronic acid and/or its salt with the halogen-containing acetic acid and/or its salt in a dissolved state. In this case, the reaction liquid may be transparent under visual observation from the viewpoint that the hyaluronic acid and/or its salt and the halogen-containing acetic acid and/or its salt are dissolved.

<Material hyaluronic acid and / or its salt>

In the present invention, "hyaluronic acid" means a polysaccharide having one or more repeating structural units composed of glucuronic acid and N-acetylglucosamine disaccharide. Further, the salt of hyaluronic acid is not particularly limited, but is preferably a food or pharmaceutically acceptable salt, and examples thereof include a sodium salt, a potassium salt, a calcium salt, a zinc salt, a magnesium salt, and an ammonium salt. Wait.

Hyaluronic acid is basically a substance comprising at least one β-D-glucuronic acid at position 1 and β-DN-acetamidine-glucosamine at the 3 position bonded 2 saccharide unit, and is substantially composed of β- D-glucuronic acid is composed of β-DN-acetamidine-glucosamine and is bonded to a plurality of 2 sugar units.

The sugar may be an unsaturated sugar, and as the unsaturated sugar, a non-reducing terminal sugar may be mentioned, and usually the unsaturation between the 4th and 5th carbons of glucuronic acid is used.

The hyaluronic acid and/or its salt (the raw material hyaluronic acid and/or its salt) used for the manufacture of the modified hyaluronic acid and/or its salt can also be used from animals. Such as those extracted from natural objects (such as cockscombs, umbilical cords, skin, joint fluids, etc.), or those obtained by cultivating microorganisms, animal cells, or plant cells (for example, fermentation methods using bacteria such as Streptococcus), Chemical or enzymatic synthesizers, etc.

As the raw material hyaluronic acid and/or its salt, any of the crude extract and the purified product may be used, but the purified product, specifically, carboxymethylation can be smoothly carried out, and the purity is 90%. The raw material hyaluronic acid and/or its salt is preferably (mass ratio) or more.

<Average molecular weight of raw material hyaluronic acid and/or its salt>

The average molecular weight of the raw material hyaluronic acid and/or its salt is preferably 4,000 or more and 4,000,000 or less, and more preferably 3,000,000 or less, from the viewpoint of smoothly performing carboxymethylation. Further, the average molecular weight of the raw material hyaluronic acid and/or its salt can be measured by the following method.

<Method for measuring molecular weight>

That is, about 0.05 g of (purified) hyaluronic acid (this product) is precisely measured, and dissolved in a sodium chloride solution having a concentration of 0.2 mol/L, and the solution correctly obtained as 100 mL and the solution 8 mL, 12 mL, and the solution are correctly measured. 16 mL was added to a sodium chloride solution at a concentration of 0.2 mol/L, and a solution of 20 mL correctly was used as a sample solution. For each sample solution and a sodium chloride solution of 0.2 mol/L concentration, the viscosity of the general test method of the Japanese Pharmacopoeia (sixteenth correction) (the first method of capillary viscosity measurement) is at 30.0 ± 0.1 ° C. Determine the specific viscosity (formula (A)) and calculate each Reduced viscosity in concentration (formula (B)). The reduction viscosity is set to the vertical axis, and the concentration (g/100 mL) of the dry matter converted by the product is set to the horizontal axis, and the pattern is drawn, and the intersection of the straight line connecting the respective points and the vertical axis is obtained. Ultimate viscosity. The ultimate viscosity determined here was introduced into the formula of Laurent (formula (C)) to calculate the average molecular weight (Torvard C Laurent, Marion Ryan, and Adolph Pietruszkiewicz, "Fractionation of hyaluronic Acid", Biochemina et Biophysica Acta., 42,476 -485 (1960), Sifang Tianqianzi, "Molecular Weight Evaluation of SEC-MALLS of Sodium Hyaluronate Preparation", National Institute of Health Research, No. 121, 030-033 (2003)).

(Formula A) specific viscosity = {(seconds of flow required for the sample solution) / (seconds of flow required for 0.2 mol/L sodium chloride solution)}-1

(Formula B) Reducing viscosity (dL/g) = specific viscosity / (relative to the concentration of dry matter converted by this product g / 100mL))

(Formula C) Ultimate viscosity (dL/g) = 3.6 × 10 ^-4 M ^0.78

M: average molecular weight

<Content of raw material hyaluronic acid and / or its salt>

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 more the hyaluronic acid and/or its salt content, the raw material hyaluronic acid and/or The higher the purity of its salt.

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

The carbazole sulfuric acid method is: adding a hyaluronic acid aqueous solution to a sodium borate ‧ sulfuric acid solution and mixing, then decomposing hyaluronic acid by heating, cooling, adding a carbazole ‧ ethanol solution and mixing, and measuring the absorbance of the sample liquid after cooling (530 nm) The method. The D-glucuronolactone which was treated in the same manner was used as a calibration curve, and the D-glucuronolactone-converted value was calculated and multiplied by 1.102 to obtain a quantitative value of glucuronic acid. The content of hyaluronic acid was calculated by multiplying the obtained quantitative value of glucuronic acid (molecular weight of hyaluronic acid/molecular weight of glucuronic acid).

<carboxymethylation>

In the present invention, "modified hyaluronic acid and/or a salt thereof" means a structure in which at least a part of hyaluronic acid and/or a salt thereof having an organic group introduced therein is different from hyaluronic acid and/or a salt thereof. Further, the "organic group" in the present invention means a group having a carbon atom.

Therefore, in the present invention, the "modified hyaluronic acid containing a carboxymethyl group and/or a salt thereof" means at least a part of hyaluronic acid and/or a salt thereof to which a carboxymethyl group is introduced.

More specifically, in the raw material-modified hyaluronic acid and/or a salt thereof, for example, a hydroxyl group constituting hyaluronic acid (refer to the following formula (1)) (in the following formula (1), N-acetyl glucosamine constituting hyaluronic acid The hydrogen atom of at least a part of the hydroxyl group at the C-4 position, the C-6 position of the sugar, and the C-2 position and the C-3 position of the glucuronic acid of the hyaluronic acid can be -CH ₂ -CO ₂ H and / or -CH ₂ -CO ₂ ^- is represented by a group). 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 the positions may be -CH ₂ -CO ₂ H And/or substituted by a group represented by -CH ₂ -CO ₂ ^- .

(wherein n represents 1 or more and 7,500 or less).

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

(wherein R ¹ to R ⁵ independently represent a group represented by a hydroxyl group, -CH ₂ -CO ₂ H, or -CH ₂ -CO ₂ ^- (only, any of R ¹ to R ⁵ represents Except for the case of a hydroxyl group, n represents 1 or more and 7,500 or less).

<pH value>

In the production method according to the present embodiment, the reaction of the raw material hyaluronic acid and/or its salt with the halogen-containing acetic acid and/or its salt is carried out under alkaline conditions from the viewpoint of improving the nucleophilicity of the hydroxyl group. Preferably, the pH of the reaction liquid (aqueous solvent) is 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).

Further, at this time, in order to adjust the reaction liquid to be alkaline, an alkaline electrolyte can be used in the reaction liquid. Examples of the alkaline electrolyte include hydroxides of alkali metals such as sodium hydroxide and potassium hydroxide, and hydroxides of alkaline earth metals such as calcium hydroxide, magnesium hydroxide and barium hydroxide. The concentration of the alkaline electrolyte in the reaction liquid is, for example, 0.2 mol/L or more and 10 mol/L or less, preferably 0.5 mol, from the viewpoint of efficiently obtaining the raw material-modified hyaluronic acid and/or a salt thereof. /L or more and 8 m / L or less.

Further, in this case, the concentration of the hyaluronic acid in the aqueous solvent is preferably 0.05 g/mL or more and 0.5 g/mL or less.

<haloacetic acid and/or its salt>

In the method for producing a raw material-modified hyaluronic acid and/or a salt thereof according to the present embodiment, the halogen-containing acetic acid and/or a salt thereof is used for introducing a carboxymethyl group into a raw material hyaluronic acid and/or a salt thereof.

The haloacetic acid can be, for example, a monohaloacetic acid and/or a salt thereof, more specifically, chloroacetic acid and/or a salt thereof, or bromoacetic acid or a salt thereof. The haloacetic acid-containing salt is preferably 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 the halogen-containing acetic acid and/or a salt thereof, the temperature of the reaction liquid is 10° C. or less (for example, more than 0° C. and 10° C.) from the viewpoint of suppressing the progress of the low molecular weight. The reaction below is preferred.

For example, in order to produce 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), a raw material modified hyaluronic acid and/or a salt thereof is used as a halogen-containing compound. The acetic acid and/or its salt is preferably bromoacetic acid and/or a salt thereof, and the reaction is carried out at a temperature of 10 ° C or lower (for example, more than 0 ° C and 10 ° C or less).

<Use amount of haloacetic acid and/or its salt>

The amount of the halogen-containing acetic acid and/or its salt to be used is usually 10% or more and 500% or less (mass ratio) of the raw material hyaluronic acid and/or its salt, and is preferably 50% or more and 200% or less (mass ratio). .

<aqueous solvent>

In the method for producing a raw material-modified hyaluronic acid and/or a salt thereof according to the present embodiment, the aqueous solvent is water or a mixture of a water-soluble organic solvent and water, from the viewpoint of high solubility of the raw material hyaluronic acid and/or a salt thereof. The liquid is preferred.

The aqueous solvent is a mixture of a water-soluble organic solvent and water. In other words, when the aqueous solvent contains both water and a water-soluble organic solvent, the ratio of the water-soluble organic solvent in the mixed solution is 60 v/v% or less (more than 0 v) from the viewpoint of improving the solubility of hyaluronic acid. More preferably, /v% and 60 v/v% or less) is 40 v/v% or less (more than 0 v/v% and 40 v/v% or less).

The water-soluble organic solvent may, for example, be an alcohol solvent such as methanol, ethanol, 1-propanol or 2-propanol, a ketone solvent such as acetone or methyl ethyl ketone, tetrahydrofuran or acetonitrile, or may be used alone or in combination. To use. Among them, a lower alcohol having 1, 2 or 3 carbon atoms such as isopropyl alcohol or ethanol is preferred.

<reaction temperature>

In the above reaction, the temperature of the reaction liquid is usually 30 ° C or lower (preferably more than 0 ° C and 30 ° C or less) from the viewpoint of being able to smoothly carry out carboxylation and suppressing the decrease in molecular weight, and is preferably 10 ° C. The following (preferably more than 0 ° C and 30 ° C or less) is more preferable. In particular, by setting the temperature of the reaction liquid to 10 ° C or lower, a high molecular weight (800,000 or more) raw material-modified hyaluronic acid and/or a salt thereof can be easily obtained.

For example, when chloroacetic acid and/or a salt thereof is used as the halogen-containing acetic acid and/or a salt thereof, carboxymethylation can be smoothly carried out, and the viewpoint of brown discoloration of the obtained raw material-modified hyaluronic acid and/or its salt can be suppressed. The temperature of the reaction liquid in the above reaction can be usually (preferably more than 0 ° C and 30 ° C or less), and preferably 1 ° C or more and 30 ° C or less.

And, for example, bromoacetic acid and/or a salt thereof is used as the halogen-containing acetic acid And / or a salt thereof, the carboxymethylation can be smoothly carried out, and the temperature of the reaction liquid in the above reaction can usually be suppressed from the viewpoint of suppressing brown discoloration of the obtained raw material-modified hyaluronic acid and/or its salt and low molecular weight. It is preferably 10 ° C or lower (preferably more than 0 ° C and 10 ° C or less), and is preferably 1 ° C or more and 10 ° C or less.

<reaction time>

In the above-mentioned reaction, the reaction time is usually 30 minutes or more and 100 hours or less, and is preferably 60 minutes or more and 60 hours or less, from the viewpoint of suppressing the molecular weight and suppressing the molecular weight.

[crosslinking]

The crosslinked product according to one embodiment of the present invention is obtained by the method for producing a crosslinked product according to the above embodiment. The crosslinked product according to the embodiment can have water swellability.

In the present invention, "water swellability" means a property of absorbing water to swell, and generally means a property of absorbing water to be colloidal. The crosslinked product according to the embodiment has a three-dimensional network structure formed by hydrogen bonding of functional groups generated by the aforementioned maintaining step, and in the 3-dimensional network structure It absorbs moisture and swells, and it can form a colloid.

Further, the crosslinked product according to the present embodiment has the same chemical structural formula, average molecular weight, and carboxymethyl group as the raw material modified hyaluronic acid and/or its salt (modified hyaluronic acid containing carboxymethyl group and/or its salt). Although the hydrogenation bond is formed, it has physical properties (water swelling property and thermal stability which will be described later) which are different from the raw material modified hyaluronic acid and/or its salt.

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

<degree of expansion>

The crosslinked product according to an embodiment of the present invention has water swellability, and the degree of swelling to water is 10 times or more and 250 times or less (mass ratio).

Further, in the present invention, "colloid" means a polymer having a three-dimensional network structure, and "water-swellable colloid" means an expansion body which is retained by absorbing moisture inside a three-dimensional network structure, more specifically It is a polymer having a property that water does not flow out of the filter paper when it is placed on the filter paper No. 707 for the Kiriyama funnel for one hour. Further, in the present invention, the "degree of expansion" means the mass of the water (the mass ratio of the crosslinked product to the water) with respect to the mass of the crosslinked product in the water-swellable colloid.

The degree of swelling of the crosslinked product according to the present embodiment is preferably 20 times or more (mass ratio), and more preferably 300 times or less (mass ratio), and is more preferable, from the viewpoint of being more stable and capable of retaining water. 250 times or less (mass ratio) is more preferably 200 times or less (mass ratio), and in the case of a hard gel, it may be, for example, 100 times or less (mass ratio). 50 times or less (mass ratio) is preferred.

<Reversible water swellability>

One of the characteristics of the crosslinked product according to the present embodiment is that it has reversible water swellability. In the present invention, "reversible water swellability" means a property which, upon addition of water, swells and becomes a colloid, and which causes the colloid to dry and remove moisture, becomes solid, and can repeatedly change from colloid to solid and from The nature of the change in solid colloid. Further, the reversible water swellability of the crosslinked product according to the present embodiment can be maintained by heating the crosslinked product or by exposing the crosslinked product to alkaline conditions.

Since the crosslinked product according to the present embodiment has reversible water swellability, the change from the colloid to the solid and the change from the solid colloid can be repeated a plurality of times, so that the workability is excellent.

<Save stability>

Since the crosslinked product according to the present embodiment is a solid, it can be stored as a solid. The hyaluronic acid or the polysaccharide such as the crosslinked product according to the present embodiment is usually stored in a state in which water is present (for example, in the form of an aqueous solution or a colloid), but it is more stable in the state of being stored in a solid state. Therefore, when the crosslinked product according to the present embodiment is stored as a solid, the storage stability is excellent.

<Enzyme-resistant decomposition>

The cross-linked product according to the embodiment has an enzyme-resistant (hyaluronic acid) fraction Solvative. More specifically, the residual ratio of the mixture after storage in 40 ° C for 24 hours in the presence of hyaluronidase (5 units/1 mg per crosslinked product) is preferably 1% or more, and is preferably in the range of 10% or more and 100% or less. More preferably, the mixture is prepared by dispersing the crosslinked product according to the present embodiment in a 50 mM phosphate buffer solution to a concentration of 0.1% by mass (solid content). Further, the enzyme decomposition resistance can be measured, for example, by the method shown in the examples of the present invention.

One of the causes of the excellent enzyme decomposition resistance of the crosslinked product according to the present embodiment is that the crosslinked product according to the present embodiment contains a carboxymethyl group. It is presumed that the crosslinked product according to the present embodiment contains a carboxymethyl group, and hyaluronic acid is difficult to recognize the hyaluronic acid skeleton contained in the crosslinked product according to the present embodiment, so that the enzyme decomposition resistance is improved.

Further, the crosslinked product according to the present embodiment can be distinguished by the presence of a peak of 3.8 ppm or more and 4.2 ppm or less in the ¹ H-NMR spectrum analysis of the crosslinked product, for example, by containing a carboxymethyl group.

<thermal stability>

The crosslinked product according to the present embodiment is excellent in thermal stability. Specifically, the residual ratio of the mixture after storage at 50 ° C for 72 hours is preferably 20% or more, and the residual ratio after storage for 96 hours is 20% or more (and the residual ratio after maintaining 168 hours is 20% or more). In the mixture, the crosslinked product of the present embodiment is dispersed in physiological saline to have a concentration of 1% by mass (solid content). Moreover, the thermal stability can be determined, for example, by the method shown in the examples of the present examples.

The crosslinked product according to the present embodiment is contained by inclusion The modified hyaluronic acid of carboxymethyl group and/or its salt contains more carboxyl groups related to hydrogen bonds than hyaluronic acid and/or its salt, so that the thermal stability is more excellent.

Further, the thermal stability of the crosslinked product according to the present embodiment is gradually lowered with time due to the hydrogen bonding described above. More specifically, the crosslinked product according to the present embodiment has a property of gradually dissociating hydrogen bonds due to heat existing in the living body (for example, before and after 37 ° C to 40 ° C), and therefore has moderate thermal stability.

[Water swellable colloid] <Content of crosslinked product>

The water-swellable colloid according to the embodiment of the present invention comprises a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof, and water, and the content of the crosslinked product is 0.4% by mass or more in terms of solid content (also It may be 0.5% by mass or more and 10% by mass or less.

The ratio of the water contained in the water-swellable colloid according to the present embodiment can be expanded by the difference in mass before and after the water-swellable colloid according to the present embodiment is dried and the moisture is removed. The mass of the colloid before drying is calculated. Further, the drying (water removal) of the water-swellable colloid according to the present embodiment can be carried out, for example, by vacuum drying, drying under reduced pressure, and freeze-drying.

The water-swellable colloid of the present embodiment preferably has a pH of 3 or more and 8 or less from the viewpoint of further improvement in thermal stability.

<thermal stability>

The water-swellable colloid of the present embodiment is excellent in thermal stability. More specifically, the water-swellable colloid of the present embodiment preferably has a residual ratio of 20% or more after storage at 50 ° C for 72 hours, and a residual ratio of 20% or more after storage for 96 hours (further storage for 168 hours) The remaining residual ratio is preferably 20% or more.

<Enzyme-resistant decomposition>

The water-swellable colloid of the present embodiment is excellent in enzyme decomposition resistance. More specifically, the water-swellable colloid of the present embodiment has a residual ratio of 1% or more after storage at 40 ° C for 24 hours in the presence of hyaluronidase (5 units per colloid 1 mL), preferably 10%. Above and above 100% is better.

[Medical Materials]

A medical material according to an embodiment of the present invention comprises a crosslinked product (for example, a water-swellable colloid) according to the above embodiment. In the medical material according to the embodiment, the crosslinked product according to the above embodiment is excellent in enzyme decomposition resistance and thermal stability, and can be stored as a solid, so that the storage stability is excellent.

In particular, the medical material according to the present embodiment includes the water-swellable colloid according to the above embodiment, and the reversible water swellability of the water-swellable colloid according to the above embodiment can be used at the time of use. Water is added to the crosslinked product according to the embodiment to prepare a water-swellable colloid, so that workability is excellent.

The medical material according to the present embodiment can be used for applications suitable for general human body suitability. As such a use, for example, a knee joint injection, a post-adhesion preventive agent, a subcutaneous injection, a drug depot, a carrier for a pharmacologically active substance, a wound covering material, an artificial skin, a surgical suture, a hemostatic agent, an artificial organ, Medical equipment, medical equipment.

<Knee joint injection>

The medical material according to the embodiment can be used as, for example, a knee joint injection. When the medical material according to the present embodiment contains the water-swellable colloid according to the above embodiment, it is excellent in appropriate elasticity, moderate thermal stability, and excellent enzyme decomposition resistance. Therefore, when the medical material according to the present embodiment is used as, for example, a knee joint injection, the water-swellable colloid of the above-described embodiment is not decomposed during the period of the living body, and can remain, and then it will be born. Since the body is decomposed, it is possible to prevent the tissues from sticking to each other and to have excellent safety.

For example, when the water-swellable colloid according to the above embodiment contains, for example, a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof having an average molecular weight of 300,000 or more and 2,000,000 or less and a carboxymethylation ratio of 1% or more, Due to its moderate softness, it can bring a good feeling when injected into the knee joint.

<Formed body>

Since the medical material according to the present embodiment includes the crosslinked product according to the above embodiment, it can be molded in accordance with the applicable portion, and can be, for example, It is colloidal, flaky, film-like, particulate, fibrous, sponge-like, tubular, and can be used in surgery. More specifically, the medical material according to the embodiment in the form of a sheet or a film can be attached to the affected part. Further, when the medical material according to the present embodiment includes the water-swellable colloid according to the above embodiment, for example, in the endoscopic surgery, the colloidal medical material according to the embodiment can be easily injected using an endoscope. Since the affected part is used, it can be suitably used as a post-adhesion preventive agent.

<After the adhesion prevention agent ‧Subcutaneous injection>

The crosslinked product (for example, a water-swellable colloid) according to the above embodiment contained in the medical material according to the present embodiment is excellent in appropriate thermal stability and excellent enzyme decomposition resistance. Therefore, when the medical material according to the present embodiment is used as, for example, a post-adhesion preventive agent or a subcutaneous injection, the water-swellable colloid of the above embodiment does not decompose and remains during the period of the living body, and then It is decomposed in the living body, so that the tissues can be prevented from sticking to each other and the safety is excellent.

<Drug long-acting agent>

The medical material according to the embodiment can be used, for example, as a drug depot. When the medical material according to the embodiment includes the water-swellable colloid according to the above embodiment, water retention, moderate thermal stability, and excellent enzyme decomposition resistance are excellent. Therefore, when the medical material according to the present embodiment is used as, for example, a drug depot, the water-swellable colloid according to the above embodiment is not decomposed during the period of the living body. It remains, and since it is decomposed in the living body, it has the effect of slowing down the auxiliary drug and is excellent in safety.

Further, in the medical material according to the present embodiment, an extender, a binder, a lubricant, a preservative, an antioxidant, a fragrance, a sweetener, a sour material, an excipient, or the like can be blended as necessary. In addition, it can also be used in combination with vitamin C, vitamin B, vitamin B12, vitamin E and other nutrients such as vitamins, nucleic acids, chondroitin sulfate, collagen and other nutrients such as minerals such as iron and zinc.

[cosmetic material]

A cosmetic according to an embodiment of the present invention comprises a crosslinked product (for example, a water-swellable colloid) according to the above embodiment.

<Water retention effect>

Further, 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 crosslinked product according to the present embodiment forms a hydrogen bond with water, it is presumed that the carboxyl group exhibits excellent water retention. Therefore, it has a high water retention effect in biological tissues such as skin. Therefore, by using the crosslinked product according to the present embodiment as a component of, for example, a cosmetic, a high moisturizing effect can be achieved.

Therefore, the crosslinked product according to the above embodiment contained in the cosmetic according to the embodiment has a high water retention effect, moderate thermal stability, and excellent enzyme decomposition resistance, so that it is used as a makeup. When used, it will gradually decompose in the living body, so it can maintain a high moisturizing effect for a long time. Further, when the cosmetic according to the embodiment contains the water-swellable colloid of the above-described embodiment, since it has an elastic property as a colloid, when it is blended in a cosmetic, it is possible to produce a peculiar touch. At the same time, by blending the active ingredient in the colloid, the active ingredient can be slowly released.

The cross-linked product (for example, a water-swellable colloid) according to the embodiment may be coated or contacted with the surface of the biological tissue for ingestion, in particular, coating or contacting the face, wrist, fingers, feet, The skin of joints and the like is preferred.

The form of the cosmetic according to the embodiment is not particularly limited, and examples thereof include a cosmetic for skin. By using the crosslinked product according to the above embodiment for a cosmetic for skin, since it has an appropriate viscosity and a high water retention effect, it is possible to impart moisture to the skin and improve the roughness of the skin.

Examples of the skin cosmetic according to the present embodiment include a cleansing liquid, a washing liquid, a lotion (for example, a whitening lotion), an emulsion (for example, a cream, a cold cream), an emulsion, a cosmetic liquid, and a mask (for example). Gel-like peeling type, cream-like wipe-off type, powder rinse type), facial cleanser, liquid foundation, lipstick, lip balm, lipstick, lip liner, blush, shaving cream, sunscreen lotion, body lotion, body lotion (including hand cream, foot cream), body cream, soap, bathing agent.

The following components may be further blended in the cosmetic according to the embodiment. As the aforementioned component, for example, a cationized polysaccharide (eg cationized hyaluronic acid, cationized hydroxyethyl cellulose, cationized guar gum, cationized starch, cationized corn paste, cationized dextran, cationized butan, cationized honey, etc.), negative ion interface Active agent (such as alkyl benzene sulfonate, polyoxyalkylene alkyl ether sulfate, alkyl sulfate, olefin sulfonate, fatty acid salt, dialkyl sulfosuccinate salt, etc.), non Ionic surfactants (such as polyoxyethylene fatty acid esters, polyoxyethylene hardened castor oil derivatives, etc.), cationic surfactants (such as alkyl trimethyl ammonium salts, dialkyl dimethyl ammonium salts, alkyl pyridines) a cationic salt, stearyl trimethylammonium chloride, etc.), an amphoteric surfactant (for example, an alkyl betaine, a alkyl amidinopropyl betaine, an imidazolinium betaine, an egg yolk lecithin, a soy lecithin, etc.), Oil (such as bismuth, hydrazine derivatives, mobile paraffin, squalane, beeswax, palm wax, olive oil, avocado oil, bitter tea oil, jojoba oil, horse oil, etc.), moisturizer (such as sodium hyaluronic acid, hydrolysis) Hyaluronic acid, acetylated hyaluronic acid, hyaluronic acid dimethyl Sterol, ceramide, lauryl glutamate di-phytosterol octyl dodecyl ester, plant glycogen, hydrolyzed eggshell membrane, trehalose, glycerol, atelopeptide collagen, sorbitol, maltitol, 1 , 3-butanediol, etc.), higher fatty acids (such as lauric acid, sebacic acid, palmitic acid, stearic acid, isostearic acid, oleic acid, etc.), higher alcohols (such as cetyl alcohol, stearyl alcohol, two Decylene, isostearyl alcohol, shark liver alcohol, etc.), polyol (such as glycerin, diglycerin, 1,3-propanediol, propylene glycol, polyethylene glycol, pentene glycerol, etc.), tackifier (such as cellulose) Ester, carboxyvinyl polymer, xanthan gum, palmitic acid dextrin, etc.), amphoteric polymer resin compound (for example, betaine dialkylaminoacrylic acid alkyl ester copolymer, etc.), cationic polymer resin compound (for example, vinylpyrrole) Pyridone / a dimethylaminoethyl methacrylate copolymer cation compound, a polydimethyldiallyl ammonium halide type cationic polymer, etc.), a preservative (for example, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, Butyl hydroxybenzoate, propyl paraben, phenoxyethanol, etc., antioxidants (such as tocopherol, BHT, etc.), metal blocking agents (such as edetate, benzoate, etc.), UV absorption Agents (for example, benzophenone derivatives, amino-benzoic acid derivatives, methoxycinnamic acid derivatives, etc.), ultraviolet light-reflecting agents (such as titanium oxide, zinc oxide, etc.), protein hydrolysates (such as keratin peptides, Collagen peptide, soybean peptide, wheat peptide, milk peptide, silk peptide, protein peptide, etc.), amino acids (such as L-arginine, glutamic acid, glycine, alanine, hydroxyguanamine Acid, cysteine, serine, L-theanine, etc., natural extracts (bitter extract, seaweed extract, eucalyptus extract, royal jelly extract, rosemary extract, alfalfa Tree extracts, etc., other functional ingredients (Coenzyme Q10, Arbutin, Polyquaternium 51, elastin, platinum nanoglia, retinyl palmitate, panthenol, allantoin, sodium lauryl glutamate, sodium ascorbyl phosphate, L-ascorbic acid 2-glycoside, Turkish 鞣Acid, citric acid, linoleic acid, tranexamic acid, etc.), phospholipid polymers, perfumes, pigments.

[Beauty materials]

A cosmetic material according to an embodiment of the present invention comprises the crosslinked product (for example, a water-swellable colloid) according to the above embodiment. By injecting the cosmetic material according to the present embodiment into, for example, the face, the head, the neck, the chest, the abdomen, the buttocks, the back, the waist, the hands, and the legs, it can be used. Achieve the beauty effect (will improve the appearance of breast enhancement, beauty, beauty, etc.). The cosmetic material according to the present embodiment has excellent enzyme decomposition resistance and moderate thermal stability.

[Examples]

Hereinafter, the present invention will be described in more detail by way of examples, but the invention is not limited to the examples.

<Example 1: Modification of modified hyaluronic acid containing carboxymethyl group (raw material modified hyaluronic acid)>

After weighing and taking out 1.04 g of sodium hydroxide in a 30 mL sample vial, 12 mL of water was added to dissolve it. Next, 2.0 g of hyaluronic acid having a molecular weight of 1.75 million was added and dissolved, and then 3.62 g of monobromoacetic acid was added thereto to dissolve, 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 liquid was added while stirring. Thereafter, the precipitate was recovered by a 400-mesh filter cloth, and then 40 mL of a 10% aqueous sodium chloride solution was added to dissolve the precipitate. Further, after the pH was adjusted with an aqueous solution of 8% hydrochloric acid, the mixture was washed three times with 100 mL of ethanol, filtered under reduced pressure, and dried under reduced pressure at 55 ° C for 3 hours to obtain a modified hyaluronic acid containing carboxymethyl group of Example 1. .

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

<Example 2: Modification of modified hyaluronic acid containing carboxymethyl group (raw material modified hyaluronic acid)>

Using a 1000 mL beaker, weighed and took out 42.6 g of sodium hydroxide, dissolved 240 mL of water, dissolved it, added 40.0 g of hyaluronic acid having a molecular weight of 1.8 million, dissolved it, and added 72.4 g of monobromoacetic acid to dissolve it at 4 ° C. It was dissolved in 16 hours. Thereafter, 10% saline was added to dilute the reaction solution until the concentration of hyaluronic acid was 3% by mass. Then, the pH of the reaction solution was adjusted to 6.3 with hydrochloric acid, and ethanol of 3 times the amount of 10% saline was added to prepare a precipitate. The precipitate was washed twice with 70% ethanol, and the residue obtained by washing once with 90% ethanol was dried under reduced pressure at room temperature for 24 hours to obtain a modified hyaluronic acid containing carboxymethyl group of Example 2.

The modified hyaluronic acid containing carboxymethyl group obtained in Example 2 had a molecular weight of 122,000 and a carboxymethylation rate of 99%.

<Example 3: Modification of modified hyaluronic acid containing carboxymethyl group (raw material modified hyaluronic acid)>

The reaction standard of 1/4 of the reaction standard used in Example 2 was used, and the same reaction as in Example 2 was carried out except that the reaction time was 3 hours, and the modification containing the carboxymethyl group of Example 3 was obtained. Hyaluronic acid.

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

<Measurement and calculation of carboxymethylation rate>

Further, the carboxymethylation ratio of the modified hyaluronic acid containing a carboxymethyl group obtained in Examples 1 to 3 was determined by the following method and the integrated value of the ¹ H-NMR spectrum.

(sample preparation)

7 mg of the sample and 1 mg of the internal standard substance sodium 4,4-dimethyl-4-cyclopentanesulfonate (DSS) were dissolved in 0.7 mL of heavy water, transferred, placed in an NMR sample tube, and capped.

(measurement conditions)

Device: Varian NMR system 400NB (Varian Technologies Japan Limited)

Observation frequency: 400MHz

Temperature: 30 ° C

Benchmark: DSS (0ppm)

Total number of calculations: 64 times

(analytical method)

The peak of N-acetinyl group (CH ₃ ) of hyaluronic acid present in the vicinity of 2.0 ppm of the integrated ¹ H-NMR spectrum and the methyl group of carboxymethyl group (-CH ₂ ) which appeared in the range of 3.8 ppm or more and 4.2 ppm or less -) The peak. The number of carboxymethyl groups and the carboxymethylation ratio (CM conversion rate) bonded to each of the two sugar repeating units of hyaluronic acid constituting the modified hyaluronic acid were determined from the integral values and the following formula.

CM conversion rate = (integral value of peaks appearing in the range of 3.8 ppm or more and 4.2 ppm or less / 2) / (integral value of peak of 2.0 ppm / 3)

<Example 4: Preparation of crosslinked product of modified hyaluronic acid containing carboxymethyl group>

The modified hyaluronic acid containing carboxymethyl group obtained in Example 1 was treated under the respective conditions shown in Table 1. First, the modified hyaluronic acid and the addition amount of each additive were adjusted so that the modified hyaluronic acid containing a carboxymethyl group was each concentration shown in Table 1, and it was stored at 4 ° C for 20 hours to be swollen and dissolved to prepare a modification containing a carboxymethyl group. A solution of hyaluronic acid. This solution was kept at -18 ° C for 16 hours. Next, this solution was washed with running water at 25 ° C for 10 minutes to obtain a composition comprising a crosslinked product of a modified hyaluronic acid containing a carboxymethyl group (Experiment Nos. 1 to 4). Further, the pH of the solution in Experiment No. 3 was 0.1, the pH of the solution in Experiment Nos. 14 to 17 was 0.2, and the pH of the solution in Experiment Nos. 1, 2, 5 to 9, 23, and 25 was 0.7. The pH of the solution in Experiment No. 4 was 0.9.

Further, the yields shown in Table 1 are values measured by the following methods. In other words, the composition obtained by each of the conditions shown in Table 1 was dispersed in pure water to have a concentration of 1% by mass to prepare a colloid, and the colloid was allowed to stand at 1 ° C for 24 hours to obtain 1 mass. The % aqueous sodium hydroxide solution was neutralized to a pH of 6. Next, the residue (colloid) was collected by suction filtration on a filter paper, washed with distilled water, dispersed in a weighed petri dish, and dried under reduced pressure. Thereafter, the mass of the culture dish was measured, and the mass of the culture dish was changed. The mass (solid content) of the residue (crosslinked product) was calculated. The yield was calculated by the following formula (3).

Yield (%) = mass of the residue (crosslinker) / mass of the composition (solid content) × 100 (3)

Further, using the modified hyaluronic acid or hyaluronic acid (unmodified) containing carboxymethyl groups of Examples 1 to 3, the conditions different from the above (maintaining temperature, concentration of the additive, concentration of the hyaluronic acid of the raw material), and the inclusion of the carboxy-containing group were obtained. The composition of the crosslinked product of modified hyaluronic acid (Experiment Nos. 5 to 25) was used, and the composition was used and the colloid was prepared in the same manner as in the above method. Further, using the crosslinked product, a thermal stability test and an enzyme decomposition resistance test were carried out in accordance with the method described later. The results are shown in Table 1. Further, in Experiment No. 8, the solution was kept at 4 ° C for 20 hours, and further kept at 25 ° C for 47 hours. Further, in Experiment No. 9, the solution was kept at -18 ° C for 20 hours, and further kept at 25 ° C for 47 hours.

<Test Example 1: Thermal Stability Test>

The crosslinked product of the modified hyaluronic acid containing carboxymethyl groups obtained under the respective conditions shown in Table 1 was dispersed in physiological saline to a concentration of 1% by mass (solid content) to prepare a mixture, and the mixture was mixed at 50 ° C. Let it sit for 7 days. The preserved material was collected by suction filtration using a 420 mesh nylon filter, and then washed with distilled water, then dispersed on a weighed petri dish and dried under reduced pressure. Thereafter, the mass of the culture dish was measured, and the mass of the residue was calculated from the mass change portion of the culture dish. The residual ratio in the thermal stability test was calculated from the following formula (4) from the mass of the crosslinked product and the mass of the residue.

Residual rate (%) in the thermal stability test = mass of the residue / mass of the crosslinked product (solid content) × 100 (4)

<Test Example 2: Enzyme-resistant decomposition test>

10 mg of the crosslinked product of the modified hyaluronic acid containing carboxymethyl groups obtained under the respective conditions shown in Table 1 was separately dispersed in 50 mM phosphate buffer to prepare a mixture of 9 mL, and hyaluronic acid enzyme (SIGMA Inc., from bovine testes) was added. In a unit (1 mL), a mixture having a concentration of 0.1% by mass (solid content) and containing 10 units of hyaluronic acid of 5 mg/crosslinker was placed, and the mixture was allowed to stand at 40 ° C for 24 hours. Next, the preserved material was suction-filtered and collected in a 420-mesh nylon filter, and then washed with distilled water, and then dispersed in a weighed petri dish and dried under reduced pressure. Thereafter, the mass of the culture dish was measured, and the mass of the residue was calculated from the mass change portion of the culture dish. The residual ratio in the enzyme decomposition resistance test was calculated by the following formula (5).

Residual rate in the enzyme decomposition resistance test (%) = mass of the residue / mass of the crosslinked product (solid content) × 100 (5)

It can be understood from Experiment Nos. 1 to 24 of Table 1 that the crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or its salt has water swellable. The properties of the colloid and the degree of swelling to water are 10 times or more and 250 times or less (mass ratio). Further, as shown in Table 1, the mixture of Experiment No. 23 disappeared in 5 days in the thermal stability test.

Further, it can be understood from the experimental numbers 1 to 24 of Table 1 that the crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or its salt and water are contained, and the content of the crosslinked product is 0.4% by mass in terms of solid content. The above water-swellable colloid of 10% by mass or less has excellent enzyme decomposition resistance and moderate heat stability.

On the other hand, the enzyme-resistant decomposition test was carried out for cross-linked hyaluronic acid Synvisc (Genzyme) and Restylane (Q-Med). More specifically, Synvisc is 1.25 g of colloid (containing 10 mg of hyaluronic acid), and Restylane is prepared by dispersing 0.5 g of colloid (containing 10 mg of hyaluronic acid) in 50 mM phosphate buffer to make 9 mL, and adding it to 50 mM phosphate buffer. When 10 mL of the mixture was prepared by using hyaluronic acid 50 units (1 mL), the enzyme decomposition resistance of the mixture was measured in the same manner as in Test Example 2, and it was confirmed that the residual ratio was 0%, and the enzyme decomposition resistance was poor. Further, the residual ratio of the crosslinked product of Experiment No. 25 (Comparative Example 1) was 0%. Since the crosslinked product of Experiment No. 25 (Comparative Example 1) was a crosslinked product of hyaluronic acid, it was understood that the enzyme decomposition resistance was poor.

<Complementary Example 1: lotion>

In the formulation, the lotion of the crosslinked product obtained in Experiment No. 1 of Example 1 was prepared in accordance with the formulation described below.

<Compound 2: Emulsion>

In this compounding example, an emulsion containing the crosslinked product obtained in Experiment No. 1 of Example 1 was prepared in accordance with the formulation described below.

<Compound Example 3: Emulsion>

In this formulation, an emulsion (lubricating emulsion) containing the crosslinked product obtained in Experiment No. 2 of Example 1 was prepared in accordance with the formulation described below.

<Complementary Example 4: Beauty liquid>

In the mixing example, a cosmetic liquid (whitening moisturizing essence) containing the crosslinked product obtained in Experiment No. 2 of Example 1 was prepared in accordance with the formulation described below.

Purified water residue

<Combination Example 5: Beauty Liquid Mask>

In the formulation, the cosmetic liquid mask (paste-like peeling-off type) in which the cross-linked product obtained in Experiment No. 1 of Example 1 was blended was prepared in accordance with the formulation described below.

<Combination Example 6: Cleansing Liquid>

In the formulation, the cleansing liquid (washing lotion type) in which the crosslinked product obtained in Experiment No. 1 of Example 1 was blended was prepared in accordance with the formulation described below.

<Combination Example 7: Sunscreen lotion>

In the present formulation, a sunscreen liquid (emulsion) containing the crosslinked product obtained in Experiment No. 1 of Example 1 was prepared in accordance with the formulation described below.

<Combination Example 8: Lip Balm>

In the present formulation, a lip balm containing the crosslinked product obtained in Experiment No. 1 of Example 1 was prepared in accordance with the formulation described below.

<Combination Example 9: Shampoo>

In the mixing example, the shampoo to which the crosslinked product obtained in Experiment No. 1 of Example 1 was blended was prepared in accordance with the formulation described below.

<Complementary Example 10: Run Conditioner>

In the formulation, the hair lotion containing the crosslinked product obtained in Experiment No. 1 of Example 1 was prepared in accordance with the formulation described below.

<Complementary Example 11: Soft Capsule>

In the present formulation, a soft capsule containing the crosslinked product obtained in Experiment No. 1 of Example 1 was prepared in accordance with the formulation described below.

<Complementary Example 12: Powder>

In the mixing example, a powder (granule) containing the crosslinked product obtained in Experiment No. 1 of Example 1 was prepared in accordance with the formulation described below.

<Complementary Example 13: Soft Capsule>

In the present formulation, a tablet containing the crosslinked product obtained in Experiment No. 1 of Example 1 was prepared in accordance with the formulation described below.

<Complementary Example 14: Colloidal Drink>

In the present formulation, a white peach colloidal beverage having a catheter bag in which the crosslinked product obtained in Experiment No. 1 of Example 1 was blended was prepared in accordance with the formulation described below.

Purified water residue

<Example 3: Post-adhesion prevention agent>

The crosslinked product obtained in Experiment No. 2 of Example 1 was rolled to a film shape having a thickness of 1 mm and molded, and after sterilization, a flaky anti-adhesion agent was obtained.

<Example 4: Subcutaneous injection>

The dried product of the crosslinked product (solid content: 1%) obtained in Experiment No. 2 of Example 1 was swollen with water for injection containing 0.9% NaCl, aseptically filled in a syringe of 1 mL, and sterilized to obtain a subcutaneous injection. .

<Example 5: Drug depot>

The dried product of the crosslinked product (solid content: 2%) obtained in Experiment No. 2 of Example 1 was swollen with water for injection containing 0.9% NaCl and 0.001% prostaglandin E1, and sterilized, and then aseptically filled in 3 mL. The syringe is used to obtain a drug depot.

<Example 6: Knee joint injection>

The dried product of the crosslinked product (0.8% in terms of solid content) obtained in Experiment No. 2 of Example 1 was swollen with water for injection containing 0.9% NaCl, sterilized, and then aseptically filled in a syringe of 2 mL to obtain a knee. Joint injection.

Claims (19)

A method for producing a crosslinked product of a modified hyaluronic acid and/or a salt thereof containing a carboxymethyl group, which comprises maintaining a solution of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof at -200 ° C or higher and 10 ° C or lower step. The method for producing a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof, wherein the concentration of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof in the solution is 1% by mass The above is 30% by mass or less. A method for producing a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to claim 2, wherein the pH of the solution is 3 or less. The method for producing a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of claims 1 to 3, wherein the average molecular weight of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof is 300,000 or more, and the carboxymethylation rate is 1% or more with respect to the 2-saccharide unit which comprises hyaluronic acid. The method for producing a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of claims 1 to 4, further comprising the step of maintaining the composition comprising the crosslinked product by the step of maintaining After the object, the composition is washed with water, and impurities other than the crosslinked product are removed. A crosslinked product of a modified hyaluronic acid and/or a salt thereof containing a carboxymethyl group, which is obtained by the production method according to any one of claims 1 to 5. A crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to claim 6, which is water swellable. A water-swellable crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof, which has a degree of swelling to water of 10 times or more and 250 times or less (mass ratio). The crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to claim 8, which comprises the above modified hyaluronic acid containing carboxymethyl group and/or a salt thereof, the above modified hyaluronic acid containing carboxymethyl group and/or The salt-based average molecular weight is 300,000 or more, and the carboxymethylation ratio is 1% or more with respect to the two-saccharide unit constituting hyaluronic acid. A crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of claims 6 to 9, which has reversible water swellability. The crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of claims 6 to 10, wherein after the mixture is stored at 50 ° C for 72 hours, the residual ratio is 20% or more. The mixture is prepared by dispersing a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of claims 6 to 10 in physiological saline to a concentration of 1% by mass (solid content). The crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or its salt according to any one of claims 6 to 11, wherein the mixture is in the presence of hyaluronic acid (5 units/1 mg per cross-linking) The residual ratio after storage for 24 hours at 40 ° C is 1% or more, and the mixture is such that the crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or its salt according to any one of claims 6 to 11 is dispersed in 50 mM phosphoric acid. In the buffer, the concentration was adjusted to 0.1% by mass (solid content). A water-swellable colloid comprising the items 6 to 12 as claimed The crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or its salt, and water, and the content of the crosslinked product is 0.4% by mass or more and 10% by mass or less in terms of solid content. The water-swellable colloid of claim 13 which has a residual ratio of 20% or more after storage at 50 ° C for 72 hours. The water-swellable colloid of claim 13 or 14 which has a residual ratio of 1% or more after storage for 24 hours at 40 ° C in the presence of hyaluronidase (5 units per colloid 1 mL). A medical material comprising a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of claims 6 to 12. The medical material of claim 16, which is used as at least one selected from the group consisting of a knee joint injection, a post-adhesion preventive agent, a subcutaneous injection, and a drug depot. A cosmetic comprising a crosslinked product of a modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of claims 6 to 12. A cosmetic material comprising the crosslinked product of the modified hyaluronic acid containing carboxymethyl group and/or a salt thereof according to any one of claims 6 to 12.